InteractiveFly: GeneBrief

RNA polymerase II 215kD subunit: Biological Overview | References

BIOLOGICAL OVERVIEW

The uninduced Drosophila hsp70 gene is poised for rapid activation. The rapid changes upon heat shock in levels and location of Heat shock factor (HSF), RNA polymerase II (Pol II) and its phosphorylated forms, and the Pol II kinase P-TEFb on hsp70 were examined in vivo by using both real-time PCR assays of chromatin immunoprecipitates and polytene chromosome immunofluorescence. These studies capture Pol II recruitment and progression along hsp70 and reveal distinct spatial and temporal patterns of serine 2 and serine 5 phosphorylation: in uninduced cells, the promoter-paused Pol II shows Ser5 but not Ser2 phosphorylation, and in induced cells the relative level of Ser2-P Pol II is lower at the promoter than at regions downstream. An early time point of heat shock activation captures unphosphorylated Pol II recruited to the promoter prior to P-TEFb, and during the first wave of transcription Pol II and the P-TEFb kinase can be seen tracking together across hsp70 with indistinguishable kinetics. Pol II distributions on several other genes with paused Pol II show a pattern of Ser5 and Ser2 phosphorylation similar to that of hsp70. These studies of factor choreography set important limits in modeling transcription regulatory mechanisms (Boehm, 2003).

Pol II is highly regulated both at the level of recruitment to promoters and in its progress through the stages of the transcription cycle. This regulation is executed through numerous associations with other proteins as Pol II enters the promoter, melts DNA, initiates transcription, begins early elongation, and eventually matures into a productive elongation complex. Pol II undergoes additional modifications, most notably phosphorylation of the CTD of its largest subunit as it progresses from its hypophosphorylated promoter entry form to the elongation phase, where it is highly phosphorylated at residues Ser2 and Ser5. These changes in phosphorylation are proposed to influence protein association, affecting not only Pol II's elongation properties but also its association with a variety of protein complexes that process pre-mRNA. Moreover, the pattern of phosphorylation is not stagnant during the elongation phases of the cycle and may be signaling specific associations. To define mechanisms involved in these processes in vivo, the rapidly and robustly activated hsp70 gene has been employed as a model (Boehm, 2003).

Technological advances of DNA-protein cross-linking and highly quantitative large-scale PCR assays were used to explore hsp70 activation kinetics of recruiting HSF, the critical Pol II kinase P-TEFb, and Pol II in vivo. The changes in Pol II were examined during the first and subsequent cycles of transcription that are triggered in response to an instantaneous heat shock of Drosophila cells. HSF recruitment occurs very rapidly, detectable at the earliest assay point of 5 s of heat shock, and reaches saturation within 75 s; this result is consistent with the rapid transcriptional response of heat shock genes and with previous, lower resolution assays of HSF recruitment. The recruitment of additional hypophosphorylated Pol II to the promoter occurs with rapidity similar to that of HSF recruitment but before an increase in Pol II phosphorylation at the promoter, which occurs by 75 s. All forms of Pol II achieve a maximal level on the promoter and gene by 5 min. The progress of Pol II across the gene can be observed, and its progress fits the known rate of elongation on Drosophila hsp70, 1.2 kb/min. Interestingly, total Pol II levels remain greater at the transcription start site than at the ORF, even during active transcription, consistent with the observation that promoter escape remains rate-limiting even during heat shock. P-TEFb, a major Pol II kinase, moves across the gene at a rate similar to that of Pol II during the first burst of transcription and thereafter remains distributed over the promoter and ORF during the full 20-min time course examined. This distribution supports a model where P-TEFb contributes to Pol II phosphorylation not only at the promoter but also during most or all of the elongation phase of transcription (Boehm, 2003).

The detection of Ser5 phosphorylation on a promoter-paused Pol II prior to gene induction corroborates a model where this phosphorylation is an early event involved in the transition from initiation of transcription to early phases of Pol II elongation. The mRNA associated with the paused Pol II molecule has previously been shown to be efficiently capped when long enough to allow access of the capping machinery. Since Ser5-P has been reported to enhance Pol II association with mRNA capping machinery and capping activity, the Ser5-P detected on paused Pol II might help to explain the efficient capping of paused RNAs. It is important to note that earlier analyses of the hsp70 gene, which determined that the paused Pol II CTD is hypophosphorylated prior to heat shock, were performed with antibodies different from those used in the present study. Importantly, the antibody generated to detect Pol IIo in those studies was directed against a peptide phosphorylated in vitro by CTK1, a yeast kinase thought to phosphorylate the CTD at serine 2. Thus, the previous analysis did not probe for the Ser5 phosphorylation reported here (Boehm, 2003).

While P-TEFb phosphorylates the CTD primarily at Ser2, it has also been shown to recognize Ser5 as a substrate. Present results suggest that the Ser5 detected in the uninduced state on hsp70 is not a result of P-TEFb activity, since P-TEFb is not detected prior to gene activation (as seen in this study). Ser5 is likely to be the substrate of the cdk7 component of TFIIH early in transcription. Indeed, cdk7 has been found in in vitro studies to be released earlier in the transcription cycle than P-TEFb. In vivo, Cdk7 is required very early in the transcription cycle and contributes to the generation of the paused Pol II on the promoter-proximal region of hsp70 (Boehm, 2003).

Analyses of the phosphorylation status of the CTD in other organisms have found Ser5-P levels to be higher at the promoter than at the ORF, a pattern similar to what was observed on hsp70 during active transcription. When total levels of Pol II are taken into account on hsp70, however, it appears that the level of Ser5-P remains constant along the gene. Comparatively, another study did not see a striking difference in total Pol II density along the genes analyzed. A third study detected more Ser5-P at the promoter than the 3' untranslated region of the human alpha-AT gene but also appeared to detect more total Pol II at the promoter region. Thus, it may be that in metazoans (or on some genes) the level of Ser5-P relative to Pol II is fairly constant along the gene. The possibility that the activity of a phosphatase may be system or gene specific is certainly plausible; for instance, heat shock of HeLa cells deactivates a CTD phosphatase (Boehm, 2003 and references therein).

Under non-heat shock conditions, total Pol II levels were greater at the 5' regions than at the ORFs for several genes that contain promoter-paused Pol II, while histone H1, which does not display a pause by nuclear run-on assay, shows no significant difference of 5' and ORF Pol II signals. Greater levels of Ser5-P were also detected at the 5' end of the genes containing paused Pol II, while levels on H1 were distributed evenly, indicating that this phosphorylation may be a general aspect of the regulatory status of a paused Pol II. This distribution of Ser5-P for the constitutively active genes Tub, GAP, and Actin5C is similar to the results of other studies which analyzed active transcription; however, Ser5-P levels on these genes are constant when standardized to total Pol II, similar to hsp70 in its active state. For these Drosophila genes, the higher level of Ser5-P at the promoter may be attributable to the presence and status of the paused Pol II, indicative of genes regulated at the level of elongation. Indeed, recent studies describe another constitutively active pause gene in human cells, dihydrofolate reductase, which shows a pattern similar to that of Ser5-P for these Drosophila genes (Boehm, 2003 and references therein).

Phosphorylation at Ser2 of the Pol II CTD may be important for processivity into active elongation and has been implicated in downstream events, including pre-mRNA splicing and 3' mRNA processing. Ser2-P levels are undetectable at +58 on hsp70 in the uninduced state, increase quickly at the 5' region upon heat shock, and appear constant through the gene in later time points (for example, 5 min). The increase in phosphorylation detected over time tracks with the recruitment of additional Pol II as well as the recruitment of P-TEFb. Taking into account total Pol II levels, there appears to be a slight increase in Ser2-P as Pol II progresses through the ORF. This correlates with the concomitant and approximately equivalent decrease in Pol IIa. Ser2-P patterns on additional genes containing a paused Pol II, when considered relative to levels of detectable total Pol II, are significantly higher in the ORF than are those in the 5' region. While these ratios may simply be a consequence of promoter-paused Pol II not being Ser2 phosphorylated, this result is similar to that of another study, where Ser2-P was only detected in the ORF. These observations led to speculation that an increase in Ser2-P may be important for cueing specific processes as Pol II proceeds through the gene. P-TEFb, the major kinase implicated in Ser2 phosphorylation, was detected concomitant with Pol II during active transcription on hsp70. While Pol II/P-TEFb ratios appear constant, a slight increase in Ser2-P occurs at the 3' end of the gene. As the presence of the kinase is not an indicator of its activity, work presently ongoing in the laboratory on P-TEFb kinase inactivation and hsp70 gene regulation should help to better understand this process (Boehm, 2003 and references therein).

Lastly, analysis of immunostaining of polytene chromosomes provides independent corroboration of the higher resolution and quantitative ChIP assays and provides insight into the formation and composition of the transcription puff. Paused Pol II on hsp70 was previously detected with this method, as was Pol II along hsp70 during heat shock. A modest detection of Ser5-P was observed on the promoter prior to heat shock. During the early stages of puff formation, Pol II resolves from promoter-bound HSF. Ser2-P and Ser5-P occupy the most decondensed regions of the puff forming a halo around the heat shock loci, while HSF is more concentrated at the chromosome core at one end of the puff. Taken together, these ChIP and immunofluorescence results provide a foundation for additional temporal and spatial assignments of specific factors relative to the phosphorylation events during the activation of transcription. Perturbation of the function or activity of specific factors using genetic and drug-based approaches will provide further insight into the mechanistic role of these factors in the recruitment and modification of transcriptional machinery and in the coupling of specific transcription processes and Pol II modifications to RNA processing events (Boehm, 2003).

Phosphorylation induces sequence-specific conformational switches in the RNA polymerase II C-terminal domain

The carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit cycles through phosphorylation states that correlate with progression through the transcription cycle and regulate nascent mRNA processing. Structural analyses of yeast and mammalian CTD are hampered by their repetitive sequences. This study identified a region of the Drosophila melanogaster CTD that is essential for Pol II function in vivo and capitalized on natural sequence variations within it to facilitate structural analysis. Mass spectrometry and NMR spectroscopy reveal that hyper-Ser5 phosphorylation transforms the local structure of this region via proline isomerization. The sequence context of this switch tunes the activity of the phosphatase Ssu72, leading to the preferential de-phosphorylation of specific heptads. Together, context-dependent conformational switches and biased dephosphorylation suggest a mechanism for the selective recruitment of cis-proline-specific regulatory factors and region-specific modulation of the CTD code that may augment gene regulation in developmentally complex organisms (Gibbs, 2017).

The carboxy-terminal domain (CTD) of Rpb1, the largest subunit in RNA polymerase II, is an essential regulator of eukaryotic gene expression. This intrinsically disordered protein (IDP), consisting of multiple tandem repeats of the consensus sequence (Y¹S²P³T⁴S⁵P⁶S⁷), acts as a scaffold for the recruitment of factors required for transcription, mRNA biogenesis and modification of the chromatin structure. Tight control over the spatial and temporal recruitment of CTD-associated factors is regulated at the molecular level in part by CTD-specific kinases and phosphatases, which generate dynamic patterns of post-translational modifications (PTMs) collectively referred to as the ‘CTD code'. While much is known about how heptads matching the consensus sequence contribute to gene expression, heptads that deviate from the consensus are found in all eukaryotes whose sequences are known. The number and complexity of these non-consensus heptads roughly correlate with developmental complexity. Expression of genes involved in multicellularity were affected by mutating non-consensus heptads of mouse cells in culture, despite data indicating that the non-consensus heptads are not essential for the viability of human cells grown in culture. Likewise, deletion of a small region encompassing several non-consensus heptads caused severe developmental defects and growth retardation in mice, demonstrating that non-consensus heptads may contribute to development and cellular differentiation (Gibbs, 2017).

Despite the wealth of information detailing CTD function, little is known about the molecular basis of the CTD code. The established view is that the intrinsically disordered nature of the CTD renders its structure and interactions non-specific, to be dictated predominantly by its PTM status. Prior structural studies, focussing on short CTD peptides composed entirely of consensus heptad repeats, revealed turn and coil structures that have been extrapolated to represent the entirety of the CTD. The repetitive amino-acid sequence comprising the CTD has been a major obstacle in studying its structure because it prevents heptad-specific interpretation of both mass spectra and NMR spectra. Therefore, recent mass spectrometry-based investigations have resorted to introducing mutations that facilitate analysis of specific regions of the CTD. However, it is unclear how best to interpret these results molecularly because the impacts of mutations on IDP structure are often difficult to predict. This study turned to Drosophila, which is unique among commonly used model organisms in that only 2 of its 42 heptads precisely match the consensus sequence. This feature has allowed use NMR and mass spectrometry to precisely monitor PTM patterns and local structural features in the context of a natural CTD sequence (Gibbs, 2017).

A model in which hyper-pSer5 does little to alter the scaling properties of the CTD2' region of Drosophila CTD; specifically the measured R_g and D_max are unaltered. Instead, dramatic structural rearrangements occur on the single heptad scale, driven by sequence context-dependent proline trans-to-cis isomerizations. A general conclusion from these observations is that these features allow the CTD to transduce homogenous PTMs into structurally and functionally diverse responses. This discovery predicts multiple potential mechanistic outcomes in the context of the CTD code (Gibbs, 2017).

The first general conclusion supported by these findings is that sequence context-dependent structural switches created through enriched cis-proline isomerization have the ability to facilitate or impair the binding of isomer-sensitive CTD interacting factors at specific regions of the CTD. The diversity of CTD sequences across eukaryotes has been recently acknowledged, though the functional significance of many non-consensus heptads has not been widely investigated. Non-consensus CTD repeats may expand the repertoire of available PTMs, thus increasing the complexity of signalling through the CTD. In the minimal system, it was observed that non-consensus motifs that contained conservative deviations from the consensus responded similarly to Dm P-TEFb and Ssu72. In contrast, more cryptic variants such as YSPNSPS produced drastically altered outcomes, depending on the modifying enzyme present, and substantial deviation from the consensus heptad sequence rendered some repeats resistant to modification by both enzymes. Even in the context of the heptads that conform the least well to the consensus, it is emphasized that the assays did not yield substantial phosphorylation of Ser2 residues, suggesting a strong specificity of Dm P-TEFb for serine residues occupying the 5-position of the heptad in vitro (Gibbs, 2017).

The findings suggest that the relationship between consensus conservation and functional specialization in the CTD may lie on a continuum. In this view, conservative sequence deviations may be tolerated by the majority of regulating enzymes, imparting only modest differences in modification kinetics and patterning. By contrast, more dramatic deviations from the consensus sequence may only support interaction with a subset of regulatory factors. While the unique functions the full set of conserved non-consensus motifs serve during transcription will need to be determined empirically, the first set of observations provide support for the emerging hypothesis that variation in CTD sequence enables differential gene regulation in the context of normal development or at the level of individual genes. In this context, the model leads to the prediction that conserved heptads maintain the ability to attract a wide range of factors involved in basic cellular processes, while non-consensus heptads enhance spatial control of interacting factor recruitment, thus creating more tightly regulated transcriptional programmes in higher eukaryotes (Gibbs, 2017).

The observation that non-consensus heptads within the Dm CTD-containing Asn7 show a preponderance for cis-pro6 in response to pSer5 is striking. These heptads are conserved from yeast to human and tend to cluster in a region just beyond the consensus repeats. For example, the human CTD has five heptads that contain Asn7, clustered between repeats 20 and 30. The results suggest that, as in Drosophila, these regions should populate a high degree of cis-proline when pSer5 marks are prevalent, as in early phases of the transcription cycle. This could restrict the binding of many known pSer5-trans-Pro6 CTD interacting factors or, alternatively, favour the association of pSer5-cis-Pro6-specific factors. Ssu72 is a pSer5-cis-Pro6-specific phosphatase known to promote the transition from hyper-pSer5 to hyper-pSer2 during transcript elongation. In line with this model, the results demonstrate that Asn7 heptads can increase the apparent Ssu72 activity targeted to a cluster of heptads in the CTD. Recent work has revealed that most CTD heptads are phosphorylated only once at any given time, suggesting ordered erasing of the pSer5 mark prior to writing of the pSer2 mark. Further, the average number of phosphates carried by yeast and human CTDs appears to be much less than the number of repeats, underpinning the transient nature of CTD modifications. The results suggest that one role of Asn7 repeats may be to pre-prime this particular region of the CTD for pSer5 dephosphorylation by Ssu72, thus assisting in the dynamic spatiotemporal control of the pSer5-to-pSer2 transition (Gibbs, 2017).

Finally, the observations reported here predict a potential mechanistic route to the establishment of rare phospho-marks. For example, it has been shown that certain CTD interacting factors, such as the histone methyltransferase Set2 recognize doubly phosphorylated repeats. Notably, little to no Ssu72 activity was observed on YSPNSPS and YSPSSSN heptads phosphorylated on Ser5. Thus these heptads remained poised for Ser2/Ser5 double phosphorylation, which may ultimately recruit specific regulatory enzymes to the CTD, such as Set2. Such a gradient in dephosphorylation following distributive phosphorylation could generate multiple epitopes for obligate co-transcriptional processes (such as capping enzyme binding pSer5 repeats) while ensuring a minimum number of epitopes are created for processes that may not be essential for each round of transcription (for example, Set2-mediated histone methylation in gene bodies). Thus the strategy employed in this study highlights the general feasibility of applying quantitative biophysical techniques to obtain mechanistic insights into the CTD code, revealing a multi-layered mechanism for the regulation of spatially and temporally ordered recruitment of factors to the CTD (Gibbs, 2017).

The C-terminal domain of RNA polymerase II is a multivalent targeting sequence that supports Drosophila development with only consensus heptads

The C-terminal domain (CTD) of RNA polymerase II (Pol II) is composed of repeats of the consensus YSPTSPS and is an essential binding scaffold for transcription-associated factors. Metazoan CTDs have well-conserved lengths and sequence compositions arising from the evolution of divergent motifs, features thought to be essential for development. On the contrary, this study shows that a truncated CTD composed solely of YSPTSPS repeats supports Drosophila viability but that a CTD with enough YSPTSPS repeats to match the length of the wild-type Drosophila CTD is defective. Furthermore, a fluorescently tagged CTD lacking the rest of Pol II dynamically enters transcription compartments, indicating that the CTD functions as a signal sequence. However, CTDs with too many YSPTSPS repeats are more prone to localize to static nuclear foci separate from the chromosomes. It is proposed that the sequence complexity of the CTD offsets aberrant behavior caused by excessive repetitive sequences without compromising its targeting function (Lu, 2019).

Transcription in eukaryotes involves coordination of RNA synthesis, RNA processing, and modulation of chromatin structure. The C-terminal domain (CTD) of the largest RNA polymerase II (Pol II) subunit, Rpb1, plays a central role by serving as a landing pad for many of the proteins involved in these processes. There is considerable variation in the amino acid sequence composition of CTDs across different evolutionary lineages. For example, the yeast CTD and the proximal half of the mammalian CTD are composed primarily of the repeating consensus heptad YSPTSPS, whereas the distal half of the mammalian CTD consists primarily of divergent motifs that differ from the consensus at one or more positions. In addition, the length of the CTD roughly scales with developmental complexity. The divergent motifs and CTD length are postulated to have evolved to provide additional layers of control for gene expression programs essential for the development of multicellular organisms, although this remains largely untested (Lu, 2019).

This study assesses the significance of sequence complexity and length of the CTD in animal development and viability by using Drosophila as a model. The Drosophila CTD contains 42 repeats, only 2 of which match the consensus heptad. Surprisingly, CTDs composed solely of YSPTSPS repeats suffice to support fly development. In addition, although consensus CTDs at approximately half wild-type lengths fully support development, consensus CTDs at wild-type lengths do not. Thus, having too many consensus heptads is deleterious, and this can be counteracted by substituting consensus heptads with divergent motifs. These results argue against the notion that highly conserved, lineage-specific divergent motifs and lengths of the CTD are essential for metazoans. To gain insight into the functional differences between various CTDs, this study analyzed their behaviors when expressed separately from the rest of Pol II. It was observe that a GFP-tagged wild-type Drosophila CTD dynamically enters transcription compartments. This defines the CTD as a signal sequence that could serve to target Pol II to sites of active transcription. Furthermore, CTDs harboring too many consensus heptads form static nuclear foci when fused to GFP and fail to support viability in the context of Pol II. These results suggest that the sequence of the CTD has evolved to balance length and composition to dynamically target Pol II to transcription compartments without resulting in aggregation (Lu, 2019).

This functional analysis of the CTD in Drosophila challenges the long-standing proposition that features of the CTD unique to multicellular organisms are essential for development. CTDs of 20 to 29 consensus heptads support Drosophila viability, development, and thermotolerance. In contrast, CTDs of 10, 42, or 52 consensus heptads do not. It is unlikely that the viability of the fully consensus CTD flies is due to compensatory mutations that have occurred elsewhere in the genome because the generation of healthy fly lines containing the 20, 24, and 29 consensus heptads using CRISPR did not require screening an inordinate number of candidates. Moreover, these results are in accordance with the RNAi rescue results, in which the selective pressure is only applied during one generation when flies are mated to a GAL4 driver line (Lu, 2019).

Remarkably, the optimal range in the number of consecutive consensus heptads that functions in Drosophila is similar to the number of consecutive consensus and near-consensus heptads found in a wide spectrum of eukaryotes ranging from yeast to plants. It is proposed that this consecutive array of consensus heptads functions as a single unit. Otherwise, it is difficult to account for why the number of consecutive heptads that is conserved within a given taxon far exceeds the sizes of the binding sites for known proteins. For example, a stretch of 15 consensus heptads is conserved between human and mouse, but the known binding sites for proteins that associate with the CTD only span three heptads or less. In addition, CTD mutant strains of yeast with varied numbers of consensus heptads spontaneously acquire additional mutations that result in CTDs approximating the natural number of 26. How this consecutive array of repeats functions as a single unit is not known. One possibility is that it provides for phosphorylation-dependent, highly cooperative regulation of factor binding. This entropically driven mechanism of regulation could provide switch-like binding and release of factors dictated by threshold levels of phosphorylation (Lu, 2019).

The divergent motifs of Drosophila likely function within the specific context established by the overall amino acid sequence of the Drosophila CTD. Otherwise, it is difficult to reconcile how all of the divergent motifs can be replaced with consensus heptads but the natural sequence of the CTD is highly conserved between distantly related species of Drosophila. Likewise, in spite of the high sequence conservation within the chordate lineage, the CTD in human cells grown in culture can be replaced with an all-consensus CTD without losing viability. Several ways in which the functions of the divergent motifs could be context-dependent are envisioned. Small angle x-ray scattering (SAXS) analysis of the Drosophila CTD reveals that it adopts a compact random coil structure. This implies the occurrence of transient intramolecular interactions. In addition, the CTD self-associates, and this self-association could contribute to the clustering of Pol II molecules that has been linked to transcription. These intra- and intermolecular interactions could extend over the entirety of the CTD, placing constraints on the sequence composition throughout the CTD. Another possibility is that divergent motifs themselves, or through interaction with other proteins, serve as binding sites for the same cadre of proteins that otherwise directly bind consensus heptads. This would explain why a fully consensus CTD functions in place of the natural Drosophila counterpart. A corollary to this hypothesis is that the divergent motifs do not provide unique regulatory functions; they simply provide alternative pathways for interactions normally provided by consensus heptads. Divergent motifs could have emerged as a consequence of constructive neutral evolution in which a chance mutation resulted in loss of consensus heptads but fortuitously produced motifs that associated with a protein providing an alternate to the lost consensus heptads. Future investigations should seek to determine whether known proteins that recognize consensus heptads also associate with divergent motifs and whether there are adaptor proteins selectively binding divergent motifs in the Drosophila CTD that, in turn, serve to recruit proteins that, in other species, bind directly to consensus heptads (Lu, 2019).

Tht finding that the functionality of the consensus CTD is dependent on the number of consensus heptads prompted an exploration of the behavior of the various CTDs when they were expressed separately from the rest of Pol II. Previous reports showed that the CTD partitions into liquid phase-separated droplets and hydrogels and the proposition that the highly transcribed regions of Drosophila polytene chromosomes known as puffs could be liquid phase-separated compartments (Lu, 2019).

GFP-tagged versions of the CTD associate with puffs, and the characteristics of these interactions are consistent with puffs being phase-separated compartments. First, treatment of salivary glands with 1,6-hexanediol caused the CTD to disperse from puffs, a behavior consistent with that of proteins partitioning into liquid phase-separated domains. Second, FRAP analysis showed that the CTD's association with puffs is dynamic and has a recovery time after photobleaching of several seconds. This is similar to the recovery times reported for the CTD when it partitions into phase-separated droplets formed by the low-complexity domain of the FUS protein. Finally, although CTDs composed of 20, 24, or 29 consensus heptads become enriched in puffs, a CTD composed of 10 consensus heptads did not. This dependence on length is consistent with the puff interaction being dependent on the valency of the CTD. Liquid phase separation in biological systems is driven by networks of weak interactions between multivalent molecules. The capacity of a protein to become enriched in a phase-separated domain depends on its capacity to interact favorably with the network. It is speculated that the CTD with 10 consensus heptads lacks sufficient valency to interact favorably with other components in the puff (Lu, 2019).

It is proposed that the CTD is functioning as a signal sequence and is serving to target Pol II to highly transcribed regions of the genome. Several molecules are known to reside at puffs that could be involved in establishing a compartment that is targeted by the CTD. One is the CTD itself because it was recently shown that the CTD self-associates into phase-separated droplets in the presence of crowding agents and that these droplets recruit Pol II (Boehning, 2018). Another is poly(ADP-ribose), whose chains are known to contribute to puff formation and have been shown to nucleate phase separation. Yet another could be FUS, which has been shown to be present at puffs and to form phase-separated droplets that can enrich the CTD. Notably, all three of these components of puffs are also found at transcribed loci in diploid cells, indicating that transcription compartments analogous to puffs could form in these cells (Lu, 2019).

In addition to its enrichment at puffs, it was observed that GFP-tagged CTD formed foci that were clearly separate from the chromosomes. FRAP analysis showed that some of these foci were dynamic whereas others were not. The recovery time for the dynamic foci was comparable with the recovery times detected at puffs. Future experiments with fluorescently labeled Pol II should reveal whether the foci formed by the CTD also include Pol II or other transcription machinery harboring the multivalent, low-complexity domains so often associated with phase separation (Lu, 2019).

The abundance of CTD foci increases dramatically when the number of consensus heptads in the CTD increases from 29 to 42 or 52. In some cases, a small number of large clumps is observed instead of multiple foci, as if the foci have coalesced. The increase in the number of foci and formation of large clumps is consistent with the known behavior of multivalent polymers, where expansion of repeat numbers can cause molecules to transition from being dynamic to forming static, dysfunctional aggregates. In contrast to the 42 consensus CTD, the Drosophila CTD, which is comparable in length, does not form large numbers of foci or clumps. This raises the possibility that divergent motifs function to counteract the tendency of the multivalent CTD to form dysfunctional aggregates. It is speculated that the formation of such aggregates could be the reason why Pol II with the 42 or 52 consensus heptads result, respectively, in defects in male fertility and pupal development (Lu, 2019).

It has been almost 4 decades since the CTD was first described, and its function remains enigmatic. Virtually everything known about the CTD's function has focused on the consensus heptads in yeast and mammalian cells grown in culture. The current results now establish Drosophila as a powerful model system for investigating CTD function. Specifically, imaging Drosophila polytene chromosomes in intact nuclei represents a democratizible system for the simultaneous visualization of the nucleoplasm and complete genome with common and accessible microscopy. The platform introduced here is amenable to exploring the partitioning, miscibility, and dynamics of any number of low-complexity domains associated with the transcription machinery with the ability to map localization to precise genomic loci, discern between heterochromatic, euchromatic, and nucleoplasmic compartments, and to tune expression levels and timing with the available suite of Drosophila genetic tools (Lu, 2019).

m⁶A RNA methylation regulates promoter- proximal pausing of RNA polymerase II

RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. This study shows that the m⁶A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m⁶A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m⁶A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m⁶A catalytic domain. Collectively, these data reveal an important link between RNAP II pausing and the m⁶A RNA modification, thus adding another layer to m⁶A-mediated gene regulation (Akhtar, 2021).

N6-methyladenosine (m6A) has recently emerged as the most-prevalent mRNA modification in eukaryotes, controlling various developmental, cellular, and molecular processes, such as pre-mRNA splicing, alternative polyadenylation, mRNA decay, and translation. Genome-wide mapping of m6A in vertebrates revealed a preferred enrichment at specific subsets of sites centered mostly around stop codons, last exons, and 3' untranslated regions (UTRs) of many mRNAs, as well as, to a lesser extent, at 5' UTRs. m6A is deposited on mRNA by a multi-subunit complex, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms-tumor-1-associating protein (WTAP in vertebrates and Fl(2)d in Drosophila), and four additional factors (RBM15/RBM15B in vertebrates, Spenito in Drosophila; VIRMA in vertebrates, Virilizer in Drosophila; ZC3H13 in vertebrates, and Xio/Flacc and HAKAI in both vertebrates and Drosophila). This complex is commonly referred to as the m6A 'writer' or methyltransferase complex (MTC). The downstream function of m6A is mediated by the so-called 'reader' proteins; among which, YTH-domain-containing protein families are the best described. Most m6As are deposited co-transcriptionally, allowing the coupling of this modification with transcription. Indeed, recent studies have shown that m6A complex members can be recruited to the chromatin by chromatin-associated proteins, post-translational modifications of histones, or transcription factors. One interesting aspect, recently uncovered, is the crosstalk between the transcription machinery and translational regulation through the m6A RNA modification. Specifically, RNA polymerase II (RNAP II) kinetics was shown to affect m6A deposition, with slower transcriptional dynamics enhancing the m6A deposition on mRNAs and impeding translation. In addition, m6A was found at many regulatory RNAs, regulating their abundance and causing indirect downstream effects on transcription and chromatin accessibility (Akhtar, 2021).

Transcription is one of the most important cellular mechanism controlling eukaryotic gene expression and follows three different highly regulated stages: initiation, elongation, and termination. After initiation and promoter clearance, RNAP II undergoes a key rate-limiting step of pausing, whereby it stalls and accumulates 30-60 nt downstream of the transcription start site. This mechanism is particularly important for developmentally and stimulus-controlled genes and serves as a checkpoint that coordinates transcription elongation, chromatin modifications, and mRNA processing (Akhtar, 2021).

Pausing has been linked to the phosphorylation state in the heptapeptide repeat of the C-terminal domain (CTD) of the large subunit of RNAP II. The phosphorylation state of CTD correlates with various stages of transcription, for example: Ser5 phosphorylation of CTD is required for mRNA capping and Ser2 phosphorylation is necessary for the recruitment of splicing factors and polyadenylation and corresponds to the elongating form of RNAP II. The transition from pausing to early elongation is critical for gene expression in higher metazoans and requires several factors. In Drosophila, paused genes predominantly fall into two categories: one is associated with GAGA factor (GAF) binding at their promoter, whereas the other binds M1BP. Together with these pause-enhancing factors, eukaryotic cells have also evolved a number of specialized elongation factors, such as polymerase associated factor 1 (PAF1), FACT, and Spt6. Various studies have underlined the significance of this transition and demonstrated its function as a key checkpoint in mRNA processing. Despite the co-transcriptional deposition of m6A, its role in regulation of RNAP II transcription is understudied (Akhtar, 2021).

This study found that the m6A MTC directly regulates the release of RNAP II from its paused state. In Drosophila cells, the complex is predominantly recruited to gene promoters, in an RNA- and transcription-dependent manner but independent of splicing. This study found predictive enrichment of motifs and binding of pause-regulating factors, such as GAF and M1BP, in regions occupied by the m6A MTC and validated their involvement in recruiting m6A MTC at the promoter. Furthermore, evidence is provided that the m6A complex components interact with the histone chaperone Spt6, previously shown to enhance elongation in Drosophila. Moreover, tethering Mettl3 to a heterologous gene promoter is sufficient to enhance the release of RNAP II toward the gene body. Lastly, it was shown that the catalytic domain of Mettl3 is essential for its recruitment to the chromatin and for promoting transcriptional effect. Collectively, these results support a model in which m6A, through its MTC, directly feeds back on the transcription machinery via the control of promoter proximal pausing of RNAP II (Akhtar, 2021).

These data uncovered a novel mechanism in which transcription-dependent recruitment of the m6A complex components positively feedbacks on the transcription machinery to promote release of RNAP II from the paused state. Although this study provides the first evidence for a role of m6A in RNAP II pausing, that link is not unexpected. First, m6A complex components and pause factors are both found at gene promoters, and the most-prevalent paused genes (i.e., developmentally regulated and stress-associated genes) tend to also be bound by the m6A MTC at their promoter. Second, inhibiting elongation by DRB, a well-known elongation inhibitor, had no effect on the recruitment of m6A MTC recruitment, suggesting that this recruitment is independent of, and happens before, transition into elongation. Third, the m6A RNA modification has a critical role during cell fate decision in early mouse development and in stem cell differentiation. These same processes are highly subjected to RNAP II pausing and might involve m6A RNA for their precise control. Interestingly, the current results suggest that m6A RNA modification affects pause regulation on many genes, demonstrating its involvement as a generic contributor in fine-tuning the transcriptional activity of paused genes. Furthermore, the effect of m6A MTC on regulation of pause escape is more pronounced on most paused genes in which m6A MTC recruitment is also most prevalent. Surprisingly, we could also observe binding of the m6A MTC complex on well-characterized enhancers, suggesting an additional uncharacterized function of m6A MTC on gene expression (Akhtar, 2021).

Another interesting aspect of the m6A in pause release comes from studies on DNA:RNA hybrids or R-loops, where m6A was shown to regulate R-loops removal (Abakir, 2020). Many of the pause sites have higher propensities of forming R-loops, and factors favorable for R-loops formation and RNAP II pausing are interconnected. These findings suggest that m6A could potentially mediate R-loop-dependent RNAP II pausing (Akhtar, 2021).

An unsupervised approach was implemented to investigate one of the key questions in the field regarding the specificity of the m6A deposition. Indeed, the degenerate consensus motif RRCAH (in which A = m6A) has been identified as a key motif for m6A deposition in several genome-wide mapping studies. However, the motif itself is more prevalent than the experimental validated frequency of m6A on RNA. This suggests that other factors have a crucial role in targeting m6A on RNA. Coherently, chromatin-associated proteins drive the deposition of m6A on RNA through m6A MTC recruitment on chromatin. One study, using acute myeloid leukemia (AML) cells identified CEBPZ-dependent recruitment of Mettl3 to promoters of a selected set of genes. Another intriguing study demonstrated H3K36me3 as a key determinant of the deposition of m6A on RNA. However, the lack of H3K36me3 at promoters and the occurrence of m6A in the 5' UTR of the genes, especially in Drosophila, suggest that additional features determine the deposition of this mark. Indeed, the distribution of m6A on RNA is different between vertebrates and Drosophila, suggesting that the deposition and ensuing regulation can be species dependent. However, the deposition of m6A in vertebrates has also been reported to be developmental-stage- and cell-type-dependent, with distinct deposition patterns, including on 5' UTR. The approach used in this study identified RNAP II, CTD-state, and pause factors (GAF and M1BP) as the most important features predictive of m6A complex binding at promoters. That is also consistent with its observed effect on RNAP II pause regulation and the transition to elongation. Consistently, the KD of kinases responsible for the phosphorylation of the CTD resulted in the decrease of Mettl3 binding at promoters. Although, that effect could potentially be mediated through other targets of those kinases. Lastly, transcriptional kinetics has also been shown to influence the deposition of this mark on RNA. All the evidence points to the existence of context-dependent features and of extensive crosstalk in guiding m6A and m6A MTC on the RNA and chromatin, respectively. Further investigations are required to identify all the factors responsible for guiding the deposition and removal of this mark in a dynamic biological process (Akhtar, 2021).

This study also identified Spt6, previously reported as elongation factor, interacting with the m6A complex. Recent studies in yeast and plant suggest an additional role of Spt6 in precisely selecting the TSS and preventing cryptic transcription starts. These results indicating a role for Spt6 in the recruitment of MTC at TSS could also implicate m6A in the fidelity of TSS selection. Furthermore, Spt6 participates in mRNA homeostasis by inducing mRNA decay through interactions with RNAP II and the Ccr4-Not deadenylation complex. m6A RNA modification has been also described to control mRNA decay, partly through Ccr4-Not. Those studies, combined with the. current findings, show another potential crosstalk between Spt6 and m6A components and support the existence of a feedback loop between mRNA synthesis and decay, allowing for rapid changes of global mRNA levels of developmental and stress regulated genes (Akhtar, 2021).

Finally, m6A was recently shown to indirectly inhibit transcription by regulating the stability of chromatin-associated RNAs (Liu et al., 2020). Although this result might seem to contradict the current findings (which, rather, point toward a positive action of m6A on transcription regulation), this potential multifaceted and context-dependent regulation is reminiscent of a similar action of the polymerase-associated factor 1 (PAF1). Indeed, depending on the cell type, PAF1 is required for either stabilizing RNAP II pausing or promoting its pause release (Akhtar, 2021).

In conclusion, the m6A-mediated control of RNAP II pausing adds a novel layer to an already highly regulated process. The feedback mechanism reported here establishes m6A as a critical regulator of the transcriptional checkpoint involving RNA processing (Akhtar, 2021).

It remains to be determined whether this function of m6A MTC is conserved across higher metazoans. Moreover, it would be interesting to demonstrate this function in a developmental context for e.g., during embryogenesis. This study, using a machine learning approach, attempted to determine the features associated with the recruitment of m6A MTC to chromatin; however, the resulting binding matrix is certainly not exhaustive. Several additional characteristics could determine the specificity of m6A deposition across the genome. In addition, it would be extremely beneficial to understand whether the chromatin recruitment of m6A MTC is a prerequisite for RNA modification or vice versa (Akhtar, 2021).

Motif 1 Binding Protein suppresses wingless to promote eye fate in Drosophila

The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. This study found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a "no-eye" phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wg^RNAi, shows a significant rescue of a reduced eye or a "no-eye" phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. Upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. These data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development (Raj, 2020).

Pol II pausing near the transcription start site has been identified as a key step in optimizing transcription of many genes in metazoans. It has been proposed that pausing allows the coupling of transcription and RNA processing. Pausing can contribute to dynamic regulation of gene expression in response to developmental and environmental signals, and can function to repress transcription. The genome-wide studies have revealed that  ~10-40% of all genes in mammalian embryonic stem cells and Drosophila have paused promoters. In Drosophila, while the phenomenon of promoter proximal pausing has been well studied in regulation of genes encoding the heat shock proteins (Hsp) and different components involved in immune response pathways, it is also proposed to play important role in regulating the gene expression during early developmental events such as patterning, sex determination etc. So far, the sequence-specific transcription factors such as GAGA factor and M1BP, and other regulators HEXIM, LARP7 (La Ribonucleoprotein 7, Transcriptional Regulator) have been implicated in dictating Pol II pausing in Drosophila. However, the biological relevance of transcriptional pausing and the exact mechanism by which the regulatory factors may contribute in pausing of Pol II is not fully understood (Raj, 2020).

M1BP regulates retinal determination and MF progression in developing eye This study tested fthe role of transcription pausing factor, M1BP during Drosophila eye development. Downregulation of M1BP levels in the developing eye was found to result in strong suppression of eye fate, however, gain-of-function of M1BP did not affect the eye fate suggesting that optimum levels of M1BP are required for Drosophila eye development. Furthermore, no domain constraint in eye suppression function was seen when M1BP levels were downregulated. In addition, when M1BP levels were downregulated (ey > M1BP^RNAi) the expression of retinal determination and differentiation genes were strongly downregulated. Interestingly, it was found that protein encoded by RD genes were downregulated in ey > M1BP^RNAi background. Therefore, M1BP may not be affecting RD gene expression directly (Raj, 2020).

During eye development, a wave of differentiation, emanates from the posterior margin of the developing eye imaginal disc, which sweeps anteriorly across the retinal primordium. The crest of this wave is referred to as the MF, which results in retinal differentiation behind it. The two signals dpp and hh plays an important role in initiation and progression of MF. Downregulation of M1BP affects retinal differentiation as well as progression of MF. It suggests that M1BP role is to promote retinal differentiation as well as MF progression. Also, M1BP downregulates the level of negative regulator(s) of the eye fate. This study screened for the genes, which may serve as target for M1BP mediated transcriptional pausing mechanism in Drosophila eye imaginal disc (Raj, 2020).

The protein encoded by Drosophila wg gene, a member of Wg/WNT signaling pathway, act short range inducer, which organizes the pattern of cells at a distance in the embryo. Since M1BP downregulation resulted in blocking retinal differentiation and MF progression, targets were sought of M1BP transcriptional pausing function using the candidate gene approach. It was found that wg-lacZ reporter, which serves as a transcriptional read out for Wg, exhibits robust induction in eye imaginal discs where M1BP levels were downregulated. This observation was further validated by qPCR approach which showed that there is a 2.2-fold increase in wg gene expression. Furthermore, in high throughput microarray screen carried out in S2R + cells, wg was also identified as a target whose expression is downregulated by M1BP using M1BP RNAi. According to microarray analysis, wg shows a 5.5-fold change when cells are treated with M1BP^RNAi (Raj, 2020).

To validate the results from qPCR approach as well induction of wg-lacZ reporter expression in ey > M1BP^RNAi eye imaginal disc, this study also employed bioinformatics analysis to determine if there are M1BP binding sites in the wingless (wg) gene. The M1BP binding sequence (YGGTCACACTR) has been reported earlier. This sequence was used for MEME analysis to screen for M1BP binding sites were found in the wg gene and regulatory region (Raj, 2020).

Wg, a ligand for evolutionarily conserved Wg/WNT signaling pathway, is known to act as a negative regulator of eye development. During Drosophila eye development, Wg activity promotes head specific fate by negatively regulating MF progression in the differentiating eye imaginal disc. Wg regulates expression of downstream gene hth, which encodes a MEIS class of transcription factor, and act as a negative regulator of eye development. This study found that in ey > M1BP^RNAi background, robust induction of wg transcription also accompanies ectopic induction of hth along with the suppression of the eye fate. Further, downregulation of wg levels, using wg^RNAi, in ey > M1BP^RNAi background rescued the eye suppression phenotype. This data clearly suggested that M1BP downregulates levels of wg, which in turn regulate expression of hth in the developing Drosophila eye (Raj, 2020).

Higher levels of Wg are known to trigger developmental cell death in the developing eye field. Interestingly, in ey > M1BP^RNAi eye discs, the eye field was significantly reduced. Since, majority of the cell death is triggered by the activation of caspase-dependent cell death, blocking caspase-dependent cell death by ectopic expression of anti-apoptotic P35 transgene in ey > M1BP^RNAi background showed rescue of eye suppression phenotype. However, these P35 mediated rescues of ey > M1BP^RNAi were not as significant as seen with wg^RNAi. This suggests that Wg might be regulating eye fate through hth induction and eye field size by triggering caspase mediated cell death. In order to rule out that these in ey > M1BP^RNAi phenotypes are not affected by reduced cell proliferation rates, levels were tested of pH3 in these developing eye fields. It was found that cell proliferation rates were not affected by this transcriptional pausing mechanism in the developing eye (Raj, 2020).

These results imply that the transcription pausing function of M1BP in regulating Wg signaling may play a critical role in Drosophila eye development. However, other factors and signaling pathways involved in regulating the M1BP function at the mechanistic level is yet to be determined. In order to further understand whether M1BP mediated transcriptional regulation is also implicated during development of other imaginal discs in Drosophila, the downregulation of M1BP function was studied in bi-Gal4 domains of wing imaginal disc. Whether this role of M1BP in regulating wg gene expression is exclusive to developing eye disc or it extends to other larval imaginal disc was tested. A bi-GAL4 driver which drives the expression of a transgene in wing imaginal disc was used. Downregulation of M1BP in bi-Gal4 expression domains of wing (bi > M1BP^RNAi) exhibits ectopic upregulation wg expression in the pouch region of the wing imaginal disc. Furthermore, M1BP expression levels are downregulated in the wing pouch region, which corresponds to the bi-Gal4 expression domain. These results suggested that the transcription pausing function of M1BP may have similar target in the eye and wing imaginal disc. Recently, HEXIM, another transcriptional regulator associated with pol II pausing, has been reported to affect wing development in Drosophila by regulating Hh signaling. In Drosophila wing imaginal disc, HEXIM knockdown causes developmental defects by inducing ectopic expression of hh and its transcriptional effector cubitus interuptus (ci), which triggers apoptosis. This suggests that the regulatory factors involved in Pol II pausing are important in maintaining the expression levels of different signaling pathways during development in Drosophila (Raj, 2020).

A number of highly conserved transcriptional pausing and elongation factors such as Spt5 precisely regulate transcription during Drosophila embryogenesis. The Spt5^W049 missense mutation causes defects in the anterior-posterior patterning and segmental patterning during embryogenesis. Interestingly, the mutant allele of Spt5 (foggy^m806) in Zebrafish also causes multiple developmental defects such as discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. These studies suggest that the regulatory mechanism in Pol II pausing during fly development are also conserved in higher organisms. The Drosophila compound eye shares similarities with the vertebrate eye at the level of genetic machinery as well as the processes of differentiation. Therefore, the information generated in Drosophila can be extrapolated to higher organisms. Since Wnt signaling is known to induce programmed cell death in patterning the vasculature of the vertebrate eye, it will be important to study what molecules other than M1BP can prevent Wg signaling from inducing cell death during early eye development (Raj, 2020).

The landscape of RNA Pol II binding reveals a stepwise transition during ZGA

Zygotic genome activation (ZGA) is the first transcription event in life. However, it is unclear how RNA polymerase is engaged in initiating ZGA in mammals. By developing small-scale Tn5-assisted chromatin cleavage with sequencing (Stacc-seq), this study investigated the landscapes of RNA polymerase II (Pol II) binding in mouse embryos. Pol II was found to undergo 'loading', 'pre-configuration', and 'production' during the transition from minor ZGA to major ZGA. After fertilization, Pol II is preferentially loaded to CG-rich promoters and accessible distal regions in one-cell embryos (loading), in part shaped by the inherited parental epigenome. Pol II then initiates relocation to future gene targets before genome activation (pre-configuration), where it later engages in full transcription elongation upon major ZGA (production). Pol II also maintains low poising at inactive promoters after major ZGA until the blastocyst stage, coinciding with the loss of promoter epigenetic silencing factors. Notably, inhibition of minor ZGA impairs the Pol II pre-configuration and embryonic development, accompanied by aberrant retention of Pol II and ectopic expression of one-cell targets upon major ZGA. Hence, stepwise transition of Pol II occurs when mammalian life begins, and minor ZGA has a key role in the pre-configuration of transcription machinery and chromatin for genome activation (Liu, 2020).

This study has developed Stacc-seq to interrogate the states of Pol II in early mammalian development. Specifically, protein A/G fused with Tn5 transposases is pre-incubated with antibodies, and then applied to recognize the targeted proteins and cleave chromatin around the binding sites. The released DNA fragments are simultaneously transposed with adaptors for sequencing. This independently developed approach uses similar strategies to CUT&Tag16, ACT-seq17, CoBATCH18 and scChIC19, and can be done in as short a time as 3.5 h. Stacc-seq differs in the pre-incubation of Tn5-protein A/G with antibodies to presumably minimize off-target chromatin tagging by Tn5, and in the optional omission of washing steps for possibly low-affinity antibodies or low-abundance targets. Stacc-seq can profile trimethylation at the 4th or 27th lysine residues of histone H3 (H3K4me3 or H3K27me3, respectively) using as few as 200 mouse embryonic stem cells (mESCs), with comparable or better performance than CUT&Tag16 and CoBATCH18. Stacc-seq can also detect Pol II binding using as few as 500 mESCs, using antibodies against RPB1 (the largest subunit of Pol II), regardless of the phosphorylation state of the CTD. In addition, Pol II Stacc-seq can be robustly applied to mouse tissues. Using spike-in DNA, Stacc-seq can be conducted in a quantitative manner, although it is more effective for samples from the same batch. Therefore, all spike-in normalization and comparison in this study were done only when the related experiments were conducted in parallel. In summary, Stacc-seq is a highly sensitive and efficient method for profiling genome-wide protein binding and histone modifications (Liu, 2020).

It is unclear how the transcription machinery engages mammalian ZGA. This study has shown that, after fertilization, Pol II initially re-engages both CG-rich promoters and distal accessible regions, which appears to reflect neither a history from gametes nor a purpose for major ZGA. This is followed by pre-configuration of Pol II, presumably to ensure accurate synchronization between chromatin maturation and the genome activation clock. One key question is how exactly the pre-configuration of Pol II occurs. Transcription factors might function as pioneering factors to recruit Pol II. On the other hand, Pol II pre-configuration also depends on minor ZGA, and the underlying mechanisms of this dependency are unclear. There are several non-exclusive possibilities: (1) in the presence of DRB or α-amanitin, Pol II disassociates from chromatin during mitosis in one-cell embryos and rebinds to its one-cell targets during the early two-cell stage, suggesting that the chromatin landscape is resistant to pre-configuration when minor ZGA is absent; (2) extended DRB treatment may exhaust free Pol II, preventing sufficient Pol II from being recruited to major ZGA sites. A large amount of Pol II may be essential to tip the transition balance towards its binding to major ZGA sites. This is perhaps analogous to the reprogramming of induced pluripotent stem cells, in which only sufficient amounts of pluripotency factors open pluripotency enhancers; (3) certain transcription products during minor ZGA, although extremely limited, may be essential for guiding Pol II pre-configuration; (4) DRB also partially inhibits the transcription of ribosomal RNA (rRNA) by Pol I, although without affecting the total amount of rRNA. Whether Pol I transcription, as part of minor ZGA, affects Pol II pre-configuration remains to be investigated. The idea that transcription factors recruit Pol II to major ZGA sites, where Pol II or minor ZGA in turn stabilizes the transition, is favored. Together, these findings reveal dynamic engagement of Pol II with chromatin upon the onset of global transcription. These data may define cornerstones for future investigations of mammalian ZGA and early development (Liu, 2020).

The integrator complex attenuates promoter-proximal transcription at protein-coding genes

Collectively, the results demonstrate that the Integrator complex mediates transcription attenuation in metazoan cells. Evidence is presented that Integrator associates with promoter-proximally paused Pol II, cleaves nascent mRNA transcripts, and directs promoter-proximal termination. This inhibitory function is broad: ∼15% of Drosophila genes and enhancers are impacted by Integrator, with receptor, growth, and proliferative pathways particularly affected. Furthermore, the mammalian Integrator complex targets genes in similar pathways for transcriptional repression, underlining the conserved nature of this behavior (Elrod, 2019).

These data resolve long-standing questions about the intrinsic stability of promoter-proximal Pol II. Genes that harbor highly unstable promoter Pol II are those where there is an active process of termination, catalyzed by the Integrator complex. The data support a model wherein the paused polymerase is inherently stable in the absence of termination factors, consistent with a wealth of biochemical characterization of elongation complexes. Thus, it is proposed that rapid turnover of promoter Pol II at specific genes results from a regulated process of Integrator-mediated RNA cleavage and active dissociation of Pol II from the DNA template (Elrod, 2019).

The mechanistic activity uncovered in this study for Integrator at protein-coding genes and enhancers parallels that described at snRNA genes, where Integrator cleaves the nascent RNA and promotes Pol II termination. Therefore, the model for Integrator function is parsimonious with its previously defined biochemical activities. Moreover, consistent with IntS9 and IntS11 subunits being paralogs of CPSF100 and CPSF73, respectively, there are many similarities between premature Pol II termination caused by Integrator and mRNA cleavage and termination by the CPA machinery. It is noted that RNA cleavage and termination at gene ends mediated by CPA factors and Xrn2 is coupled with polyadenylation to protect the released mRNA. Likewise, Integrator-catalyzed cleavage of snRNAs is coupled to proper 3' end biogenesis. In contrast, termination driven by Integrator at protein-coding and enhancer loci does not appear to be dependent on Xrn2, and the RNA products are typically degraded rapidly. These results indicate that the Integrator endonuclease activity can be deployed for different purposes at different loci, with the outcome governed by the locus-specific recruitment of termination, RNA processing, or RNA decay machineries. Moreover, a recent study reports that Integrator limits the activation of the metallothionein A (MtnA) gene in Drosophila cells during copper stress (Tatomer, 2019). MtnA was not detected as a target of Integrator in unstressed cells, suggesting that Integrator occupancy and/or activity can be altered in response to the cellular environment (Elrod, 2019).

It has been established that cleavage and termination by the CPA machinery is greatly facilitated by pausing of Pol II, as is snRNA 3' end formation by Integrator. Current models invoke a kinetic competition between Pol II elongation and termination, wherein slowed transcription elongation provides a greater window of opportunity for termination to occur. Consistent with these models, this study found that promoter-proximally paused Pol II is an optimal target for Integrator-mediated cleavage and termination at mRNA and eRNA loci. The findings thus suggest a novel function for promoter-proximal pausing, wherein slowed elongation provides a regulatory opportunity that enables gene attenuation. Likewise, it is suggested that pausing further downstream, as Pol II approaches the first nucleosome, could present an additional target for Integrator-mediated termination (Elrod, 2019).

Integrator-repressed genes, which exhibit very low levels of productive elongation, have chromatin characteristics that are common at enhancers. In particular, these genes display low levels of active histone modifications H3K4me3 and H3K36me3, with an enrichment in H3K4me1. Like at Integrator-repressed genes, transcription at enhancers is known to be non-productive, with a highly unstable Pol II that yields only short, rapidly degraded RNAs. Remarkably, depletion of Integrator and increased productive elongation at promoters and enhancers is coupled with the deposition of H3K4me3. Thus, th data support models wherein the chromatin features surrounding a TSS reflect the level and productivity of transcription at the locus rather than specifically demarcating the coding versus non-coding potential of the region (Elrod, 2019).

Taken together, the role described in this study for Integrator in determining the fate of promoter Pol II sheds new light on Integrator function in development and disease states. Mutations in Integrator have been associated with a myriad of diseases, with each of the 14 Integrator subunits implicated in one or more disorders. Intriguingly, many of these disease states are not characterized by defects in splicing and are often associated with disruption in normal development. Thus, the human genetics foretold that Integrator functions extend well beyond snRNA processing. Accordingly, this study finds that Integrator targets a set of stimulus- and developmentally-responsive genes to potently repress their activity. It will be interesting in future work to tease out the specific roles of the individual Integrator subunits in gene regulation, in the hopes of exploiting this knowledge for therapeutic benefit (Elrod, 2019).

Evolutionarily conserved principles predict 3D chromatin organization

Interaction domains in Drosophila chromosomes form by segregation of active and inactive chromatin in the absence of CTCF loops, but the role of transcription versus other architectural proteins in chromatin organization is unclear. This study finds that positioning of RNAPII via transcription elongation is essential in the formation of gene loops, which in turn interact to form compartmental domains. Inhibition of transcription elongation or depletion of cohesin decreases gene looping and formation of active compartmental domains. In contrast, depletion of condensin II, which also localizes to active chromatin, causes increased gene looping, formation of compartmental domains, and stronger intra-chromosomal compartmental interactions. Condensin II has a similar role in maintaining inter-chromosomal interactions responsible for pairing between homologous chromosomes, whereas inhibition of transcription elongation or cohesin depletion has little effect on homolog pairing. The results suggest distinct roles for cohesin and condensin II in the establishment of 3D nuclear organization in Drosophila (Rowley, 2019).

Inter- and intra-chromosomal interactions among DNA-bound proteins establish patterns of chromatin organization detectable by Hi-C. The original low-resolution genome-wide Hi-C maps described the segregation of active and inactive chromatin into A and B compartments. Later, higher-resolution maps identified domains characterized by preferential intra- versus inter-domain contacts. Interaction domains have been described in different organisms and are commonly referred to as topologically associating domains (TADs). In addition to these features, intense point-to-point loops have been detected by high-resolution Hi-C in mammals. The anchors of these loops are enriched in CTCF and cohesin, and predominantly contain CTCF motifs in convergent orientation (Rowley, 2019).

CTCF loops are an important component of chromatin organization in vertebrates, yet plants and invertebrates either lack a homolog or CTCF does not appear to form stable loops. Instead, chromosomal domains in these organisms, including Drosophila, correspond to the transcriptional state of specific sequences in the genome. Borders between these domains form at discontinuities between active and inactive regions containing proteins and histone modifications characteristic of their transcriptional state. This pattern of 3D organization is similar to that observed in mammals after depletion of CTCF or Rad21 and has been studied in detail in Drosophila, where analyses of high-resolution Hi-C data show that chromatin is predominately organized by the fine-scale segregation of active and inactive chromatin into A and B compartmental domains (Rowley, 2017). Indeed, transcriptional state alone can be used to computationally simulate the experimental Hi-C interaction pattern at 1-kb resolution with great accuracy (Rowley, 2017). In further support for a role of transcription or factors associated with the transcriptional state of genes in chromatin organization, inhibition of transcription initiation and subsequent degradation of RNA polymerase II (RNAPII) using triptolide disrupts Drosophila compartmental domains and their interactions. Interestingly, the extent of disruption of 3D organization correlates with the levels of RNAPII after triptolide treatment. Drosophila Hi-C maps also show a few hundred punctate signals corresponding to specific point-to-point interactions, but these loops are not associated with CTCF. Instead, the loop anchors are enriched for developmental enhancers, Pc, and Rad21. It is unclear whether these Pc loops are formed by cohesin-mediated loop extrusion as it has been proposed for CTCF loops in mammals (Rowley, 2019).

In addition to inter- and intra-chromosomal interactions, Drosophila chromosomes participate in extensive pairing with their homologs. Pairing between homologs is responsible for the transvection phenomenon, which involves interactions between enhancers and promoters of genes located in two homologous chromosomes. Analysis of the extent of this pairing typically makes use of fluorescence in situ hybridization (FISH) probes hundreds of kilobases long, making it difficult to determine whether pairing occurs at discrete loci or in large regions. Several proteins have been shown to affect homolog pairing including condensin II, the levels of which are regulated by the SCFSlimb ubiquitin ligase. Depletion of Slimb increases levels of condensin II and decreases homolog pairing, while depletion of condensin II increases homolog pairing, suggesting that condensin II antagonizes chromosome pairing. While the role of condensin II in this aspect of nuclear organization is well known, its relationship to other aspects of chromosome organization is largely unexplored (Rowley, 2019).

This study examined the contribution of condensin II, cohesin, and the distribution of RNAPII to the establishment of various features of Drosophila 3D chromatin organization. Furthermore, analysis of homologous pairing interactions using Hi-C data suggests that pairing occurs at discrete loci with an average length of 6.4 kb enriched for architectural proteins. The results highlight the importance and distinct roles of RNAPII or other components of the transcription complex, cohesin, and condensin II in the establishment of nuclear organization (Rowley, 2019).

These results support a model of chromatin organization where RNAPII and cohesin promote interactions within genes to create small gene domains. Interactions between adjacent gene domains result in the formation of active compartmental domains, and interactions among these domains give rise to the characteristic plaid pattern of Hi-C heatmaps often referred to as the A compartment. The frequency of interactions within and between genes and A compartmental domains correlates with the amount of RNAPII and cohesin, which co-localize extensively in the genome. Because of this, the allocation of a specific sequence to the A compartment should not be done in absolute terms. Rather, sequences in the A compartment have different positive eigenvector values that correlate with the amount of RNAPII and cohesin. Contiguous sequences lacking RNAPII and cohesin have a negative eigenvector value and form B compartmental domains. Interactions among B compartmental domains in Drosophila are more infrequent compared to those among A compartmental domains, that is, the plaid pattern of Hi-C heatmaps in Drosophila arises in large part due to interactions between A compartments. However, sequences within B compartmental domains interact as frequently as those located in A domains. These interactions may arise as a consequence of proteins present in silenced genes. Alternatively, or in addition, interactions within B compartmental domains may result from interactions between adjacent A domains, which enclose B domains within loops similar to those formed by CTCF/cohesin in vertebrates. This is supported by results showing that inhibition of transcription initiation with triptolide or using the heat shock response, which result in the loss of A compartmental domains, also result in decreased interaction frequencies within B domains (Rowley, 2019).

These findings suggest that, whereas interaction frequency of sequences in active genes correlates with transcription elongation, it is likely that the presence of RNAPII, or other components of the transcription/elongation complexes, is a better candidate to explain the correlation between transcription and 3D organization. Inhibition of transcription results in dramatic changes to chromatin domains in Drosophila, yet transcription inhibition was reported to have little effect in mammalian embryonic nuclei. It is speculated that transcription inhibition studies in mammalian cells could be affected by the prevalence of CTCF loop domains. These loops may tether chromatin together such that inhibition of transcription for short periods of time is insufficient to disrupt chromatin organization. Meanwhile, in organisms that lack CTCF loops, such as Drosophila and prokaryotes, the larger effect of transcription inhibition may be due to the lack of point-to-point chromatin tethering by CTCF loops. It would be interesting to analyze whether absence of transcription or depletion of RNAPII with inhibitors such as triptolide have a stronger effect in cells depleted of CTCF (Rowley, 2019).

Previous results have shown a role for condensin II in chromatin structure during interphase. Condensin II colocalizes extensively with Drosophila architectural proteins, but in spite of the similar distribution, some observations suggest a distinct role for Cap-H2 in chromatin biology with respect to other architectural proteins. For example, all architectural proteins, including Rad21, are re-distributed during the heat shock response and they accumulate at enhancer sequences. However, the amount of enhancer-bound Cap-H2 and the number of occupied enhancers decreases after temperature stress. These observations may be explained by the opposing roles that condensin II and cohesin play in mediating intra-chromosomal interactions. Condensin II is present in active chromatin but it antagonizes the formation of gene domains and A compartmental domains, and condensin II depletion results in an increase to long-range A-A compartmental interactions. These results are in line with recent observations indicating that chromosome volume, as detected by Oligopaint, increases in Cap-H2 knockdown Drosophila cells (Rosin, 2018). The mechanisms by which these two SMC motors play opposing role in chromatin interactions is unclear. Presumably, their function in chromatin 3D organization is related to their ability to extrude loops, as was proposed for cohesin in mammals. Condensin has also been shown to extrude loops in vitro (Ganji, 2018), and it would be interesting to understand whether its role, opposite to that of cohesin, is based on different potential extrusion mechanisms between these two complexes. Thus, condensin II could antagonize cohesin interactions by directly inhibiting these same interactions or by promoting different interactions (Rowley, 2019).

Drosophila chromosomes participate in extensive homologous chromosome pairing, but the details of the mechanisms underlying this phenomenon are not well understood. Analysis of Hi-C data support a button model of pairing, where the buttons are short pairing sites likely corresponding to binding sites for specific proteins, rather than large domains. These pairing sites are enriched in architectural proteins, including Rad21 and Cap-H2. Although depletion of Rad21 only has no effect on pairing, it is possible that some architectural proteins may promote pairing while others act as anti-pairers, as is the case for Cap-H2. The general antagonistic role of condensin II in the establishment of interactions between homologs as well as short- and long-range intra-chromosomal contacts suggests common mechanisms responsible for these apparently different phenomena (Rowley, 2019).

In situ dissection of domain boundaries affect genome topology and gene transcription in Drosophila

Chromosomes are organized into high-frequency chromatin interaction domains called topologically associating domains (TADs), which are separated from each other by domain boundaries. The molecular mechanisms responsible for TAD formation are not yet fully understood. In Drosophila, it has been proposed that transcription is fundamental for TAD organization while the participation of genetic sequences bound by architectural proteins (APs) remains controversial. This study investigated the contribution of domain boundaries to TAD organization and the regulation of gene expression at the Notch gene locus in Drosophila. Deletion of domain boundaries was found to result in TAD fusion and long-range topological defects that are accompanied by loss of APs and RNA Pol II chromatin binding as well as defects in transcription. Together, these results provide compelling evidence of the contribution of discrete genetic sequences bound by APs and RNA Pol II in the partition of the genome into TADs and in the regulation of gene expression in Drosophila (Arzate-Mejia, 2020).

This study provides evidence that discrete genetic sequences occupied by APs and RNA Pol II are potent chromatin insulators that actively partition the genome into Topological Domains. Furthermore, partial disruption or complete removal of the domain boundaries alter genome topology, transcription, and the recruitment of APs and RNA Pol II (Arzate-Mejia, 2020).

Whether domain boundaries are autonomous discrete genetic elements mediating the formation of TADs is a subject of intense debate. The collection of CRISPR-Cas9 mediated deletions of domain boundaries at the Notch locus provide evidence on the existence of autonomous genetic elements bound by APs and Pol II that act as chromatin insulators essential for TAD formation (Arzate-Mejia, 2020).

A 300-bp sequence comprising the entire intergenic region between kirre and Notch is a modular chromatin insulator constituting a domain boundary. Non-overlapping portions of the intergenic region, with binding sites for specific APs and RNA Pol II, act as discrete modules that restrain interactions of the kirre and the Notch genes, with the removal of all modules necessary for TAD fusion. The topological effects observed upon boundary deletion are remarkably consistent with cytological data from the Notch mutant facet-strawberry (faswb) where deletion of a ~0.9-kb region spanning the 5' region of Notch results in loss of an interband and fusion of the 3C7 band containing Notch with the upstream band. Also, reporter assays in transgenic flies and cytological evidence support an autonomous role for the 5' intergenic region of Notch as a chromatin insulator as the ectopic insertion of this sequence is sufficient and necessary to split a band into two, forming an interband in polytene chromosomes. In the case of the intragenic enhancer boundary, deletion of a ~2-kb region results in a dramatic increase of ectopic interactions between Notch domains and loss of a ~1-Mb domain downstream of Notch. Therefore, evidence from cytological studies in the fly and the in nucleus Hi-C data from CRISPR mutants conclusively demonstrates that domain boundaries are essential for TAD formation (Arzate-Mejia, 2020).

Recent reports have suggested a prominent role for transcription as the main driver for domain organization in Drosophila. Also, a role for RNA Pol II in mediating domain formation has been recently proposed. The data provide important observations that support a role for RNA Pol II in boundary activity and therefore in TAD formation in Drosophila. First, re-analysis of public Hi-C data from early stages of Drosophila embryogenesis suggests that the 5' boundary of Notch is established before Zygotic Genome Activation (nuclear cycle 13), and therefore, before transcription at the locus. The appearance of TAD boundaries at Notch strongly correlates with the early acquisition of chromatin accessibility (nuclear cycle 11) and with the binding of proteins like RNA Pol II (nuclear cycle 12), the general transcription factor TBP, and the pioneering factor Zelda. Second, transcription inhibition early in development results in a decrease in intra-domain interactions within the Notch locus. However, the boundaries and the domains at Notch are still detected, which correlates with the retention of RNA Pol II at domain boundaries suggesting that RNA Pol II is key for TAD formation. In support of this, it was observed that deletion of the Promoter Proximal Region of Notch (5pN-Δ102) resulted in a major decrease in transcription within the D1 domain (>80%) but just in discrete topological changes mainly detected as a reduction in intra-domain interactions for the D1 domain, consistent with the topological effects observed at the locus upon transcription inhibition. Importantly, loss of the Promoter Proximal Region of Notch resulted in the reduction but not loss of RNA Pol II binding, which implies that the remaining RNA Pol II could be sufficient to sustain boundary activity. In support of this, the fusion of the D1 domain of Notch with the upstream TAD correlates with complete loss of RNA Pol II at the 5' end of Notch. A similar trend was observed when removing the intronic boundary of Notch, with the fusion of Notch domains strongly correlating with loss of RNA Pol II binding at the region adjacent to the deleted boundary and in exon 6. Furthermore, the formation was observed of a new TAD spanning the full Notch locus despite a significant loss of transcription along the gene. Therefore, although transcription plays a role in mediating intra-domain interactions, the data suggest that discrete, accessible genomic sequences occupied by RNA Pol II, could have a major role in shaping Drosophila genome organization independent of transcription (Arzate-Mejia, 2020).

Architectural Proteins in Drosophila can mediate long-range interactions, however their role in shaping TADs has remained elusive. The data suggest a role of architectural protein binding sites (APBSs) in boundary activity in part throughout RNA Pol II recruitment. For example, non-overlapping regions of the 5' boundary have a differential effect on RNA Pol II recruitment, which correlates with the presence of different APBSs. In particular, it was observed that deletion of a ~200-bp region containing just a CTCF motif (5pN-Δ183) have a stronger effect in RNA Pol II recruitment than deletion of the Promoter Proximal Region (5pN-Δ102) which contains a binding site for M1BP.Then, in this case the CTCF DNA-binding motif seems important to either directly or indirectly recruit RNA Pol II. In support of this, mutation of the CTCF motif results in loss of binding of nuclear proteins. It esd also observed that loss of both CTCF and M1BP binding sites in the 5pN-Δ343 mutant correlates with the maximal decrease of CTCF and RNA Pol II occupancy, complete loss of insulation and TAD fusion, implying that domain boundaries can be resilient to the loss of RNA Pol II binding through the presence of multiple APBSs. In support of a role for DNA-binding APs in boundary activity through RNA Pol II recruitment, it has been reported that depletion of the pioneering factor Zelda results in loss of RNA Pol II recruitment, deficient local insulation and fusion of adjacent TADs (Arzate-Mejia, 2020).

TAD boundaries can block unspecific regulatory communication, however, their role in gene regulation has been recently subject to intense debate. The data support that TADs have an important function in gene regulation in Drosophila. It was found that deletions spanning the 5' boundary of Notch, consistently results in loss of transcription within the D1 domain likely as a combination of reduced RNA Pol II occupancy at the 5' end of the gene, loss of insulation between adjacent TADs and gain of ectopic interactions. Deletion of the B2 boundary also results in a reduction in Notch transcription. Interestingly, boundary disruption leads to loss of RNA Pol II binding at exon 6 and at the 5' region of Notch, suggesting that it influences RNA Pol II recruitment to the Notch, locus probably by direct physical interaction. Then, in this case, reduction in transcription could be a consequence of disrupting physical interactions between regulatory elements that affect RNA Pol II recruitment, rather than the consequence of insulation loss. Furthermore, loss of the mega-domain due to deletion of the B2 boundary affects gene regulation of the genes located within the domain. Therefore, the evidence shows that disruption of TAD organization by alteration of boundaries impacts gene expression (Arzate-Mejia, 2020).

Finally, an important observation from these experiments is that deletion of TAD boundaries and accompanying changes in gene transcription as well as changes in the recruitment of CTCF and RNA Pol II at domain boundaries, do not abolish the intra-TAD specific organization of Notch since subdomains are preserved despite TAD fusion. These suggest that additional mechanisms contribute to folding the genome into smaller domains, possibly by aggregation of regions with similar chromatin features (Arzate-Mejia, 2020).

In conclusion, the data demonstrate the existence of discrete genetic sequences with boundary activity that influence genome organization into Topological Associated Domains and the regulation of gene expression. Other domain boundaries with a similar chromatin composition and APs occupancy could behave similarly (Arzate-Mejia, 2020).

Based on the results, a mechanism is proposed for boundary formation through the binding of APs that results in recruitment of RNA Pol II. In such a model, a boundary is robust to APs depletion as far as RNA Pol II binding is maintained. Finally, it is envisioned that genome organization in Drosophila is dependent on two mechanisms: one driven by self-association of regions with similar transcriptional or epigenetic profiles and one that partitions the genome into interaction domains driven by genetic elements acting as chromatin insulators (Arzate-Mejia, 2020).

Mediator and RNA polymerase II clusters associate in transcription-dependent condensates

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. This study used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. It is suggested that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo (Cho, 2018).

A conventional view of eukaryotic gene regulation is that transcription factors, bound to enhancer DNA elements, recruit coactivators such as the Mediator complex, which is thought to interact with RNA polymerase II (Pol II) at the promoter. This model is supported by a large body of molecular genetic and biochemical evidence, yet the direct interaction of Mediator and Pol II has not been observed and characterized in living cells. Using superresolution and light-sheet imaging, the organization and dynamics of endogenous Mediator and Pol II in live mouse embryonic stem cells (mESCs) was studied. Whether Pol II and Mediator interact in a manner consistent with condensate formation was directly tested, their biophysical properties were quantitatively characterized, and the implications of these observations for transcription regulation in living mammalian cells was considered (Cho, 2018).

To visualize Mediator and Pol II in live cells, mouse embryonic stem cell lines were generated with endogenous Mediator and Pol II labeled with Dendra2, a green-to-red photoconvertible fluorescent protein. Live-cell superresolution imaging was performed and Mediator was found to form clusters with a range of dynamic temporal signatures. Mediator exists in a population of transient small (~100 nm) clusters with an average lifetime of 11.1 ± 0.9 s, comparable to that of transient Pol II clusters observed in this study and previously in differentiated cell types. In addition, it was observed that both Mediator and Pol II form a population of large (>300 nm) clusters (~14 per cell), each comprising ~200 to 400 molecules, that are temporally stable (lasting the full acquisition window of the live-cell superresolution imaging) (Cho, 2018).

The extent to which these clusters depend on the stem cell state was tested. The mESCs were subjected to a protocol to differentiate them into epiblastlike cells (EpiLCs) within 24 h. Differentiation had no apparent effect on the population of transient clusters, consistent with previous observations that transient clusters persist in differentiated cell types. However, both the size and the number of stable clusters decreased along the course of differentiation, suggesting that these stable clusters are prone to change as cells differentiate (Cho, 2018).

Focused was placed on the stable clusters of Mediator and Pol II and whether they are colocalized was investigated. mESCs were generated with endogenous Mediator and Pol II tagged with JF646-HaloTag and Dendra2, respectively. Direct imaging of both JF646-Mediator and Dendra2-Pol II showed bright spots of large accumulations in the nucleus, which corresponded to stable Pol II clusters according to subsequent superresolution imaging of Dendra2-Pol II in the same nuclei. The same observations were made with Dendra2-Mediator. Of 143 Mediator clusters imaged by dual-color light-sheet imaging, 129 (90%) had a colocalizing Pol II cluster. It was concluded that these Mediator and Pol II clusters colocalize in live mESCs (Cho, 2018).

Previous studies have shown that high densities of Mediator are located at enhancer clusters called super-enhancers (SEs) and that some are disrupted by loss of the BET (bromodomain and extraterminal family) protein BRD4 (Drosophila homolog: fs(1)h), which is a cofactor associated with Mediator. This study found that treatment of mESCs with JQ1, a drug that causes loss of BRD4 from enhancer chromatin, dissolved transient and stable clusters of both Mediator and Pol II clusters (Cho, 2018).

After transcription initiation, Pol II transcribes a short distance (~100 base pairs), pauses, and is released to continue elongation when phosphorylated by CDK9. It was hypothesized that inhibition of CDK9 might selectively affect the Pol II stable clusters. It was observed that upon incubation with DRB (5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole), Pol II stable clusters dissolved but Mediator stable clusters remained. Quantification of Mediator-Pol II colocalization revealed that incubation with DRB progressively decreased the fraction of Mediator stable clusters that colocalized with Pol II. This effect could be reversed when DRB was washed out; the colocalization fraction recovered completely. These results imply that the association between Mediator and Pol II clusters may be hierarchical, with upstream enhancer recruitment controlling both clusters but downstream transcription inhibition selectively affecting Pol II clusters (Cho, 2018).

The long-term dynamics of stable clusters were characterized by using lattice light-sheet imaging in live mESCs. It was observed that clusters can merge upon contact. The time scale of coalescence was very rapid, comparable to the full volumetric acquisition frame rate (15-s time interval). The added-up intensity of the two precursor clusters was close to that of the newly merged cluster. These biophysical dynamics are reminiscent of those of biomolecular condensates in vivo (Cho, 2018).

In addition to coalescence, in vivo condensates had rapid turnover of the molecular components, as shown by fast recovery in fluorescence recovery after photobleaching (FRAP) assays, and were sensitive to a nonspecific aliphatic alcohol, 1,6-hexanediol. FRAP analyses of clusters revealed very rapid dynamics and turnover of their components: 60% of the Mediator and 90% of Pol II components were exchanged within ~10 s within clusters. Moreover, the treatment of mESCs with 1,6-hexanediol resulted in the gradual dissolution of both Mediator and Pol II clusters. Together, these results suggest that the stable clusters are in vivo condensates of Mediator and Pol II (Cho, 2018).

It was hypothesized that a phase separation model with induced condensation at the recruitment step of Mediator to enhancers would qualitatively account for the observations in this study. The model implies that the condensates are chromatin associated and colocalize with enhancer-controlled active genes. Therefore these two specific implications were tested. The diffusion dynamics of Mediator clusters were tracked by computing their mean squared displacement as a function of time (n = 6 cells). On short time scales, the cluster motion was subdiffusive, with an exponent α = 0.40 ± 0.12. This is the same exponent found in the subdiffusional behavior of chromatin loci in eukaryotic cells. The same diffusional parameters were also observed when tracking a chromatin locus labeled by dCas9-based chimeric array of guide RNA oligonucleotides (CARGO) in mESCs. It is concluded that clusters diffuse like chromatin-associated domains (Cho, 2018).

It was hypothesized that clusters were in close physical proximity to actively transcribed genes that can be visualized by global run-on nascent RNA labeling with ethynyl uridine (EU). The run-on results showed that 2 min after DRB washout, virtually all Mediator clusters observed were proximal or overlapping with nascent RNA accumulations, as imaged by Click labeling of EU in fixed cells. Yhe MS2 endogenous RNA labeling system was employed to investigate whether active transcription could be observed at Esrrb, one of the top SE-controlled genes in mESCs. Bright foci were observed consistent with nascent MS2-labeled gene loci, and the gene loci were confirmed by dual-color RNA fluorescence in situ hybridization (FISH) targeting the MS2 sequence and intronic regions of Esrrb. Intronic FISH on 125 Esrrb loci from 82 fixed cells showed that 93% of Esrrb loci had a stable Mediator cluster nearby (within 1 µm) but only ~22% of the loci colocalized with a stable Mediator cluster, suggesting that the Mediator-bound enhancer only occasionally colocalizes with the gene. The variability in colocalization may be explained by a dynamic 'kissing' model, where a distal Mediator cluster colocalizes with the gene only at certain time points (Cho, 2018).

By dual-color three-dimensional (3D) live-cell imaging with lattice light-sheet microscopy, it was found that some Mediator clusters were up to a micrometer away from the active Esrrb gene locus but in some instances directly colocalized with the gene. In addition, the dynamic interaction between Mediator clusters and the gene locus was directly observed, supporting the dynamic kissing model. Tracking of loci in all six cells indicated that colocalization below the resolution limit of 300 nm occurred at ~30% of the time points. However, even when they were not overlapping, the Mediator cluster and the gene loci moved as a pair through the nucleus, consistent with two adjacent regions anchoring to the same underlying chromatin domain. It is proposed that Mediator clusters form at the Esrrb SE and then interact occasionally and transiently with the transcription apparatus at the Esrrb promoter (Cho, 2018).

This study has found that Mediator and Pol II form large stable clusters in living cells and has shown that these clusters have properties expected for biomolecular condensates. The condensate properties were evident through coalescence, rapid recovery in FRAP analysis, and sensitivity to hexanediol. In a model of phase separation on the basis of scaffold-client relationships, it is possible that enhancer-associated Mediator forms a condensate and provides a 'scaffold' for 'client' RNA Pol II molecules. The model proposed whereby large Mediator clusters at enhancers transiently kiss the transcription apparatus at promoters has a number of implications for gene control mechanisms. The presence of large Mediator clusters at some enhancers may allow Mediator condensates to contact the transcription apparatus at multiple gene promoters simultaneously. The large size of the Mediator clusters may also mean that the effective distance of the enhancer-promoter DNA elements can be in the same order as the size of the clusters (>300 nm), larger than the distance requirement for direct contact. It is speculated that such clusters may help explain gaps of hundreds of nanometers that are found in previous studies measuring distances between functional enhancer-promoter DNA elements. Such cluster sizes also imply that some long-range interactions could go undetected in DNA interaction assays that depend on much closer physical proximity of enhancer and promoter DNA elements (Cho, 2018).

Mediator and RNA polymerase II clusters associate in transcription-dependent condensates

Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. This study demonstrates that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes (Sabari, 2018).

Phase separation of fluids is a physicochemical process by which molecules separate into a dense phase and a dilute phase. Phase-separated biomolecular condensates, which include the nucleolus, nuclear speckles, stress granules, and others, provide a mechanism to compartmentalize and concentrate biochemical reactions within cells. Biomolecular condensates produced by liquid-liquid phase separation allow rapid movement of components into and within the dense phase and exhibit properties of liquid droplets such as fusion and fission. Dynamic and cooperative multivalent interactions among molecules, such as those produced by certain intrinsically disordered regions (IDRs) of proteins, have been implicated in liquid-liquid phase separation (Sabari, 2018).

Enhancers are gene regulatory elements bound by transcription factors (TFs) and other components of the transcription apparatus that function to regulate expression of cell type-specific genes. Super-enhancers (SEs) -- clusters of enhancers that are occupied by exceptionally high densities of transcriptional machinery -- regulate genes with especially important roles in cell identity. DNA interaction data show that enhancer elements in the clusters are in close spatial proximity with each other and the promoters of the genes that they regulate, consistent with the notion of a dense assembly of transcriptional machinery at these sites. This high-density assembly at SEs has been shown to exhibit sharp transitions of formation and dissolution, forming as the consequence of a single nucleation event and collapsing when concentrated factors are depleted from chromatin or when nucleation sites are deleted. These properties of SEs led to the proposal that the high-density assembly of biomolecules at active SEs is due to phase separation of enriched factors at these genetic elements. This study has provided experimental evidence that the transcriptional coactivators BRD4 and MED1 (a subunit of the Mediator complex) form condensates at SEs. This establishes a new framework to account for the diverse properties described for these regulatory elements and expands the known biochemical processes regulated by phase separation to include the control of cell-identity genes (Sabari, 2018).

SEs regulate genes with prominent roles in healthy and diseased cellular states. SEs and their components have been proposed to form phase-separated condensates, but with no direct evidence. This study demonstrates that two key components of SEs, BRD4 and MED1, form nuclear condensates at sites of SE-driven transcription. Within these condensates, BRD4 and MED1 exhibit apparent diffusion coefficients similar to those previously reported for other proteins in phase-separated condensates in vivo. The IDRs of both BRD4 and MED1 are sufficient to form phase-separated droplets in vitro, and the MED1-IDR facilitates phase separation in living cells. Droplets formed by MED1-IDR are capable of concentrating transcriptional machinery in a transcriptionally competent nuclear extract. These results support a model in which transcriptional coactivators form phase-separated condensates that compartmentalize and concentrate the transcription apparatus at SE-regulated genes and identify SE components that likely play a role in phase separation (Sabari, 2018).

SEs are established by the binding of master TFs to enhancer clusters. These TFs typically consist of a structured DNA-binding domain and an intrinsically disordered transcriptional activation domain. The activation domains of these TFs recruit high densities of many transcription proteins, which, as a class, are enriched for IDRs. Although the exact client-scaffold relationship between these components remains unknown, it is likely that these protein sequences mediate weak multivalent interactions, thereby facilitating condensation. It is proposed that condensation of such high-valency factors at SEs creates a reaction crucible within the separated dense phase, where high local concentrations of the transcriptional machinery ensure robust gene expression (Sabari, 2018).

The nuclear organization of chromosomes is likely influenced by condensates at SEs. DNA interaction technologies indicate that the individual enhancers within the SEs have exceptionally high interaction frequencies with one another, consistent with the idea that condensates draw these elements into close proximity in the dense phase. Several recent studies suggest that SEs can interact with one another and may also contribute in this fashion to chromosome organization. Cohesin, an SMC (structural maintenance of chromosomes) protein complex, has been implicated in constraining SE-SE interactions because its loss causes extensive fusion of SEs within the nucleus. These SE-SE interactions may be due to a tendency of liquid-phase condensates to undergo fusion (Sabari, 2018).

The model whereby phase separation of coactivators compartmentalizes and concentrates the transcription apparatus at SEs and their regulated genes raises many questions. How does condensation contribute to regulation of transcriptional output? A study of RNA Pol II clusters, which may be phase-separated condensates, suggests a positive correlation between condensate lifetime and transcriptional output. What components drive formation and dissolution of transcriptional condensates? These studies indicate that BRD4 and MED1 likely participate, but the roles of DNA-binding TFs, RNA Pol II, and regulatory RNAs require further study. Why do some proteins, such as HP1a, contribute to phase-separated heterochromatin condensates and others contribute to euchromatic condensates? The rules that govern partitioning into specific types of condensates have begun to be studied and will need to be defined for proteins involved in transcriptional condensates. Does condensate misregulation contribute to pathological processes in disease, and will new insights into condensate behaviors present new opportunities for therapy? Mutations within IDRs and misregulation of phase separation have already been implicated in a number of neurodegenerative diseases. Tumor cells have exceptionally large SEs at driver oncogenes that are not found in their cell of origin, and some of these are exceptionally sensitive to drugs that target SE components. How is it possible to take advantage of phase separation principles established in physics and chemistry to more effectively improve understanding of this form of regulatory biology? Addressing these questions at the crossroads of physics, chemistry, and biology will require collaboration across these diverse sciences (Sabari, 2018).

Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture

Genome-wide studies has identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and developmental enhancers (dCP). It is hypothesized that the two enhancer classes are occupied by distinct architectural proteins, affecting their enhancer-promoter contacts. It was determined that both enhancer classes are enriched for RNA Polymerase II, CBP, and architectural proteins but there are also distinctions. hkCP enhancers contain H3K4me3 and exclusively bind Cap-H2, Chromator, DREF and Z4, whereas dCP enhancers contain H3K4me1 and are more enriched for Rad21 and Fs(1)h-L. Additionally, the interactions of each enhancer class were mapped utilizing a Hi-C dataset with <1 kb resolution. Results suggest that hkCP enhancers are more likely to form multi-TSS interaction networks and be associated with topologically associating domain (TAD) borders, while dCP enhancers are more often bound to one or two TSSs and are enriched at chromatin loop anchors. The data support a model suggesting that the unique architectural protein occupancy within enhancers is one contributor to enhancer-promoter interaction specificity (Cubenas-Potts, 2017).

This study characterize the protein occupancy, chromatin interactions and architecture profiles for the two enhancer classes found in Drosophila. Each enhancer class has distinct H3K4 methylation states, is bound by both common and distinct architectural proteins, and is involved in distinct types of chromatin interactions. First, it was established that hkCP enhancers exclusively bind CAP-H2, Chromator, DREF and Z4, while dCP enhancers do not and are preferentially enriched for but not exclusively bound by Fs(1)h-L and Rad21. In addition, hkCP enhancers are more likely than dCP enhancers to associate with multiple TSSs, which promotes a higher transcriptional output. Finally, hkCP enhancers preferentially associate with topologically associating domain (TAD) borders, whereas dCP enhancers are enriched at chromatin loop anchors present inside TADs. Interestingly, enhancers activated by both core promoters exhibit more hkCP enhancer like characteristics, indicating that the both CP enhancers may represent an intermediate among the distinctive hkCP and dCP enhancers. Altogether, these results provide strong correlative evidence, supporting a model suggesting that architectural proteins are critical regulators of enhancer-promoter interaction specificity and that the interactions between enhancers and promoters significantly contribute to the generation of 3D chromatin architecture (Cubenas-Potts, 2017).

The importance of architectural proteins in regulating enhancer-promoter interactions in Drosophila is supported by the observation that the vast majority of architectural protein sites present in the genome correspond to enhancers and promoters. Historically, architectural proteins were identified as insulators, which were functionally demonstrated to block enhancer-promoter interactions. The insulator function of architectural proteins correlates with their enrichment at TAD borders. However, several lines of evidence, including ChIA-PET analysis of CTCF- and cohesin-mediated interactions in mammals, suggest that these architectural proteins help mediate long range contacts among regulatory sequences. In Drosophila this study observed that nearly all of the Group 1 and Group 2 architectural protein sites are associated with enhancers or promoters defined by STARR-seq, TSSs or CBP peaks, suggesting that architectural proteins help mediate enhancer-promoter interactions. Notably, Group 3 architectural proteins include the classic insulator proteins CTCF, CP190, Mod(mdg4) and SuHw, and at least 25% of their peaks cannot be explained by enhancers or promoters. It is interesting to speculate that the non-enhancer-promoter sites may be involved in more classical insulator functions or contributing to the chromatin architecture of inactive regions of the genome (Cubenas-Potts, 2017).

The conclusion that architectural proteins are critical regulators of the specificity between enhancers and promoters is supported by two main lines of evidence. First, the current results demonstrate a strong correlation between each enhancer class and distinct architectural protein subcomplexes. Functional evidence supporting this conclusion comes from mutational analyses of the DRE motif in the distinct enhancer classes, which likely recruits DREF and the other hkCP enhancer associated architectural proteins. Zabidi (2015) demonstrated that the tandem DRE motif alone was sufficient to enhance expression of the housekeeping core promoter and that mutation of DRE motifs within an hkCP enhancer reduced its promoter interactions in a luciferase assay. Furthermore, addition of a DRE motif to a dCP enhancer changed its promoter specificity. Because DREF and potentially BEAF-32 bind to the DRE motif, these results strongly support a model suggesting that the differential occupancy of Cap-H2, Chromator, DREF and Z4 in the two enhancer classes is a critical regulator of their specific interactions with the core promoter types. However, the data cannot discount the notion that unique transcription factor binding at proximal TSSs also contribute to the specificity of enhancer-promoter interactions. Although hkCP enhancer identity is most highly correlated with CAP-H2, Chromator, DREF and Z4 localization, these four architectural proteins are not found in isolation within hkCP enhancers. BEAF-32 and CP190 are also strongly enriched in hkCP enhancers, which are also associated with high occupancy APBSs and TAD borders. Thus, the full architectural protein complement at hkCP enhancers is far more complex than the four hkCP-specific architectural proteins. In addition, architectural proteins that are truly unique to dCP enhancers were not detected. Because dCP enhancers exhibit higher cell type specificity, it cannot be discounted that there are additional dCP enhancers present in the Drosophila genome that were not identified by STARR-seq and thus, excluded from this analysis. From these studies, it is unclear if the enrichment of Fs(1)h-L and Rad21, particularly because Fs(1)h-L and Rad21 are present in hkCP enhancers at lower levels, or the absence of BEAF-32, CAP-H2, Chromator, CP190, DREF and Z4 truly distinguishes the architectural protein complexes found at dCP enhancers. In the future, careful biochemical analyses will be required to gain a comprehensive understanding of the complete organization of architectural protein subcomplexes associated with each enhancer class (Cubenas-Potts, 2017).

hkCP enhancers are associated with multi-TSS chromatin interactions and TAD borders. The promoter-clustering by hkCP enhancers results in a dose-dependent increase in transcriptional output for the interacting genes. Thus, one likely molecular mechanism by which hkCP enhancers promote robust transcriptional activation is by increasing the local concentration of RNA Polymerase II and general transcription factors (GTFs) by bringing multiple TSSs into close proximity. It is interesting that the hkCP enhancers, which form promoter clusters, are associated with TAD borders. It is speculated that the hkCP enhancer interactions involve inter-TAD contacts within the A-type compartment, indicative of the formation of transcription factories (70). From this analysis, it is unclear if the hkCP enhancers alone are sufficient for the formation of the 3D interactions or the neighboring TSSs and their associated transcription factors are also contributing to these contacts. It is hypothesized that the genes recruited to the factories contain the housekeeping promoter motifs (DRE, Ohler 1, Ohler 6 and TCT) and that the hkCP enhancer residents Cap-H2, Chromator, DREF and Z4, are critical to the formation of these 3D contacts (Cubenas-Potts, 2017).

dCP enhancers are more likely to be present within TADs and are enriched on the subTAD-like chromatin loop anchors. dCP enhancers do not form promoter clusters, but are more likely to interact with individual TSSs. One possible explanation for this observation is that the genes interacting with dCP enhancers require the binding of sequence-specific transcription factors, and increasing the concentration of GTFs and RNA polymerase II is not an effective mechanism to promote transcriptional output. The chromatin loop association is consistent with dCP enhancers forming a strong contact with a single TSS. However, it is acknowledged that dCP enhancers are likely one of multiple molecular mechanisms contributing to chromatin loop formation. Surprisingly, the chromatin loops that were observed in Drosophila are distinct from the chromatin loops described in humans. A recent study reported approximately 10,000 chromatin loops in the genome of GM12878 lymphoblastoid cells, but this study detected only 458 chromatin loops in Drosophila utilizing a similar method. The reason why there are so few chromatin loops in Drosophila compared to humans is unclear. It is possible that chromatin loops represent a more precise level of architecture within TADs between specific enhancers and promoters in mammals, but because TADs are significantly smaller in flies (median size 32.5 kb compared to 880 kb in mice, the chromatin loops are not as prominent or easily detected in the Drosophila genome. Notably, it appears that the chromatin loops are generated by different architectural proteins in the two species. The chromatin loops in humans are anchored by convergent CTCF motifs, while the results presented in this study demonstrate that the chromatin loop anchors in Drosophila are depleted of CTCF. Because the chromatin loops in Drosophila show a strong enrichment for Fs(1)h-L, a Brd4 homolog, and the architectural proteins Rad21, Nup98, TFIIIC and Mod(mdg4), it is possible that a combination of transcription and architectural proteins is required for chromatin loop formation in flies, which may be different from mammals . Altogether, it is clear that dCP enhancers are involved in individual contacts with TSSs and are likely one mechanism by which chromatin loops form in Drosophila (Cubenas-Potts, 2017).

Surprisingly, only ~20% and ~12.5% of all hkCP enhancer and ~7.5% and ~8.5% of dCP enhancer interactions involve a TSS or enhancer on the opposite anchor, respectively. The biological significance of the enhancer to non-TSS association is unclear. One possible explanation is that current methods for identifying statistically significant interactions are not sufficiently robust and that many of the enhancer to non-TSS interactions are not representative of biologically significant contacts. However, it cannot be discounted that the non-TSS interactions mediated by enhancers are real and the biological significance of these contacts remains to be determined. Throughout this analysis, the patterns of TSS interactions were compared with each enhancer class instead of drawing conclusions about the absolute number of TSSs bound per enhancer, minimizing the impact of any non-specific interactions within the data. Additional molecular studies for the various type of enhancer interactions (enhancer to promoter, enhancer to non-TSS, etc.) will be required to evaluate the various biological contributions of each (Cubenas-Potts, 2017).

This study found that the functional differences between enhancers that activate housekeeping versus developmental genes are reflected in their chromatin and architectural protein composition, and in the type of interactions they mediate. hkCP enhancers are marked by H3K4me3, associate with TAD borders, and mediate large TSS-clustered interactions to promote robust transcription. This class of enhancers contain the architectural proteins CAP-H2, Chromator, DREF and Z4. In contrast, dCP enhancers are marked by H3K4me1, associate with chromatin loop anchors and are more commonly associated with single TSS-contacts. dCP enhancers are depleted of the hkCP-specific architectural proteins and show an enrichment for Fs(1)h-L and Rad21. The results support a model suggesting that the unique occupancy of architectural proteins in the distinct enhancer classes are key contributors to the types of interactions that enhancers can mediate genome-wide, ultimately affecting enhancer-promoter specificity and 3D chromatin organization. In the future, further characterization of the broadly defined housekeeping and developmental enhancers into smaller subclasses may yield additional levels of regulation and formation of unique architectural protein and transcription factor protein complexes as key mediators of long range chromatin contacts (Cubenas-Potts, 2017).

The Hox proteins Ubx and AbdA collaborate with the transcription pausing factor M1BP to regulate gene transcription

In metazoans, the pausing of RNA polymerase II at the promoter (paused Pol II) has emerged as a widespread and conserved mechanism in the regulation of gene transcription. While critical in recruiting Pol II to the promoter, the role transcription factors play in transitioning paused Pol II into productive Pol II is, however, little known. By studying how Drosophila Hox transcription factors control transcription, this study uncovered a molecular mechanism that increases productive transcription. The Hox proteins AbdA and Ubx target gene promoters previously bound by the transcription pausing factor M1BP, containing paused Pol II and enriched with promoter-proximal Polycomb Group (PcG) proteins, yet lacking the classical H3K27me3 PcG signature. AbdA binding to M1BP-regulated genes results in reduction in PcG binding, the release of paused Pol II, increases in promoter H3K4me3 histone marks and increased gene transcription. Linking transcription factors, PcG proteins and paused Pol II states, these data identify a two-step mechanism of Hox-driven transcription, with M1BP binding leading to Pol II recruitment followed by AbdA targeting, which results in a change in the chromatin landscape and enhanced transcription (Zouaz, 2017).

Understanding Hox transcriptional networks is central to understanding their wide repertoire of functions, yet observing where they bind in the genome does not explain why they bind there. In using a homogenous cell‐based system devoid of endogenous Hox expression to conditionally express the Hox protein Ubx or AbdA, this study has demonstrated that Drosophila Hox proteins target proximal promoters genome‐wide, which is conserved (for Ubx at least) in developing embryos. While studies into Hox genomic binding have historically focussed on enhancer elements in spatially and temporarily controlling individual gene expression, genome‐wide promoter enrichment of Hox proteins is known to occur for mouse HoxB4 in hematopoietic stem cells, mouse Hoxa2 in the second branchial arches and for zebrafish Hoxb1a in early embryogenesis. However, why Hox proteins target the promoter‐proximal region has been little explored. A major advantage of the Drosophila S2 cell system is that the conditional Hox expression system allows studying in fine detail the sequence of events occurring upon promoter binding and the impact on gene expression (Zouaz, 2017).

The promoters targeted by both AbdA and Ubx in Drosophila are essentially promoters containing either GAF or M1BP. GAF controls mainly development and morphogenic genes, whereas M1BP controls genes mainly involved in basic cellular processes (Li, 2013), and this distinction in gene ontology is reflected in the genes whose promoters are targeted by AbdA. As AbdA and Ubx also target enhancer regions, it cannot be ruled out that the observed promoter binding is the result of enhancer~promoter interaction. However, given that the majority of genes controlled by M1BP do not have distal enhancers (Zabidi, 2015), it is unlikely that this is the case for M1BP‐targeted promoters. Both GAF and M1BP are important and distinct Drosophila Pol II pausing factors, a role that proved important in understanding the nature of promoters targeted by AbdA and Ubx, since the majority of all promoters targeted by the Hox proteins contained poised Pol II. GAF binding sites have previously been shown enriched at Ubx targets, although a link between Hox and GAF in regulating gene transcription was not demonstrated. Similarly, in S2‐AbdA cells, AbdA binding at GAF‐regulated promoters has little clear‐cut effect on poised Pol II status, although the amount of elongating Pol II and gene transcription appears reduced. It was at M1BP‐bound promoters where AbdA was found to have an effect on Pol II pausing, whereby AbdA binding results in a reduction in poised Pol II giving rise to increased productive transcription. Taken together with the findings that both Ubx and AbdA target nearly identical promoters, that AbdA and M1BP synergise in reporter gene expression, that both Ubx and AbdA interact with M1BP in embryos and AbdA colocalises with M1BP on polytene chromosomes, these data suggest functional cooperation between M1BP and AbdA/Ubx. To this end, demonstrating that M1BP expression is essential in inhibiting autophagy in the larval fat body, an Exd‐independent cellular function shared by all Drosophila Hox proteins where the loss of expression of all Hox genes is essential for autophagy induction, suggests that M1BP may function with Hox proteins in their generic function of autophagy inhibition (Zouaz, 2017).

Similar to the distinct mechanisms at play in pausing Pol II at M1BP‐ and GAF‐controlled promoters, these data suggest that distinct mechanisms of release of paused Pol II exist at the two classes of poised Pol II promoters: additional factors that are not present in S2 cells are likely required to permit Hox‐induced productive transcription at GAF‐controlled promoters since little evidence is foundthat AbdA binding affects Pol II pausing, whereas at M1BP‐controlled genes, AbdA binding is sufficient to increase gene transcription through the release of paused Pol II (Zouaz, 2017).

Testing the association of AbdA ChIP peaks in S2‐AbdA cells with those of numerous publicly available histone‐modifying proteins and histone marks in S2 cells, this study found that the M1BP‐ and GAF‐poised Pol II promoters targeted by AbdA were enriched for PcG proteins and H3K4me3. The finding that AbdA‐enhanced transcription at M1BP promoters was more consistently concomitant with a loss of promoter PcG protein binding than at GAF‐controlled promoter, suggests that the emerging role for PcG proteins in maintaining a poised Pol II state can be perturbed by Hox binding. Indeed, it is noteworthy that of the PcG proteins tested here, it is promoter‐bound dRing that is most affected upon AbdA binding, suggesting that, like in vertebrates where Ring1 plays a major role in restraining the poised Pol II at promoters, dRing may play a major role in tethering the poised Pol II state in Drosophila. Given that no clear effect on PcG binding occurs at GAF‐controlled promoters, even when these genes are repressed upon AbdA binding, it reinforces the notion that contrary to M1BP targets, the control of gene expression by AbdA at GAF genes is unlikely to occur through the regulation of poised Pol II status (Zouaz, 2017).

Where PcG proteins are linked to maintaining gene repression, trithorax group proteins (trxG) are the PcG antagonists, responsible for maintaining gene expression. As a transcription factor, GAF has been traditionally classified as a trxG protein although it displays repressive activity and can recruit PcG complexes. As such, GAF can be classified as a member of the growing family of genes that display both PcG and trxG phenotypes, the so‐called enhancers of trithorax and polycomb (ETP) family. This study shows that M1BP colocalises with PcG proteins at promoters in S2 cells and phenotypically enhances the PcG homeotic phenotype of extra male sex combs on the second and third pairs of legs. Indeed, M1BP-/Pc- transheterozygous males have an average of 5.3 legs displaying sex combs, which is more than most combinations of PcG mutant transheterozygotes, demonstrating the large increase in penetrance of the Pc phenotype upon M1BP mutation. As such, M1BP would be genetically classified as a PcG gene. However, PcG genes, by definition, display homeotic phenotypes due to the derepression of Hox genes when mutated and so since this study observed neither increased derepression of the upstream Hox gene responsible for sex comb development, Scr, upon M1BP mutation nor Hox expression in fat body cells following RNAi, M1BP cannot thus be classified as a PcG gene. Given that M1BP is a transcription factor involved in gene expression (Li, 2013), the hypothesis is therefore favoured that, like GAF, M1BP is likely to be a member of the ETP family. How ETP proteins can enhance the phenotypes of both repressors (PcG) and activators (trxG) has long remained a mystery. Demonstrating here that GAF and M1BP colocalise with PcG at poised Pol II promoters with the loss of PcG at those genes displaying increased expression upon AbdA binding, may go a long way to better understand how transcription factors and transcriptional repressors intricately cooperate to regulate gene transcription (Zouaz, 2017).

In summary, this work identifies a novel mechanism for Pol II pausing release mediated by AbdA: at genes bound by M1BP, targeting of AbdA results in the specific loss of PcG proteins, the release of poised Pol II and increases in H3K4me3 histone marks, which results in promoting productive transcription. Identified in S2 cells where Hox PBC‐class cofactors are absent, this mechanism may more generally apply to Hox‐generic functions that are independent of PBC‐class cofactors, such as the repression of autophagy in the Drosophila fat body or sex comb development in Drosophila males. It may also apply to PBC‐dependent Hox target gene regulatien by cooperating with Hox PBC‐bound genomic regions located remote from the promoter. Further work aimed at studying Hox PBC‐bound enhancers together with poised Pol II status, and promoter‐proximal Hox and PcG binding, should provide further insight into how enhancer‐bound protein complexes influence the basic mechanisms of transcription regulated through poised Pol II. Uncovering such a Hox‐driven mechanism of gene regulation by sequence‐specific transcription factors, PcG proteins and poised Pol II in the developing animal would have been fraught with difficulties, not least of which is the quagmire of PcG proteins being essential global repressors of all Hox genes (Zouaz, 2017).

dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes

dDsk2 (Ubqln2/Ubiquilin) is a conserved extraproteasomal ubiquitin receptor that targets ubiquitylated proteins for degradation. This study reports that dDsk2 plays a nonproteolytic function in transcription regulation. dDsk2 interacts with the dHP1c complex, localizes at promoters of developmental genes and is required for transcription. Through the ubiquitin-binding domain, dDsk2 interacts with H2Bub1 (monoubiquitylated H2B), a modification that occurs at dHP1c complex-binding sites. H2Bub1 is not required for binding of the complex; however, dDsk2 depletion strongly reduces H2Bub1. Co-depletion of the H2Bub1 deubiquitylase dUbp8/Nonstop suppresses this reduction and rescues expression of target genes. RNA polymerase II is strongly paused at promoters of dHP1c complex target genes and dDsk2 depletion disrupts pausing. Altogether, these results suggest that dDsk2 prevents dUbp8/Nonstop-dependent H2Bub1 deubiquitylation at promoters of dHP1c complex target genes and regulates RNA polymerase II pausing. These results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications (Kessler, 2015).

This study reports that the extraproteasomal ubiquitin receptor dDsk2 interacts with the dHP1c complex, localizes at promoters and is required for transcription. Binding sites of the dHP1c complex are marked by H2Bub1 and the results suggest that, through the UBA domain, dDsk2 binds H2Bub1. However, reducing H2Bub1 levels affects binding of the complex only weakly, indicating that the interaction with H2Bub1 has only a minor contribution to recruitment. Actually, dDsk2 contains a single ubiquitin-binding site of low affinity (Kd~400 μM), which is in contrast to most ubiquitin receptors that contain several ubiquitin-binding sites that act synergistically to provide high-affinity binding. Similarly, the interaction of dDsk2 with H2Bub1 is of low specificity since selective recognition of ubiquitylated substrates is largely based on the recognition of the linkage type, length and anchoring site of a polyubiquitin chain, and, consequently, requires the presence of several ubiquitin-binding sites. In fact, the UBA domain of dDsk2 recognizes a monoubiquitylation in PTEN with a similar affinity as in H2B. On the other hand, binding of the dHP1c complex likely involves the recognition of specific DNA sequences since it depends on the zinc-finger proteins WOC and ROW. Noteworthy, dHP1c complex-binding sites are significantly enriched in a specific DNA sequence motif. However, although the interaction with H2Bub1 is weak, binding of the complex unexpectedly depends on dDsk2. Besides, dHP1c is dispensable for WOC and ROW binding, as well as for dDsk2 binding). These effects do not appear to be the consequence of changes in gene expression levels since dDsk2 mRNA levels do not significantly change on WOC, ROW or dHP1c depletion. Furthermore, dDsk2 depletion upregulates ROW and weakly downregulates dHP1c mRNA levels. Finally, it was reported that dHP1c interacts with RNA pol II, suggesting that binding of the dHP1c complex might depend on RNA pol II. However, arguing against this possibility, it was observed that binding of the complex at promoters is resistant to treatment with Actinomycin D. Altogether, these results suggest that WOC, ROW and dDsk2 constitute the actual binding module of the complex, being fully interdependent for binding to chromatin and required for binding of dHP1c. In this regard, the slight reduction of ROW and dHP1c protein levels observed on dDsk2 depletion is most likely due to their inability to bind chromatin, as described previously for dHP1c in ROW and WOC knockdowns, as well as for other chromatin-associated proteins when their binding to chromatin is impaired (Kessler, 2015).

These results suggest that the main function of dDsk2 in the dHP1c complex is to prevent H2Bub1 deubiquitylation by dUbp8/Nonstop, as H2Bub1 levels are strongly reduced on dDsk2 depletion and recovered after dUbp8/Nonstop co-depletion. Protection against deubiquitylation has also been reported for Rad23 and appears to be a common feature of many ubiquitin receptors. Simultaneous dDsk2 and dUbp8/Nonstop depletion also restores expression of target genes, whereas it has no effect on recruitment of the complex at promoters. Furthermore, overexpression of the ΔUBA–dDsk2 construct, which misses the UBA domain that mediates interaction with H2Bub1 in vitro, reduces H2Bub1 levels at promoters and downregulates expression of target genes. Altogether, these results strongly suggest that the contribution of dDsk2 to transcription regulation is mainly based on this protective function (Kessler, 2015).

dHP1c complex target genes show features associated with strong RNA pol II pausing. The results support a contribution of dDsk2 to pausing since its depletion reduces RNA pol II occupancy preferentially at TSS and strongly decreases NELF-E levels. dDsk2 depletion downregulates expression and, in good agreement, total RNA pol II occupancy across target genes is reduced. Interestingly, the majority of NELF target genes are also downregulated on NELF depletion in S2 cells51, and NELF potentiates gene expression in the Drosophila embryo. Actually, ~80% of dHP1c complex target genes that change expression in NELF knockdown conditions are downregulated. Furthermore, NELF-E depletion shows a similar reduction of total RNA pol II occupancy across target genes. Altogether, these observations suggest that disrupting RNA pol II pausing does not generally increase productive transcription, but results in reduced total RNA pol II occupancy and decreased expression. It is possible that premature pause release interferes with RNA pol II activation into elongation, resulting in abortive transcription that, in turn, could affect RNA pol II recruitment and/or re-initiation. Notice, however, that dDsk2 depletion does not affect the extent of histone acetylation detected at target promoters, suggesting that they retain the transcriptional active chromatin state (Kessler, 2015).

Notably, co-depletion of dUbp8/Nonstop, which rescues H2Bub1, also rescues the pausing defect caused by dDsk2 depletion and expression levels are restored, suggesting that dynamic regulation of H2Bub1 levels at promoters of dHP1c target genes plays a role in RNA pol II pausing. In this regard, work performed in yeast suggested that transcriptional activation involves sequential cycles of H2B ubiquitylation and deubiquitylation and that Ubp8 promotes Ctk1-dependent phosphorylation of Ser2 in the CTD33, a modification that is required for activation into the elongating Pol IIoser2 form. Nevertheless, H2Bub1 deubiquitylation at TSS does not appear to be sufficient by itself to induce pause release since dBre1 depletion, which also reduces H2Bub1 at TSS, has no significant effect in pausing. In this regard, it must be noted that dBre1 travels with the elongating RNA pol II along coding regions to induce H2Bub1, which stimulates Facilitates-Chromatin-Transcription (FACT) activity and, thus, facilitates elongation. On the other hand, dUbp8/Nonstop activity is mainly restricted to promoters since its depletion in dBre1-deficient cells has little effect in H2Bub1 levels at coding regions. On the contrary, dUbp8/Nonstop depletion in dDsk2-deficient cells strongly rescues H2Bub1 levels at coding regions. Interestingly, whereas dUbp8/Nonstop depletion restores expression of target genes in dDsk2-depleted cells, it has only a slight effect in dBre1-deficient cells. Altogether, these results suggest that dBre1 depletion impairs elongation and, thus, might prevent the release of paused RNA pol II by disturbing its actual engagement into elongation. Further work is required to better understand the mechanisms that regulate RNA pol II pausing, the actual contribution of dDsk2 and whether it involves H2Bub1 and/or additional factors also targeted by ubiquitylation (Kessler, 2015).

The dHP1c complex appears to have a particularly important contribution to nervous system development and function since target genes are enriched in related functions and knockdown conditions preferentially affect gene expression in the nervous system. Actually, WOC and ROW are highly expressed in the nervous system during embryo and larval development, and mutant larvae show brain defects. Furthermore, in humans, the WOC homologue DXS6673E/ZNF261 has been implicated in X-linked mental retardation. Interestingly, mutations in the human Dsk2 homologues (Ubqln-1/2) have been associated with Alzheimer's disease as well as other neurodegenerative diseases. Noteworthy, Ubqln-1/2 are detected in both the nucleus and the cytoplasm, and the development and progression of neurofibrillary tangles in Alzheimer's disease brains associate with an altered nuclear Ubqln-1 content. Whether the role of dDsk2 in transcription regulation is conserved in humans and contributes to disease remains to be determined (Kessler, 2015).

In summary, these results indicate that the ubiquitin receptor dDsk2 plays a nonproteolytic function in the regulation of H2Bub1 and RNA pol II pausing at promoters of dHP1c complex target genes. Ubiquitin receptors have been previously reported to play nonproteolytic functions in DNA repair and transcription elongation. Furthermore, in response to DNA damage, human Rad23B was found to interact with ubiquitylated p53, localize at chromatin and accumulate at the p21 promoter. In addition, in mouse embryonic stem cells, several components of the NER complex, including Rad23B, have been shown to act as an Oct4/Sox2 co-activator complex that associates with chromatin and is required for stem cell maintenance. Recruitment of NER factors to active promoters has also been reported in HeLa cells in the absence of DNA damage. However, in these cases, the precise function of the ubiquitin receptor has not been elucidated. In this regard, these results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications and transcription initiation. It must also be noted that ubiquitylation participates in the regulation of multiple genomic functions and that the number of proteins containing ubiquitin-binding domains is large, ~100 in humans. Therefore, a role of ubiquitin-binding proteins as epigenetic regulators of chromatin emerges as a distinct possibility (Kessler, 2015).

TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, human RNAPII promoter-proximal pausing was reconstituted in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID (Fant, 2020).

RNA polymerase II (RNAPII) transcribes all protein-coding and many non-coding RNAs in the human genome. RNAPII transcription initiation occurs within the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes typically pause after transcribing 20-80 bases, and paused polymerases represent a common regulatory intermediate. Accordingly, paused RNAPII has been implicated in enhancer function, development and homeostasis, and diseases ranging from cancer to viral pathogenesis. Precisely how RNAPII promoter-proximal pausing is enforced and regulated remains unclear; however, protein complexes, such as NELF and DSIF, increase pausing, whereas the activity of CDK9 (P-TEFb complex) correlates with pause release (Fant, 2020).

Although much has been learned about RNAPII promoter-proximal pausing and its regulation, the underlying molecular mechanisms remain enigmatic. One reason for this is the complexity of the human RNAPII transcription machinery, which includes the ∼4.0 MDa PIC and many additional regulatory factors. Another underlying reason is that much current understanding derives from cell-based assays, which are indispensable but cannot reliably address mechanistic questions. For instance, factor knockdowns or knockouts cause unintended secondary effects and the factors and biochemicals present at each gene in a population of cells cannot possibly be defined. In vitro assays can overcome such limitations, but these have typically involved nuclear extracts, which contain a similarly undefined mix of proteins, nucleic acids, and biochemicals. To circumvent these issues, this study sought to reconstitute RNAPII promoter-proximal pausing entirely from purified human factors (no extracts). Success with this task enabled addressing some basic mechanistic questions and opens the door for future studies to better define the contribution of specific factors in RNAPII promoter-proximal pause regulation (Fant, 2020).

Structural data indicate that TFIID lobe C subunits TAF1 (see Drosophila Taf250) and TAF2 bind promoter DNA downstream of the TSS (Louder, 2016; Patel, 2018). Past studies revealed that insertion of 10-bp DNA at the +15 site relative to the TSS disrupted RNAPII pausing at the HSP70 gene in Drosophila S2 cells (Kwak, 2013). This led to a 'complex interaction' model for pausing, in which a promoter-bound factor(s) establishes an interaction (directly or indirectly) with the paused RNAPII complex. In agreement with this model, a TFIID requirement was observed for RNAPII promoter-proximal pausing in vitro, which is further supported by PRO-seq data in TAF-depleted human and Drosophila S2 cells. Additional evidence for TFIID-dependent regulation of RNAPII pausing derives from correlations among paused genes and DNA sequence elements bound by TFIID. Defects in TFIID function are linked to numerous diseases, including cancer and neurodegenerative disorders. Its requirement for RNAPII promoter-proximal pause regulation may underlie these and other biological functions (Fant, 2020).

Biochemical reconstitution of RNAPII promoter-proximal pausing provides a level of mechanistic control that is simply not possible with cell-based assays; consequently, it was discovered that RNAPII pausing is an inherent property of the human PIC and that TFIID is a key PIC factor that establishes pausing. The results also reveal NELF, DSIF, and P-TEFb as auxiliary factors that, although not required for pausing, enable robust regulation of this common transcriptional intermediate state. Time course experiments indicated that polymerases in the paused region remained active and generated elongated transcripts over time. Experiments with P-TEFb showed enhanced release of paused intermediates, providing further evidence that polymerases in the paused region were active and competent for elongation. However, some transcripts remained in the pause region after the 10-min reactions, even with added P-TEFb. This result is also consistent with current models that invoke alternative outcomes for promoter-proximal paused RNAPII, including premature termination, arrest, or a more stable paused intermediate. Addressing the mechanisms and factors that regulate these distinct outcomes could be explored in future studies (Fant, 2020).

Despite its advantages, the reconstituted in vitro transcription assay does not match the complexity of regulatory inputs that converge upon active promoters in a living cell. To test the TFIID requirement for promoter-proximal pausing in cells, it was possible to rapidly deplete TFIID lobe C subunits TAF1 and TAF2 using Trim-Away, and genome-wide changes in nascent transcription were assessed with PRO-seq. Consistent with the in vitro data, global transcription increased at protein-coding genes upon TAF1/2 knockdown, with evidence for enhanced pause release. PRO-seq reads increased at 5' ends and downstream of promoter-proximal pause sites at thousands of genes in TAF1/2-depleted cells. These data are consistent with increased pause release and increased re-initiation, two processes that are coupled in metazoan cells. Unexpectedly, however, increased pause release did not yield similar genome-wide increases in gene body reads. Instead, the PRO-seq data revealed a sharp reduction in reads downstream of promoter-proximal pause sites, at around +300 from the TSS in both human and Drosophila cells. These results implicate additional regulatory mechanisms, downstream of the pause site, that may terminate or arrest RNAPII. Although future studies are needed to identify the factors involved, it is noted that the Integrator complex was recently shown to cleave nascent transcripts downstream of pause sites at hundreds of genes in Drosophila cells (Tatomer, 2019). Because promoter-proximal pausing helps ensure proper capping of transcripts at their 5' ends, downstream regulatory mechanisms may become important when RNAPII promoter-proximal pausing is disrupted (Fant, 2020).

A TFIID requirement for RNAPII promoter-proximal pausing implies that other pause regulatory factors may function directly or indirectly through TFIID. Although additional mechanistic aspects remain to be addressed, it is notable that pause regulatory factors, including P-TEFb and MYC, interact (directly or indirectly) with TFIID; moreover, TFIID is conformationally flexible and likely undergoes structural reorganization during RNAPII transcription initiation and pause release. Such structural transitions may contribute to TFIID-dependent regulation of RNAPII pausing. Whereas nucleosomes likely affect promoter-proximal pausing, they are not required, based upon our results and data in Drosophila and mammalian systems. TFIID possesses multiple domains that bind chromatin marks associated with transcriptionally active loci, including H3K4me3, which suggests TFIID function is regulated in part through epigenetic mechanisms. Future studies should help establish whether specific chromatin marks contribute to TFIID-dependent regulation of RNAPII pausing, potentially by affecting TFIID promoter occupancy or by impacting TFIID structure and function (Fant, 2020).

The Integrator complex cleaves nascent mRNAs to attenuate transcription

Cellular homeostasis requires transcriptional outputs to be coordinated, and many events post-transcription initiation can dictate the levels and functions of mature transcripts. To systematically identify regulators of inducible gene expression, high-throughput RNAi screening of the Drosophila Metallothionein A (MtnA) promoter was performed. This revealed that the Integrator complex, which has a well-established role in 3' end processing of small nuclear RNAs (snRNAs), attenuates MtnA transcription during copper stress. Integrator is an evolutionarily conserved complex that contains 14 subunits and regulates RNA processing and gene transcription by associating with the C-terminal domain of RNA polymerase II large subunit. Integrator complex subunit 11 (IntS11) endonucleolytically cleaves MtnA transcripts, resulting in premature transcription termination and degradation of the nascent RNAs by the RNA exosome, a complex also identified in the screen. Using RNA-seq, >400 additional Drosophila protein-coding genes whose expression increases upon Integrator depletion. This study focused on a subset of these genes and confirmed that Integrator is bound to their 5' ends and negatively regulates their transcription via IntS11 endonuclease activity. Many noncatalytic Integrator subunits, which are largely dispensable for snRNA processing, also have regulatory roles at these protein-coding genes, possibly by controlling Integrator recruitment or RNA polymerase II dynamics. Altogether, these results suggest that attenuation via Integrator cleavage limits production of many full-length mRNAs, allowing precise control of transcription outputs (Tatomer, 2019).

In response to physiological cues, environmental stress, or exposure to pathogens, specific transcriptional programs are induced. These responses are often coordinated, rapid, and robust, in part because many metazoan genes are maintained in a poised state with RNA polymerase II (RNAPII) engaged prior to induction. In addition to promoter-proximal pausing, there are many regulatory steps post transcription initiation that dictate the characteristics and fate of mature transcripts. For example, alternative splicing and/or 3' end processing events can lead to the production of multiple isoforms from a single locus, and these transcripts can have distinct stabilities, translation potential, or subcellular localization (Tatomer, 2019).

It is particularly important that genes produce full-length functional mRNAs and mechanisms such as telescripting, involving U1 snRNP, actively suppress premature cleavage and polyadenylation events in eukaryotic cells. Nevertheless, many promoters are known to generate short unstable RNAs. This suggests that premature transcription termination may often occur, thereby limiting RNAPII elongation and production of full-length mRNAs (for review, see Kamieniarz-Gdula and Proudfoot 2019). Moreover, this process can be regulated. For example, it was recently shown that the cleavage and polyadenylation factor PCF11 stimulates premature termination to attenuate the expression of many transcriptional regulators in human cells (Kamieniarz-Gdula, 2019). Potentially deleterious truncated transcripts generated by premature termination are often removed from cells by RNA surveillance mechanisms, including by the RNA exosome. However, the full repertoire of cellular factors and cofactors that control the metabolic fate of nascent RNAs, especially during the early stages of transcription elongation, is still unknown (Tatomer, 2019).

An unbiased genome-scale RNAi screen was performed in Drosophila cells to reveal factors that control the output of a model inducible eukaryotic promoter. Transcription of Drosophila Metallothionein A (MtnA), which encodes a metal chelator, is rapidly induced when the intracellular concentration of heavy metals (e.g., copper or cadmium) is increased. This increase in transcriptional output is dependent on the MTF-1 transcription factor, which relocalizes to the nucleus upon metal stress and binds to the MtnA promoter. The RNAi screen identified MTF-1 and other known regulators of MtnA transcription, but also surprisingly identified the Integrator complex as a potent inhibitor of MtnA during copper stress. Integrator harbors an endonuclease that cleaves snRNAs and enhancer RNAs, and this study has found that Integrator can likewise cleave nascent MtnA transcripts to limit mRNA production. Using RNA-seq, hundreds of additional Drosophila protein-coding genes were found whose expression increases upon Integrator depletion. Focused studies on a subset of these genes confirmed that Integrator can cleave these nascent RNAs, thereby limiting productive transcription elongation. Altogether, it is proposed that Integrator-catalyzed premature termination can function as a widespread and potent mechanism to attenuate expression of protein-coding genes (Tatomer, 2019).

Altogether, the data indicate that the Integrator complex can attenuate the expression of protein-coding genes by catalyzing premature transcription termination. The IntS11 endonuclease cleaves a subset of nascent mRNAs, which ultimately triggers degradation of the transcripts by the RNA exosome along with RNAPII termination. It is suggested that many protein-coding genes are negatively regulated via this attenuation mechanism, and the Drosophila MtnA promoter highlights context-specific regulation by Intgerator. Transcription of MtnA is induced by copper or cadmium stress, and yet this study finds that Integrator is robustly recruited to the MtnA promoter only under copper stress conditions. This is not because the Integrator complex is generally diassembled or 'poisoned' by cadmium, as Integrator continues to regulate the outputs of other protein-coding genes. It is instead proposed that context-specific regulation of this locus may be related to the fact that cadmium is a strictly toxic metal, while copper is required for the function of a subset of enzymes and must be maintained in a narrow concentration range. Therefore, homeostatic control of MtnA is required to maintain copper levels, while cells need to maximally produce MtnA in the presence of cadmium. It is thus proposed that regulation of MtnA levels by Integrator during copper stress is for fine-tuning purposes, perhaps to limit maximal transcriptional induction and/or facilitate transcriptional shut-off once copper stress has passed. The results suggest that the Integrator complex can be recruited to gene loci only when needed, thereby ensuring tight control over transcriptional output (Tatomer, 2019).

In addition to cleaving MtnA transcripts, Integrator cleaves multiple other RNA classes in metazoan cells, including enhancer RNAs (Lai, 2015), snRNAs (Baillat, 2005), telomerase RNA (Rubtsova, 2019), and some herpesvirus microRNA precursors (Cazalla, 2011; Xie, 2015). Using RNA-seq, this study has expanded this list of Integrator target loci and identified hundreds of additional protein-coding genes that are negatively regulated by Integrator. Focused is placed on a set of Integrator-dependent genes; Integrator was found to catalyze premature transcription termination of these genes, consistent with prior studies that suggested roles for Integrator in termination (Skaar, 2015; Shah, 2018; Gomez-Orte, 2019). Some of these genes (CG8620, Pepck1, and Sirup) have promoter-proximal RNAPII that rapidly turns over, which may indicate that Integrator can aid in clearing paused or stalled RNAPII. Once Integrator has cleaved the nascent mRNAs, this study finds that they are rapidly degraded from their 3' ends by the RNA exosome. This may be critical for enabling subsequent rounds of transcription (especially at the MtnA locus), perhaps because the small RNAs can form stable RNA-DNA hybrids (R-loops) that block transcription initiation or elongation (Tatomer, 2019).

Endonucleolytic cleavage is critical for Integrator regulation at snRNA and protein-coding genes, but the data indicate that these loci have different dependencies on Integrator subunits. Genetic studies indicate that Integrator subunits 4, 9, and 11 (which form the Integrator cleavage module) are most important for snRNA processing, while the non-catalytic Integrator subunits (all of which currently lack annotated molecular functions) play minor roles. In contrast, large increases in mRNA expression were observed when many of the non-catalalytic subunits were depleted (especially IntS1, IntS2, IntS5, IntS6, IntS7, and IntS8). IntS13 was recently shown to be able to function independently from other Integrator subunits at enhancers (Barbieri, 2018), suggesting the existence of submodules or 'specialized' complexes that may enable the activity and function of Integrator to be distinctly regulated depending on the gene locus and cellular state. Future work will reveal the subunit requirements of Integrator complexes at distinct loci and clarify the interplay between IntS11 endonuclease activity and other Integrator subunits. For example, the non-catalytic subunits may be critical for the formation and targeting of the complex to specific loci and/or controlling RNAPII dynamics (Tatomer, 2019).

Finally, it is noted that the metazoan Integrator complex has parallels with the yeast Nrd1-Nab3-Sen1 (NNS) complex that (1) terminates transcription at both mRNA and snRNA loci and (2) interacts with the RNA exosome. Interestingly, the underlying molecular mechanisms of transcription termination carried out by these two complexes are quite distinct. NNS uses the Sen1 helicase to pull the nascent transcript out of the RNAPII active site, while Integrator likely promotes termination by taking advantage of its RNA endonuclease activity and providing an entry site for a 5'-3' exonuclease. There is currently conflicting data on whether the canonical 'torpedo' exonuclease Rat1/Xrn2 is involved in termination at snRNA genes as only subtle termination defects have been observed at these loci when Rat1/Xrn2 is depleted from cells. Notably, Cpsf73 has been shown to behave as both an endonuclease and exonuclease, raising the possibility that IntS11 could support a 'Rat1/Xrn2-like' function and mediate termination. Future studies that compare and contrast the Integrator and NNS complexes, especially how their recruitment and termination activities are controlled, will shed light on this important facet of gene regulation. In summary, transcription attenuation through premature termination was first described decades ago in bacteria, and the current work indicates that the metazoan Integrator complex can function analogously to limit expression from protein-coding genes (Tatomer, 2019).

Thermodynamic model of gene regulation for the Or59b olfactory receptor in Drosophila

Complex eukaryotic promoters normally contain multiple cis-regulatory sequences for different transcription factors (TFs). The binding patterns of the TFs to these sites, as well as the way the TFs interact with each other and with the RNA polymerase (RNAp), lead to combinatorial problems rarely understood in detail, especially under varying epigenetic conditions. The aim of this paper is to build a model describing how the main regulatory cluster of the olfactory receptor Or59b drives transcription of this gene in Drosophila. The cluster-driven expression of this gene is represented as the equilibrium probability of RNAp being bound to the promoter region, using a statistical thermodynamic approach. The RNAp equilibrium probability is computed in terms of the occupancy probabilities of the single TFs of the cluster to the corresponding binding sites, and of the interaction rules among TFs and RNAp, using experimental data of Or59b expression to tune the model parameters. The model reproduces correctly the changes in RNAp binding probability induced by various mutation of specific sites and epigenetic modifications. Some of its predictions have also been validated in novel experiments (Gonzalez, 2019).

Transcription-generated torsional stress destabilizes nucleosomes

As RNA polymerase II (Pol II) transcribes a gene, it encounters an array of well-ordered nucleosomes. How it traverses through this array in vivo remains unresolved. One model proposes that torsional stress generated during transcription destabilizes nucleosomes ahead of Pol II. This study describes a method for high-resolution mapping of underwound DNA, using next-generation sequencing, and shows that torsion is correlated with gene expression in Drosophila cells. Accumulation of torsional stress, through topoisomerase inhibition, results in increased Pol II at transcription start sites. Whereas topoisomerase I inhibition results in increased nascent RNA transcripts, topoisomerase II inhibition causes little change. Despite the different effects on Pol II elongation, topoisomerase inhibition results in increased nucleosome turnover and salt solubility within gene bodies, thus suggesting that the elongation-independent effects of torsional stress on nucleosome dynamics contributes to the destabilization of nucleosomes (Teves, 2013).

The stalled Pol II accumulates immediately downstream of the TSS after inhibition of either Topo I or Topo II, but the elongating Pol II, as measured by nascent-RNA production, is affected differently. Topo I inhibition results in increased nascent-RNA levels near the 5′ ends of genes. In contrast, Topo II inhibition affects nascent-RNA levels of only highly expressed genes, whereas expression of most genes remains unaltered, thus suggesting that only Topo I acts within gene bodies to regulate Pol II elongation kinetics. Despite the changes in expression and Pol II kinetics after topoisomerase inhibition, accumulation of torsional stress results in increased nucleosome turnover within gene bodies genome wide, providing direct evidence for an in vivo influence of DNA torsion on nucleosome dynamics that is separable from Pol II elongation–associated dynamics. The data support a model whereby the transient wave of positive torsion downstream of Pol II destabilizes genic nucleosomes to allow progression, and the transient negative torsion stabilizes nucleosome formation behind Pol II to maintain chromatin structure. In this way, a delicate balance between nucleosomal destabilization, maintenance and Pol II progression is achieved (Teves, 2013).

It has been generally accepted that inhibition of topoisomerases results in an immediate halt in transcription, owing to accumulation of torsional stress. Early studies have shown that transcription of rDNA requires topoisomerase activity, and in vitro transcription assays using viral RNA polymerases are greatly inhibited when Topo I activity is absent. This study has shown that topoisomerase inhibition alters Pol II kinetics, which is a potential mechanism for decreased expression. Topo I inhibition alters both the initiating and elongating form of Pol II, and after a short inhibition period it results in little change in expression as measured by changes in complete nascent RNA transcripts. This result seems contradictory to the dogma that topoisomerase inhibition leads to transcription inhibition. However, various other studies inhibiting only Topo I have yielded mixed effects on transcription. Topo I inhibition has been shown to increase Pol II and nascent RNA near the TSS, but not at the 3′ end of the Dhfr gene in cultured CHO cells, and to increase the release of paused Pol II and antisense transcription of the HIF1A gene in human cells. These studies suggest that the highly conserved Topo I may also have a conserved effect on Pol II kinetics. In contrast to Topo I inhibition, Topo II inhibition resulted in altered Pol II elongation kinetics of only the most highly expressed genes, thus confirming that Topo I is the main relaxer of transcription-generated torsional strain, and Topo II acts together with Topo I in highly expressed genes (Teves, 2013).

It is noteworthy that the torsion-induced increase in nucleosome turnover is observed only beyond the first nucleosome downstream of the TSS (+1 nucleosome). In fact, relative to the genome-wide average, the +1 nucleosome and promoter nucleosomes showed decreased turnover upon topoisomerase inhibition. The +1 nucleosome has the highest density of stalled Pol II, thus suggesting a specialized interaction between Pol II and the +1 nucleosome. Indeed, MNase-protected fragments spanning the +1 nucleosome and the footprint of Pol II are enriched immediately downstream of the TSS. It is possible, therefore, that under topoisomerase inhibition, the interaction between Pol II and the +1 nucleosome is further stabilized, thus resulting in increased stalled Pol II at the TSS and decreased turnover at the +1 nucleosome after topoisomerase inhibition. Furthermore, the decreased turnover of promoter nucleosomes may reflect the altered Pol II initiation kinetics after topoisomerase inhibition. But how are the effects of torsional stress generated at TSSs propagated beyond the +1 nucleosome? Mathematical modeling of transcription-generated torsion suggests that transcription of 5 bp is sufficient to propagate a wave of positive supercoils at a rate two orders of magnitude faster than the rate of Pol II elongation. Single-molecule experiments on supercoil dynamics have shown that this propagation has two modes: the slower mechanism of diffusion that occurs in short-range distances and the much faster mode of 'hopping' in which supercoils are propagated in long distances under millisecond time frames. Therefore, it is possible that the effect of increased turnover downstream of the +1 nucleosome is a result of torsional stress being propagated in long-range distances through the hopping mechanism. In this way, topological changes to DNA during transcription can act as the medium that connects events at the TSSs to ones far downstream within gene bodies (Teves, 2013).

Architecture of an RNA polymerase II transcription pre-initiation complex

The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft (Murakami, 2013).

This study has revealed a central principle of the PIC: the association of promoter DNA only with the GTFs and not with pol II. Promoter DNA is suspended above the pol II cleft, contacting three GTFs -- TFIIB, TFIID (TBP subunit), and TFIIE -- at the upstream end of the cleft (TATA box) and contacting TFIIH (Ssl2 helicase subunit) at the downstream end. In between, the DNA is free and available for action of the helicase, which untwists the DNA to introduce negative superhelical strain and thereby promote melting at a distance (Murakami, 2013).

This principle of the PIC is a consequence of the rigidity of duplex DNA. The promoter duplex must follow a straight path, whereas bending through ~90° is required for binding in the pol II cleft. Only after melting can the DNA bend for entry in the cleft. Melting is thermally driven, induced by untwisting strain in the DNA above the cleft. A melted region is short-lived and must be captured by binding to pol II, which occurs rapidly enough because the DNA is positioned above the cleft. The GTFs therefore catalyze the formation of a stably melted region (transcription bubble) in two ways, by the introduction of untwisting strain (by the helicase) and by positioning promoter DNA (Murakami, 2013).

Untwisting strain is distributed throughout the DNA above the pol II cleft, so melting may occur at any point, but only a melted region adjacent to TFIIB is stabilized by binding to pol II. The reason is again the rigidity of duplex DNA, and the requirement for a sharp bend adjacent to TFIIB to penetrate the pol II cleft. A single strand of DNA must extend from the point of contact with TFIIB, ~13 bp downstream of the TATA box, through the binding site for the transcription bubble in pol II. TFIIB may also interact with the single strand to stabilize the bubble (Murakami, 2013).

These conclusions are based on results from both cryo-EM and XL-MS, which served to validate one another: Segmentation and labeling of electron density, based on fitting pol II and other known structures, was consistent with all but three of 266 cross-links observed. The PIC structure is also consistent with partial structural information from x-ray crystallography (pol II-TFIIB, pol II-TFIIS, TFIIA-TBP-TFIIB-DNA, and Tfb2-Tfb5), from nuclear magnetic resonance (Tfb1-Tfa1 and Tfa2-DNA), and from EM (core and holo TFIIH). This consistency provides cross-validation, both supporting this PIC structure and establishing the relevance of the partial structural information. Further consistency was found with the results of FeBABE cleavage mapping of complexes formed in yeast nuclear extract; the locations of proteins along the DNA in the PIC structure and those determined with FeBABE cleavage differ by no more than 5 bp. This PIC structure also agrees with results of protein-DNA cross-linking in a reconstituted human transcription system; positions of TFIIE and TFIIH differ between the two studies by ~20 and 10 bp. The location of Ssl2 in this structure, ~30 bp downstream from the TATA box, supports the proposal, made on the basis of previous DNA-protein cross-linking analysis, that helicase action torques the DNA to introduce untwisting strain and thereby to promote melting at a distance (Murakami, 2013).

Dynamics of heat shock factor association with native gene loci in living cells

Direct observation of transcription factor action in the living cell nucleus can provide important insights into gene regulatory mechanisms. Live-cell imaging techniques have enabled the visualization of a variety of intranuclear activities, from chromosome dynamics to gene expression. However, progress in studying transcription regulation of specific native genes has been limited, primarily as a result of difficulties in resolving individual gene loci and in detecting the small number of protein molecules functioning within active transcription units. This study reports that multiphoton microscopy imaging of polytene nuclei in living Drosophila salivary glands allows real-time analysis of transcription factor recruitment and exchange on specific native genes. After heat shock, this study has visualized the recruitment of RNA polymerase II (Pol II) to native hsp70 gene loci 87A and 87C in real time. Heat shock factor (HSF), the transcriptional activator of hsp70, is localized to the nucleus before heat shock and translocates from nucleoplasm to chromosomal loci after heat shock. Assays based on fluorescence recovery after photobleaching show a rapid exchange of HSF at chromosomal loci under non-heat-shock conditions but a very slow exchange after heat shock. However, this is not a consequence of a change of HSF diffusibility, as shown here directly by fluorescence correlation spectroscopy. The results provide strong evidence that activated HSF is stably bound to DNA in vivo and that turnover or disassembly of transcription activator is not required for rounds of hsp70 transcription. It is concluded that transcriptional activators display diverse dynamic behaviours in their associations with targeted loci in living cells. This method can be applied to study the dynamics of many factors involved in transcription and RNA processing, and in their regulation at native heat shock genes in vivo (Yao, 2006).

The rapid recovery pattern of HSF under non-heat shock (NHS) and slow recovery under heat shock (HS) corresponds in vivo to the marked difference in the DNA-binding affinity of HSF monomers (NHS) and trimers (HS). It is therefore proposed that a transcription activator's exchange dynamics on its targets may simply reflect the dissociation rate constant of the protein-promoter complex. The low affinity of some activators leads to their transient binding and has been suggested to cause the probabilistic assembly of transcriptional machinery. The high affinity of other activators leads to their stable binding, and this in turn is conducive to the formation of stable coactivator assemblies and the efficient recruitment of Pol II for repeated cycles of transcription. The exchange dynamics of some activators may involve other mechanisms; for instance, NF-kappaB, which has high affinity for DNA, was found to exchange rapidly at the tandem-repeat target gene loci. In addition, chromatin remodelling might have a function in these processes (Yao, 2006).

The slow exchange of activated HSF at the hsp70 promoter presents a sharp contrast with the rapid recruitment and elongation of RNA polymerase II at hsp70 genes during HS. During a 2-min transcription cycle (that is, the time it takes a Pol II molecule to transcribe the hsp70 gene, more than 20 Pol II molecules have begun the transcription of each hsp70 gene; however, very little new HSF has bound to the gene as shown by FRAP. Therefore, the data do not support the 'activation by destruction' hypothesis that the recruitment of new polymerase requires the ubiquitin-proteasome system (UPS) to turn over the 'spent' activator on the promoter. Moreover, more than the total amount of intracellular HSF would be degraded during a short period of heat shock if 'activation by destruction' were true for every round of heat shock gene transcription. HSF is an acidic, strong activator, like many positive regulatory factors, and hsp70 transcription resembles that of many other genes. Recent results on the yeast Gal4 activator have shown that it, too, is stably bound to its regulatory sites during gene activation. Therefore two independent and complementary approaches on the two widely studied acidic activators have revealed their stable binding to DNA during gene activation. Alternative models for activator function that propose activator recycling as a key component, such as hit and run, chaperone-assisted disassembly or UPS-mediated turnover, can apply to some but clearly not all transcriptional activators (Yao, 2006).

The stable binding of HS-activated HSF and the transient binding of ligand-activated GR collectively show the diverse 'action modes' of transcription activators: both stably bound and transiently bound activators can support gene transcription. How individual activators function in these two modes on their respective gene targets remains to be seen, with the underlying mechanisms yet to be determined. Importantly, the dynamic behaviour of coactivators, Pol II transcription and RNA-processing machinery at native mRNA genes is largely unknown in living cells, and the described experimental approach will be applicable to further investigations (Yao, 2006).

Number of nuclear divisions in the Drosophila blastoderm controlled by onset of zygotic transcription

The cell number of the early Drosophila embryo is determined by exactly 13 rounds of synchronous nuclear divisions, allowing cellularization and formation of the embryonic epithelium. The pause in G2 in cycle 14 is controlled by multiple pathways, such as activation of DNA repair checkpoint, progression through S phase, and inhibitory phosphorylation of Cdk1, involving the genes grapes, mei41, and wee1. In addition, degradation of maternal RNAs and zygotic gene expression are involved. The zinc finger Vielfaltig (Vfl) controls expression of many early zygotic genes, including the mitotic inhibitor fruhstart. The functional relationship of these pathways and the mechanism for triggering the cell-cycle pause have remained unclear. This study shows that a novel single-nucleotide mutation in the 3' UTR of the RNA polymerase RNPII215 gene leads to a reduced number of nuclear divisions that is accompanied by premature transcription of early zygotic genes and cellularization. The reduced number of nuclear divisions in mutant embryos depends on the transcription factor Vfl and on zygotic gene expression, but not on grapes, the mitotic inhibitor Fruhstart, and the nucleocytoplasmic ratio. It is proposed that activation of zygotic gene expression is the trigger that determines the timely and concerted cell-cycle pause and cellularization (Sung, 2012).

Embryos from germline clones of the lethal mutation X161 (in the following, designated as mutant embryos) showed a reduced cell number but otherwise developed apparently normally until at least gastrulation stage. Cell specification along the anterior-posterior and dorsoventral axes proceeded as in wild-type, as demonstrated by the seven stripes of eve expression, mesoderm invagination, and cephalic furrow formation. The reduced cell number can be due to a lower number of nuclear divisions prior to cellularization or to loss of nuclei in the blastoderm. To distinguish these possibilities, time-lapse recordings were performed of mutant embryos in comparision to wild-type. To measure the cell-cycle length, the nuclei in these embryos were fluorescently labeled. Three types of embryos were observed: (1) with 13 nuclear divisions with an extended interphase 13 (28 min versus 21 min in wild-type), (2) with 12 nuclear divisions, and (3) with partly 12 and partly 13 nuclear divisions with an extended interphase 13. Because a severe nuclear fallout phenotype was not observed, it is concluded that the reduced cell number in gastrulating embryos is due to the reduced number of nuclear divisions. Consistent with these observations, the number of centromeres and centrosomes was normal in mutant embryos (Sung, 2012).

In wild-type embryos, interphase 14 is different from the preceeding interphases, in that the plasma membrane invaginates to enclose the individual nuclei into cells. In X161 embryos with patches in nuclear density, furrow markers showed more advanced furrows in the part with a lower number of divisions, indicating a premature onset of cellularization. Furthermore, in time-lapse recordings, the speed of membrane invagination was measured, with no obvious difference found between X161 and wild-type embryos. Additionally, cellularization was investigated by live imaging with moesin-GFP labeling F-actin. Clear accumulation of F-actin at the furrow canals was observed in wild-type embryos after about 20 min in interphase 14, but not in interphase 13. In X161 embryos with 12 nuclear divisions, a comparable reorganization was observed already in interphase 13 after about 25 min. This analysis shows that both the cell-cycle pause and cellularization are initiated in X161 embryos earlier than in wild-type embryos (Sung, 2012).

To identify the mutated gene in X161, the lethality and blastoderm phenotype was mapped. The X161 gene was separated from associated mutations on the chromosome by meiotic recombination and mapped to a region of four genes by complementation analysis with duplications and deficiencies. Sequencing of the mapped region and complementation tests with two independent RPII215 loss-of-function alleles, RPII215(1) and RPII215[G0040], and a transgene comprising the RPII215 locus revealed the large subunit of the RNA polymerase II as the mutated gene. A single point mutation was identified in the 3' UTR of RPII215 about 40 nt downstream of the stop codon. This region in the 3' UTR is not conserved and does not show any obvious motifs (Sung, 2012).

To test whether the mutation in the noncoding region affects transcript or protein expression, mRNA levels were quantified by reverse transcription and quantitative PCR and protein levels by whole-mount staining and immunoblotting with extracts of manually staged embryos. mRNA levels were found to be the same in wild-type and X161. In contrast, immunohistology and immunoblotting revealed reduced RPII215 protein levels. In summary, the analysis shows that the X161 point mutation within the 3' UTR affects mainly RPII215 protein levels. The precocious onset of cellularization raised the hypothesis that the timing of zygotic gene expression may be affected in the X161 embryos. To establish the expression profiles of selected maternal and zygotic genes, nCounter NanoString technology was used with embryos staged by the nuclear division cycle. Embryos expressing histone 2Av-RFP were manually selected 3 min after anaphase of the previous mitosis or at midcellularization (Sung, 2012).

Expression of ribosomal proteins was analyzed. They did not change much and were not different in wild-type and mutant embryos, confirming the robustness of the method. Zygotic genes, whose expression strongly increases during the syncytial cycles, showed an earlier upregulation in X161 than in wild-type embryos. Comparing the profiles by plotting the ratio of the expression levels, a clear difference was revealed in cycle 12, with a factor of up to ten, indicating that zygotic genes are precociously expressed in X161 embryos. The premature expression of early zygotic genes was confirmed by whole-mount in situ hybridization for slam and frs mRNA (Sung, 2012).

Next, expression profiles were analyzed of RNAs subject to RNA degradation. Transcripts representative for the two classes of degradation were selected, depending on zygotic gene expression, and on egg activation. Degradation of string, twine, and smaug transcripts in interphase 14 depends of zygotic gene expression. In X161 mutants, the mRNA of these three genes was degraded already in cycle 13, slightly sooner than in wild-type. The profiles of string and twine RNA were confirmed by RNA in situ hybridization. Consistent with the precocious RNA degradation in X161, Twine and String protein levels decreased already in interphase 13 of X161 embryos. Finally, the profile was analyzed of mRNAs whose degradation depends on egg activation. No consistent pattern or clear difference was detected between the profiles of wild-type and X161 mutants. The data show that zygotic gene expression starts earlier in X161 than in wild-type and that degradation of mRNAs follows zygotic gene expression (Sung, 2012).

The cell cycle may be paused prematurely by altered levels of maternal factors, such as CyclinB, grapes, and twine, or by precociously expressed zygotic genes, such as frs and trbl. To distinguish these two options, mutant embryos with suppressed zygotic gene expression were analyzed. Embryos injected with the RNA polymerase II inhibitor α-amanitin develop until mitosis 13 but then fail to cellularize and may undergo an additional nuclear division, depending on injection conditions. Using this assay, whether zygotic genes are required for the reduced number of nuclear divisions was tested in X161 mutants. If the precocious cell-cycle pause were due, for example, to reduced levels of CyclinB mRNA, α-amanitin injection should not change the reduced number of divisions. All injected mutant embryos passed through at least 13 nuclear divisions, similar to injected wild-type embryos, whereas injection of water resulted in a mixed phenotype of 12 and 13 nuclear divisions, comparable to uninjected X161 embryos. This experiment demonstrates that the reduced division number in X161 embryos requires zygotic gene expression (Sung, 2012).

The expression of many early zygotic genes is controlled by the zinc-finger protein Vfl (also called Zelda). Tests were performed to see whether the precocious cell-cycle pause in X161 mutants is mediated by vfl-dependent genes. Analysis of X161 vfl double-mutant embryos revealed that, in contrast to X161 mutants, the cell cycle undergoes at least 13 divisions. Activation of zygotic gene expression was further analyzed by staining for Vfl and activated RPII21. Staining of both in presyncytial stages of X161 mutants was detected already in cycle 5. No specific staining for the activated RPII215 was detected in X161 vfl double-mutant embryos, and no difference in Vfl staining in syncytial embryos was detected in wild-type and X161 embryos. These findings show that the genes relevant for the precocious cell-cycle pause in X161 mutants are vfl target genes. A zygotic gene involved in cell-cycle control is frs, which is sufficient to induce a pause of the cell cycle. Analysis of X161 frs double-mutant embryos showed, however, that the number of nuclear divisions was not changed as compared to X161 single mutants. This indicates that frs is not the only cell-cycle inhibitor expressed in the early embryo. Proteins mediating the DNA repair checkpoint, such as Grapes/Chk1, are required for the cell-cycle pause. Passing normally through the nuclear division cycles, the cell cycle shows striking abnormalities in nuclear envelope formation and chromosome condensation in interphase 14 in embryos from grapes females. Tests were performed to see whether the timing of the transition in cell-cycle behavior in grapes embryos depends on the onset of zygotic transcription by analyzing X161 grapes double-mutant embryos. Some of the X161 grapes double mutants were found to show the defects in nuclear envelope formation and chromatin condensation already in interphase 13, indicating that the requirement of grapes for chromatin structure shifted from interphase 14 to 13. These data suggest that the activation of grapes and the DNA checkpoint depends on the onset of zygotic gene expression (Sung, 2012).

A factor controlling the number of nuclear divisions is the ploidy of the embryo, given that haploid embryos undergo 14 instead of 13 nuclear divisions prior to cellularization. Based on this and on related observations, it has been proposed that the nucleocytoplasmic (N/C) ratio controls the trigger for MBT. To address the functional relationship of X161 and the N/C ratio, haploid X161 embryos were analyzed. A mixture was observed in the number of nuclear divisions between 12 and 14 in fixed embryos. Embryos were even observed containing three patches with nuclear densities corresponding to 12, 13, and 14 nuclear divisions. About half of the embryos underwent 12 nuclear divisions, similar to X161 embryos. These data suggest that ploidy acts independently of general onset of zygotic transcription, which is consistent with the observation that only a subset of zygotic genes are expressed with a delay in haploid embryos. Consistent with this report, cellularization starts for a first time temporarily in interphase 14 in haploid embryos and for a second time in interphase 15. These observations suggest that the N/C ratio in Drosophila specifically affects cell-cycle regulators such as frs, for example, but not general zygotic genome activation and onset of cellularization (Sung, 2012).

In summary, the data support the model that activation of the zygotic genome controls the timing of the MBT. First, onset of MBT is sensitive to changes in RNA polymerase II activity. Second, the changes in zygotic gene expression in X161 embryos occur earlier than the changes in zygotic RNA degradation, Cdc25 protein destabilization, or activation of grapes. Third, the X161 mutant phenotype depends on zygotic transcription and on the transcription factor Vfl, showing that the precocious cell-cycle pause and onset of cellularization cannot be due to changes in maternal factors, such as higher expression of CyclinB. Although the altered levels of RNA polymerase II in X161 mutants probably affect expression of many genes during oogenesis, these changes seem not to matter in functional terms, given the overall normal morphology and specific mutant phenotype. It is conceivable that transcriptional repressors are expressed or translated in eggs in lower levels. In the embryo, such lower levels of repressors would allow the trigger for onset of zygotic gene expression to reach the threshold earlier than in wild-type embryos. The first signs of zygotic transcription are detected already during the presyncytial stages, before nuclear cycle 8/9. This may be the time when the trigger for MBT is activated (Sung, 2012).

RNA polymerase is poised for activation across the genome

Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter1, followed by initiation of RNA synthesis and the transition to productive elongation. In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region. To determine the importance of polymerase stalling for transcription regulation, a genome-wide search was carried out for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. The data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues (Muse, 2007).

Promoter-proximal pausing was first described at the Drosophila heat shock genes (for example, Hsp70), where Pol II is recruited to the promoter and initiates RNA synthesis before gene activation but stalls after elongating 20-50 nucleotides into the gene. Escape of the engaged but stalled polymerase from the Hsp70 promoter region is regulated and is rate-limiting for gene expression. Subsequently, nearly a dozen Drosophila (for example, Hsp26, Hsp27 and βTub), viral (HIV), and mammalian (including Myc, Junb and Igk) promoters have been shown to possess stalled polymerase. However, stalling is currently thought to occur at only a small number of promoters, and the full spectrum of genes affected by Pol II stalling has yet to be investigated using a genome-wide approach in any organism (Muse, 2007).

Stalled Pol II is observed at the uninduced Hsp70 promoter in Drosophila S2 cells by chromatin immunoprecipitation (ChIP). Strong Pol II signal is present near the Hsp70 promoters and decreases precipitously at probes within the genes. Pol II occupancy at the Hsp70 promoter is greater than that at nearby promoters, including the aurora kinase (aur) gene, whose expression is considerably higher than that of Hsp70. Thus, ChIP analysis of uninduced Hsp70 illustrates two hallmarks of stalled Pol II: much higher Pol II signal near the promoter than within the gene, and absence of correlation between Pol II occupancy and the levels of gene expression (Muse, 2007).

To identify other genes with stalled Pol II, chromatin immunoprecipitation microarray (ChIP-chip) experiments were carried out using tiling oligonucleotide microarrays encompassing the Drosophila genome. An antibody was used against the Pol II Rpb3 subunit to detect Pol II regardless of the phosphorylation status of the Pol II Rpb1 C-terminal domain (CTD). ChIP-chip data was analyzed with previously described computational methods to identify annotated promoters occupied by polymerase. Of the unique promoters represented on both the ChIP-chip and RNA expression arrays, 5,403 promoters were bound by Pol II and 7,702 were unbound (Muse, 2007).

Among bound genes, many showed significant Pol II signals across the gene, whereas others had Pol II signal concentrated near the promoter. To identify genes with polymerase distribution consistent with stalled Pol II, namely those genes with high promoter-proximal polymerase signals accompanied by low Pol II signals within the gene, the difference was calculated between the average polymerase signals in these regions for all 5,403 bound genes. Many genes had similar average signals within the promoter and downstream regions, indicative of rather uniform Pol II binding across the gene. Although the calculated values for most genes fit within a normal Gaussian distribution, a substantial number of outliers were found that showed promoter-proximal enrichment of polymerase (PPEP) and were thus good candidates for polymerase stalling. Notably, Drosophila genes that are known to harbor stalled Pol II show PPEP (for example, Hsp26, Hsp27 and βTub (Muse, 2007).

There was no correlation between the average Pol II signal near the promoter of genes with PPEP and the RNA expression levels observed, suggesting that the amount of Pol II recruited to these promoters does not directly dictate levels of gene expression. By comparison, genes with more uniform Pol II binding showed a correlation between Pol II and expression levels. These results are in agreement with recent ChIP-chip data from human cells that identified subsets of genes at which Pol II levels did not correlate with RNA expression. However, Pol II signals in the downstream regions of both groups correlated with RNA expression (uniform Pol II binding). Transcripts from genes with PPEP were present at levels that ranged from barely detectable to substantially expressed, consistent with prior reports that promoter-proximal stalling serves not only to fully repress transcription but also to attenuate transcription of active genes (Muse, 2007).

Permanganate footprinting of a number of genes with PPEP confirmed that Pol II enrichment at these promoters resulted from stalling during early elongation. Permanganate reacts with single-stranded thymine residues, like those in an open transcription bubble, revealing both the presence and the location of a transcriptionally engaged but stalled polymerase. Permanganate hyper-reactivity was observed within the promoter-proximal region of all genes with PPEP analyzed (Muse, 2007).

To probe the mechanisms causing Pol II enrichment at candidate promoters, it was asked whether NELF, a known regulator of polymerase stalling, played a role at genes with PPEP. In support of this idea, ChIP with an antibody to NELF showed pronounced NELF occupancy of promoters with PPEP. Pol II Rpb3 ChIP-chip was carried out on partial genomic arrays (~20% of Drosophila genome) using cells that were mock-treated or depleted of NELF by RNAi. A modest duration was used of NELF-RNAi that markedly decreases NELF protein levels but does not lead to substantially altered gene expression profiles (Muse, 2007).

NELF depletion had a profound effect on polymerase signals at genes with PPEP. Moreover, the decrease in Pol II signal observed occurred only in the promoter region and not within the body of the gene. Analysis of the difference between average Pol II signals within the promoter and downstream regions for the 1,100 bound genes present on these arrays showed 200 genes with PPEP in mock-treated cells (18.2%), but only 85 genes with PPEP in the NELF-depleted sample. Thus, NELF-dependent stalling led to promoter-proximal enrichment of polymerase at nearly 60% of the candidate genes. Stalling at the remaining 85 genes may be unaffected by NELF depletion because of relatively tighter NELF retention at these genes or, alternatively, it might be NELF independent (Muse, 2007).

Querying the Gene Ontology database with a list of genes with PPEP, a significant overrepresentation was found of genes that respond to stimuli. Notably, nearly a third of are candidate genes are involved in development. Supporting a role of polymerase stalling in development, recent work has implicated stalling at the Drosophila sloppy paired 1 (slp1) gene in the regulation of cell fate specification. Furthermore, the genes involved in the processes of cell differentiation and cell communication were significantly enriched in the PPEP gene list, which also included many rapidly induced genes involved in the Toll-signaling, MAP-kinase, defense and immune-responsive pathways. Gene Ontology queries carried out with randomly selected sets of 1,000 Drosophila genes did not show significant enrichment in specific Gene Ontology categories (Muse, 2007).

To test the idea that Pol II stalled at the newly identified genes with PPEP could be released upon gene induction, advantage was taken of the fact that key players in the response to ultraviolet (UV) irradiation have PPEP. Before UV exposure, the UV-inducible genes W (also known as hid), CG12171 and Hsp70 had substantial enrichment of Pol II at their promoters compared to the downstream regions. Ultraviolet exposure activated transcription of these genes and led to a substantial decrease in stalled Pol II, as observed by permanganate mapping, as well as to a shift of Pol II signal downstream into the genes. Thus, activation of these UV-inducible genes involves the regulated release of stalled Pol II (Muse, 2007).

In conclusion, genome-wide analysis identified hundreds of Drosophila genes that possess stalled Pol II, indicating that this method of transcription regulation is much more widespread than previously appreciated. It has been shown that, in addition to heat shock-inducible promoters, a number of constitutively expressed genes have stalled Pol II, and Pol II stalling might thus be a common phenomenon. This work fully confirms that prediction and shows that NELF plays a key role in maintaining polymerase stalled near a large number of promoters. Notably, Pol II stalls near the promoters of many genes that, like Hsp70, respond to environmental or developmental stimuli, suggesting that the rapid release of stalled Pol II facilitates efficient, integrated responses to the dynamically changing environment. A stalled Pol II in the promoter-proximal region could help to establish an active chromatin structure around these genes and maintain them poised for activation. Moreover, the prevalence of promoter-proximal stalling at developmental-control genes suggests that stalling plays a fundamental role in development (Muse, 2007).

Transcriptional pausing controls a rapid antiviral innate immune response in Drosophila

Innate immune responses are characterized by precise gene expression whereby gene subsets are temporally induced to limit infection, although the mechanisms involved are incompletely understood. This study shows that antiviral immunity in Drosophila requires the transcriptional pausing pathway, including negative elongation factor (NELF) that pauses RNA polymerase II (Pol II) and positive elongation factor b (P-TEFb), which releases paused Pol II to produce full-length transcripts. A set of genes was identified that is rapidly transcribed upon arbovirus infection, including components of antiviral pathways (RNA silencing, autophagy, JAK/STAT, Toll, and Imd) and various Toll receptors. Many of these genes require P-TEFb for expression and exhibit pausing-associated chromatin features. Furthermore, transcriptional pausing is critical for antiviral immunity in insects because NELF and P-TEFb are required to restrict viral replication in adult flies and vector mosquito cells. Thus, transcriptional pausing primes virally induced genes to facilitate rapid gene induction and robust antiviral responses (Xu, 2012).

P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development

Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. This study used a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-alpha. It was also demonstrated that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits (see Med19), providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. It is concluded that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos (Dahlberg, 2015).

The established function of P-TEFb is to phosphorylate the RNA Pol II CTD as well as the elongation factors DSIF and NELF, allowing Pol II to enter into productive elongation. This study demonstrates that embryos from which a substantial amount of the P-TEFb maternal load has been reduced show specific gene expression and morphological phenotypes. Some non-paused genes are more sensitive to diminished P-TEFb levels than many paused genes, consistent with recent P-TEFb inhibitor studies. This provides in vivo evidence that also non-paused genes transit through a P-TEFb-dependent checkpoint (Dahlberg, 2015).

P-TEFb inhibition or knock-down leads to a global decrease in Ser2 phosphorylation. In cellularizing Drosophila embryos, a similar reduction is seen in Ser2 phosphorylation. However, the global effect on Ser2 phosphorylation does not explain the selective gene expression changes. High transcription does not explain the sensitivity to P-TEFb depletion either, since term and CG7271 are expressed at similar levels to slam and bnk. Instead, the P-TEFb down-regulated genes that this study has identified are non-paused, which would suggest that they require P-TEFb continuously for efficient release into elongation. However, the state of pausing is not the only determinant for sensitivity to P-TEFb depletion since bnk, slam and nullo are also non-paused, rapidly and highly induced in pre-cellular embryos and regulated by the transcription factor Zelda, but not affected by P-TEFb knock-down. Moreover, it is possible that among the many paused genes in the early embryo, some that are also down-regulated by P-TEFb depletion could be detected by further investigation. Moreover, although both rho and eve are highly paused genes in the embryo, expression of rho, but not eve, is increased upon Cdk9 knock-down. Therefore, as yet unidentified features of P-TEFb-regulated genes confer sensitivity to reduced P-TEFb levels (Dahlberg, 2015).

Surprisingly, the ratio of Pol II at the promoter versus the 3' end at P-TEFb down-regulated genes does not change in Cdk9 embryos. Since no difference in Pol II distribution along the tested genes could be noted, Pol II appears to be released into elongation to the same extent in P-TEFb and wild-type embryos. Rather, it appears that compared to unaffected genes, less Pol II associates with genes whose expression is reduced in P-TEFb embryos. Thus, lowered Pol II occupancy may explain diminished transcription of some genes in P-TEFb embryos. Consistent with this idea, analysis of global run-on sequencing (Gro-seq) data suggests that the Sry-α and CG7271 genes are regulated at the Pol II recruitment step, and not by release from pausing in early embryos. Furthermore, Pol II ChIP-seq has shown that none of the P-TEFb down-regulated genes ever display pausing during development (Dahlberg, 2015).

Why is there less Pol II associated with some genes in P-TEFb embryos? It is possible that P-TEFb regulates expression of transcription factors during oogenesis that are needed for Pol II recruitment to P-TEFb-regulated genes. One such candidate is Zelda, which is required for zygotic genome activation. Since zelda embryos show phenotypes similar to P-TEFb and also regulates the P-TEFb-sensitive genes Sry-α, term, and CG7271, tests were performed to see whether maternal transcript levels of zelda are affected in P-TEFb embryos. In situ hybridization of P-TEFb depleted embryos showed that zelda mRNA levels are comparable to wild type embryos, suggesting that P-TEFb is not controlling maternal expression of zelda. P-TEFb might control transcription of other maternal factors that play a role in Pol II recruitment to P-TEFb-sensitive genes. Another possibility is that P-TEFb has a more direct function in recruiting Pol II to a specific set of promoters. This alternate function of P-TEFb and SEC could be evolutionarily conserved, since knocking down the SEC component ELL2 in mouse embryonic stem cells affects Pol II occupancy at the non-paused Cyp26a1 gene. A recent study showed that whereas a majority of genes in mouse embryonic stem cells accumulate Pol II at the promoter after P-TEFb inhibition, around 20% showed a decrease in Pol II promoter occupancy. Yet one more possibility is that cross talk between pausing and initiation explains why these genes and P-TEFb down-regulated genes in the Drosophila embryo have reduced Pol II occupancy. Inhibiting release into elongation may feed back on transcription initiation and result in decreased Pol II levels (Dahlberg, 2015).

A fraction of P-TEFb is present in the Super Elongation Complex (SEC). The SEC components ELL and Lilliputian (dAFF4) have previously been shown to be required for embryo development and segmentation. This study demonstrates that P-TEFb and these SEC components display similar morphological and gene expression phenotypes in early embryos, providing in vivo evidence that P-TEFb function is mediated at least in part as a component of the SEC. However, lilli mutant embryos are different in some respects from P-TEFb knock-down embryos. Expression of some genes, including ftz, is reduced in lilli mutant embryos, but not by P-TEFb depletion. This could be because this lilli allele is a stronger loss of function mutation that reduces Lilli protein levels more than P-TEFb is reduced by short hairpin miRNAs (shmiRNAs). Alternatively, this SEC component has functions that do not require P-TEFb. Importantly, many other chromatin and transcriptional regulators that were knocked-down using shmiRNAs did not share embryonic phenotypes with P-TEFb and SEC components, demonstrating the specificity of the phenotype and allowing for the identification of factors that contribute to P-TEFb and SEC function in the embryo (Dahlberg, 2015).

The Mediator complex was purified based on its ability to mediate activated transcription by bridging upstream transcription factors with Pol II, but additional functions for Mediator have emerged recently. The Mediator subunit MED26 interacts with Eaf, a member of the SEC, and recruits elongation factors to promoters in mammalian cells. It has also been shown that mammalian MED23 can recruit P-TEFb by interacting with Cdk9, and that HIF1a can recruit the SEC via the CDK8 Mediator subunit in response to hypoxia. Thiss study found that depletion of several Drosophila Mediator subunits phenocopy P-TEFb embryos and result in identical gene expression change. These results are consistent with a Mediator-SEC interaction that is important for gene transcription in vivo, and indicate that Mediator and SEC function together not only to control elongation, but also in recruiting Pol II to some developmental genes (Dahlberg, 2015).

In many organisms, germ cells are specified early during embryo development. In order to prevent these cells from differentiating into somatic cells, mRNA expression is transiently, but globally, repressed. A common strategy to specifically prevent Pol II transcription has evolved that involves inhibition of Ser2 phosphorylation. In Drosophila, polar granule component (pgc) is the germ plasm factor that represses Pol II transcription and Ser2 phoshorylation in the pole cells, by preventing P-TEFb from associating with chromatin. This study found that pgc is expressed and that pole cells are generated in P-TEFb embryos, despite loss of cells from the embryo posterior at later stages (Dahlberg, 2015).

In C. elegans, the PIE-1 protein binds to CycT and prevents Ser2 phosphorylation in the germline blastomeres. In contrast to somatic cells, loss of Cdk9 from mature germ cells has little effect on Ser2 phosphorylation, whereas Cdk12 loss abolishes Ser2 phosphorylation. Interestingly, Cdk12 and Ser2 phosphorylation are not required for C. elegans germline development and function, whereas Cdk9 is essential. Thus, P-TEFb has substrates other than the Pol II CTD that are needed for C. elegans germline function (Dahlberg, 2015).

Interestingly, this study detected elevated levels of Pol II Ser 2 phosphorylation in pre-cellular P-TEFb embryos, indicating that P-TEFb is not responsible for Ser2 phosphorylation in the Drosophila female germline. shmiRNAs targeting Cdk12 and CycK were used in the germline, but these females failed to produce eggs, demonstrating that Cdk12 is required for oogenesis. Thus, there are both similarities and differences between the C. elegans and Drosophila germline, but in both organisms P-TEFb appears to function differently in germ cells and somatic cells (Dahlberg, 2015).

Staining of nuclei and the plasma membrane in P-TEFb embryos demonstrated cellularization defects, including multinucleated cells, and showed that cells are also lost from the posterior after cellularization. Damaged nuclei or nuclei with cell cycle defects can trigger a similar phenotype, nuclear fallout, thereby preventing them from becoming somatic nuclei. It is possible that P-TEFb depletion causes cell cycle perturbations that result in the observed phenotype, although no obvious chromosome segregation defects were detected in the embryo posterior. Another possibility is that gene expression is perturbed in the embryo posterior by P-TEFb depletion, causing the nuclei to detach from the cortex (Dahlberg, 2015).

A rather small number of zygotically transcribed genes are known to control cellularization. This study has identified an additional Zelda, SEC, and P-TEFb regulated gene that could be involved in this process. The gene terminus (term) was identified based on its blastoderm-specific expression. Expression becomes restricted to the posterior in cellularized embryos, at the same time as cell loss was observed in the embryo posterior in P-TEFb embryos. It was discovered that shmiRNA knockdown of Term or deletion of a large genomic region that includes Term is lethal to embryos and results in morphological defects, including a failure to form a cellular blastoderm. Over-expression of Term similarly causes morphological deformations of early embryos. These results show that Term is essential for early development, and indicate that Term may play a role in cellularization. However, the term phenotypes are different from the lack of cells observed in the posterior of cellularized P-TEFb embryos. The idea is therefore favored that the P-TEFb phenotype is caused by multiple gene expression changes (Dahlberg, 2015).

The molecular function of Term is unexplored. It encodes a 428 amino acid (aa) protein with a single C2H2-type zinc-finger. Term is closely related to CG7271, 423 out of the 428 aa are identical. Term also shows 28% aa homology to the CG6885 gene product, in which the zinc-finger is also conserved. These three genes have very similar gene expression profiles with transcription restricted to early zygotic activation. However, none of these genes are conserved outside the Drosophila genus, suggesting that species within this clade have adopted them to perform an essential early embryonic function (Dahlberg, 2015).

A large fraction of the genes involved in early embryo patterning, both the ones controlling anterior-posterior development and those involved in dorsal-ventral patterning, contain a promoter-proximal paused Pol II. Recent Gro-seq experiments have indicated that the majority of these are regulated by release from pausing. Given the function of P-TEFb in releasing Pol II from pausing into active elongation, it could be expected that these paused genes would be susceptible to reduced P-TEFb amounts. Indeed, mitotic Cdk9 clones in imaginal discs demonstrated various patterning defects and reduced expression of Hox genes that contain paused Pol II. However, inhibiting P-TEFb activity with flavopiridol showed that highly paused genes are less susceptible to P-TEFb inhibition than non-paused genes, indicating that they experience release from pausing less frequently than non-paused genes. This study found that the majority of segmentation and dorsal-ventral genes are expressed in a relatively normal embryonic pattern, despite the morphological changes and loss of cells from P-TEFb knock-down embryos. Instead, this study found that some non-paused genes are most sensitive to reduced P-TEFb levels (Dahlberg, 2015).

The results are consistent with a model for metazoan gene transcription where all genes require P-TEFb-mediated escape from pausing, and where non-paused genes rely most heavily on rapid release into elongation. In this model, non-paused genes will be most sensitive to diminished P-TEFb levels. The results are also in line with studies of the SEC in mammalian cells, which showed that P-TEFb and SEC components are enriched on highly transcribed genes that are rapidly induced. SEC depletion resulted in decreased Pol II occupancy of both paused and non-paused genes. Although P-TEFb and the SEC may directly regulate Pol II recruitment to rapidly transcribed genes in conjunction with the Mediator complex, P-TEFb-regulated release from pausing could also feedback on transcription initiation (Dahlberg, 2015).

The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila

RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. This study shows that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. It was shown that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network reveals physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrates that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, these results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin (Eberle, 2015).

Approximately 30% of the genome of Drosophila melanogaster is heterochromatic and is made up of transposons, transposon fragments and repetitive sequences with different degrees of complexity. The heterochromatin contains high levels of heterochromatin-specific proteins, such as Heterochromatin Protein 1a (HP1a), and is enriched in certain patterns of post-translational modifications of the histone tails. Heterochromatin formation involves a cascade of histone modifications that are targeted to specific regions of the genome by complex protein-protein and protein-nucleic acid interactions. In the switch from euchromatin to heterochromatin, acetylated H3K9 (H3K9ac) is deacetylated by histone deacetylases such as RPD3/HDAC1. H3K9 is subsequently methylated by histone methyltransferases, and the methylated H3K9 (H3K9me) acts as a binding site for HP1a. The properties of the heterochromatin can spread along the chromatin fiber, and HP1a plays a central role in this process. The ability of HP1a to dimerize, to interact with the methyltransferase SU(VAR)3-9, and to bind H3K9me provides the basis for the spreading of heterochromatin. An additional level of complexity in the establishment of heterochromatic states is provided by the fact that HP1a can also bind RNA in both D. melanogaster and Schizosaccharomyces pombe. Recent studies on Swi6, the HP1a ortholog of S. pombe, have shown that the interaction of Swi6 with RNA interferes with the binding of Swi6 with H3K9me (Eberle, 2015).

Small non-coding RNAs are essential components of the regulation of chromatin packaging in different organisms. Fission yeast uses siRNAs to silence heterochromatic sequences through the recruitment of the H3K9 methyltransferase Clr4. RNAi-dependent mechanisms of heterochromatin assembly exist also in plants, where siRNAs direct de novo DNA methyltransferases to specific genomic sequences. Animal cells use instead the piRNA pathway to trigger heterochromatin assembly and transposon silencing in the germ line. In D. melanogaster, non-coding RNAs transcribed from transposon-rich regions are processed into piRNAs, and a 'Piwi-piRNA guidance hypothesis' has been recently proposed for the recruitment of SU(VAR)3-9 and HP1a to heterochromatin. The Piwi-piRNA system is active during early development and it directs the initial establishment of heterochromatin states not only in the germ line but also in somatic cells. Recent studies suggest that after embryogenesis, the patterns of heterochromatin packaging are maintained through cell divisions via piRNA-independent mechanisms (Eberle, 2015).

An important player in the regulation of non-coding RNAs is the exosome, a multiprotein complex with ribonucleolytic activity. In D. melanogaster, the core of the exosome associates with two catalytic active subunits, RRP6 and DIS3. In the cell nucleus, the exosome is involved in the processing of many non-coding RNAs, including pre-rRNAs, and in the quality control of mRNA biogenesis. The exosome ribonucleases also degrade a large variety of unstable, non-coding RNAs in various organisms including S. cerevisiae, plants, and animals. Moreover, recent studies have revealed that RRP6 participates in the regulation of enhancer RNAs and in the degradation of unstable transcripts synthesized at DNA double-strand breaks (Eberle, 2015).

The exosome has been functionally linked to the methylation of H3K9 in heterochromatin. In S. pombe, RRP6 participates in the assembly of centromeric heterochromatin through an RNAi-independent mechanism, and collaborates with the RNAi machinery to silence developmentally regulated loci and retrotransposons. Much less is known about the possible links between RRP6 and heterochromatin in animals. This study found that a fraction of RRP6 is associated with heterochromatin in the genome of D. melanogaster, and physical interactions has been identifiedf between RRP6 and several heterochromatin factors, including HP1a, SU(VAR)3-9, and RPD3. These results show that SU(VAR)3-9 promotes the targeting of RRP6 to transposon-rich heterochromatic loci. In these loci, RRP6 contributes to maintaining the structure of the heterochromatin by degrading non-coding RNAs that would otherwise compromise the packaging of the chromatin (Eberle, 2015).

This study shows that RRP6 interacts physically with HP1a and SU(VAR)3-9, and that RRP6 is associated with a subset of heterochromatic regions of the genome. Less RRP6 is bound to the heterochromatin in cells with reduced levels of SU(VAR)3-9, which indicates that SU(VAR)3-9 contributes to the targeting of RRP6 to heterochromatin. Although the RNAi experiments do not reveal whether the effect of SU(VAR)3-9 knockdown on RRP6 occupancy is direct or indirect, the fact that RRP6 and SU(VAR)3-9 colocalize and can be co-immunoprecipitated suggests that SU(VAR)3-9 facilitates the recruitment of RRP6 to the heterochromatin, or stabilizes the interaction of RRP6 with other chromatin components, through a physical interaction (Eberle, 2015).

This study has focused on RRP6, and the existence of multiple exosome subcomplexes in cells of D. melanogaster makes it difficult to establish whether the entire exosome has a role in the heterochromatin. However, two observations suggest that this is the case. Firstly, the simultaneous depletion of both catalytic subunits of the exosome, RRP6 and DIS3, gave additive effects on the levels of chromatin-associated RNAs and on the association of HP1a to heterochromatic RNAs. Secondly, it was previously shown that a fraction of RRP4, a core exosome subunit, is also associated with chromatin. Altogether, these observations suggest that the entire exosome, not RRP6 alone, is targeted to heterochromatic loci through an interaction with SU(VAR)3-9 (Eberle, 2015).

Depletion of RRP6 or simultaneous depletion of RRP6 and DIS3 led to a local increase in heterochromatic transcripts associated with subtelomeric and pericentromeric regions, without a significant increase in the density of RNA Pol-II at those regions. This suggests that under normal conditions the RRP6 and DIS3 degrade pervasive RNAs that are transcribed from the heterochromatin. Direct MNase assays and PLA-based assays designed to measure the compaction of the chromatin revealed that the depletion of the exosome ribonucleases loosens the structure of the heterochromatin in the regions that accumulate heterochromatic non-coding RNAs, without affecting the levels of H3K9 methylation or the association of SU(VAR)3-9 with the chromatin. In S. pombe, deletion of the rrp6 gene leads to a derepression of heterochromatin, and this effect is partly due to the fact that in the absence of RRP6 activity, aberrant RNA species accumulate in S. pombe and recruit the siRNA machinery in competition with the RNAi-dependent pathways of H3K9 methylation. The situation is different in D. melanogaster, as no change in H3K9me2 or SU(VAR)3-9 recruitment occurred when RRP6 and DIS3 were depleted (Eberle, 2015).

What is then the mechanism by which the exosome ribonucleases influence the compaction of the heterochromatin in D. melanogaster? The HP1a ortholog in S. pombe, Swi6, is an RNA-binding protein, and non-coding RNAs can cause the eviction of Swi6 from the S. pombe heterochromatin by competing with H3K9me for Swi6. The HP1a protein of D. melanogaster interacts with several RNA-binding proteins and can bind directly to RNA. This study has shown that depletion of RRP6 and DIS3 results in increased levels of non-coding transcripts associated with heterochromatin in D. melanogaster cells. HP1a-RIP signals at selected heterochromatic loci are also increased in cells depleted of RRP6 and DIS3. Altogether, these observations are consistent with a model in which RRP6, and perhaps also DIS3, participate in the degradation of heterochromatic non-coding RNAs that, if stabilized, would outcompete the binding of HP1a to the methylated H3K9 and would thereby disrupt the packaging of the heterochromatin (Eberle, 2015).

Heterochromatin domains are characterized by high levels of H3K9me2 and by the presence of HP1a and SU(VAR)3-9. These results show that RRP6 interacts with SU(VAR)3-9 and that this interaction is important to tether RRP6 to the heterochromatin. Transcripts derived from sporadic transcription of heterochromatic repeat sequences are kept at low levels by RRP6 degradation. Failure to degrade such transcripts results in increased levels of chromatin-associated transcripts, increased binding of HP1 to the chromatin-associated transcripts, and chromatin decondensation (Eberle, 2015).

Specialized protein-protein interactions target RRP6 to different chromatin environments RRP6 and the exosome act on many different types of transcripts and participate in many essential biological processes. The existence of multiple mechanisms to target RRP6 to different types of transcripts, or even to different nuclear compartments, is thus not unexpected. The association of the exosome-or exosome subunits- with genes transcribed by RNA polymerase II (Pol-II) is mediated by interactions with different types of proteins. Co-immunoprecipitation experiments in D. melanogaster identified SPT5 and SPT6, two transcription elongation factors, as interaction partners for the exosome, which led to the proposal that the exosome is tethered to the transcription machinery during transcription elongation. In D. melanogaster, the exosome is also tethered to protein-coding loci through interactions with the hnRNP protein HRP59/RUMP. In human cells, a NEXT complex containing MTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7 mediates an interaction between the exosome and Pol-II transcripts through the nuclear cap-binding complex. In many cases, these intermolecular interactions target the exosome to genomic loci that produce relatively stable transcripts, for instance protein-coding transcripts or stable non-coding RNAs. In these loci, the role of the exosome is primarily linked to RNA surveillance, not turnover (Eberle, 2015).

Much less is known about the mechanisms that target the exosome or its individual subunits to non-protein coding RNAs in the heterochromatin. This study of the RRP6 interactome in cells of D. melanogaster has revealed interactions between RRP6 and heterochromatin factors, and has established an important role for SU(VAR)3-9 in determining RRP6 occupancy. Depletion of SU(VAR)3-9 has a profound effect on the association of RRP6 with a subset of chromatin regions, including many transposon loci. The present findings suggest that these regions, that can be referred to as 'SUV-dependent', produce transcripts that are actively degraded by RRP6. SU(VAR)3-9 has less impact on the targeting of RRP6 to euchromatic protein-coding genes, where interactions with the Pol-II machinery and with mRNA-binding proteins play instead a decisive role. Altogether, the picture that emerges from many studies is that specialized protein-protein interactions target RRP6 to specific genomic environments where RRP6 participates in the processing, surveillance or degradation of specific RNA substrates (Eberle, 2015).

ELAV links paused Pol II to alternative polyadenylation in the Drosophila nervous system

Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes. A particularly dramatic form of APA occurs in the developing nervous system of flies and mammals, whereby various developmental genes undergo coordinate 3' UTR extension. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation sites, thereby fostering the formation of exceptionally long 3' UTRs. This study presents evidence that paused Pol II promotes recruitment of ELAV to extended genes. Replacing promoters of extended genes with heterologous promoters blocks normal 3' extension in the nervous system, while extension-associated promoters can induce 3' extension in ectopic tissues expressing ELAV. Computational analyses suggest that promoter regions of extended genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ChIP-seq assays identify ELAV in the promoter regions of extended genes. This study provides evidence for a regulatory link between promoter-proximal pausing and APA (Oktaba, 2014).

ELAV is an RNA-binding protein that has been shown to bind to U-rich regions in target mRNAs, including neuroglian and erect wings. Recently, the Hox gene Ultrabithorax (Ubx) was shown to be bound by ELAV through similar elements to regulate alternative splicing, but ELAV was not found to bind to predicted binding sites in the Ubx 3' UTR. Similarly, this study also failed to identify specific ELAV recognition sequences within extended 3' UTRs. The present study investigated how ELAV is selectively recruited to appropriate targets during neurogenesis (Oktaba, 2014).

The activities of synthetic reporter genes were exanubed in transgenic embryos to determine whether extended 3' UTRs are sufficient for the selective recruitment of ELAV in vivo. Transgenes contain the Drosophila synthetic core promoter (DSCP) attached to a GFP coding sequence followed by the entire extended 3' UTR of elav, one of the targets of ELAV. If elav 3' UTR sequences are sufficient to recruit ELAV, then this transgene should produce mRNAs containing extended 3' UTRs (Oktaba, 2014).

Expression of 3' UTR sequences was monitored via double labeling assays with GFP coding sequences to distinguish transgene mRNAs from endogenous elav transcripts. Expression of the transgene was confirmed by colocalization of GFP with a probe directed against the short 3' UTR. However, colocalization of GFP with extended sequences was not observed, indicating that mRNAs produced from the transgene lack 3' extensions. The only signals containing 3' extensions corresponded to endogenous elav mRNAs (Oktaba, 2014).

Additional experiments were done to determine why the transgene fails to produce extended transcripts. The possibility that the GFP coding sequence somehow inhibits expression of extended sequences by creating GFP transgenes lacking proximal poly(A) signals was excluded. Such constructs no longer depended on ELAV for 3' extension and were found to produce mRNAs containing extended 3' UTR sequences when expressed in ectopic tissues lacking ELAV (Oktaba, 2014).

To test whether promoter sequences play a role in ELAV recruitment, the DSCP sequence was swapped with a 333 base pairs (bp) genomic DNA fragment encompassing the native elav promoter region, consisting of 92 bp upstream and 241 bp downstream of the (TSS). Strikingly, colocalization of GFP and extension sequences was observed indicating expression of the elav 3' UTR extension, as seen for the endogenous locus (Oktaba, 2014).

To confirm that 3' extension depends on native promoter regions of extended genes, a construct bearing the fully extended brat 3' UTR downstream of GFP was also tested, using three different promoters: the DSCP, the native promoter producing the short form of brat, and the native promoter producing the extended form of brat. Only the brat promoter associated with endogenous extension mediated expression of transgenic transcripts containing 3' UTR extensions. These observations suggest that the promoter regions of extended genes are essential for the ELAV-mediated expression of 3' UTR extensions (Oktaba, 2014).

The preceding results suggest that promoter sequences are important for the synthesis of 3' extensions in the developing nervous system. Their importance was determined by examining nonneural tissues. Ectopic ELAV can drive 3' UTR extension in ectopic tissues from endogenous loci. Attempts were made to determine whether ectopic ELAV could also induce ectopic 3' extensions from transgenic DNAs (Oktaba, 2014).

Both the GFP-elav transgene and ELAV protein were expressed in muscle cells using a Mef2-Gal4 driver. In this context, mRNA expression from the reporter is easily distinguished from endogenous elav expression, which occurs only in the nervous system. The DSCP fails to generate 3' UTR extensions, and only endogenous elav transcripts in the CNS were detected. In contrast, the GFP-elav transgene containing the native elav promoter produced transcripts with extended 3' UTRs in muscle tissue. Quantification of transgene expression in dissected muscle tissue using quantitative PCR (qPCR) shows that both promoters drive robust transgene expression (GFP signal), but only the native promoter drives expression of extension sequences. Similarly, the second brat promoter, but not the DSCP, was also able to drive expression of an extended brat 3' UTR in muscle cells (Oktaba, 2014).

Whether the promoter sequence from one extended gene could promote extension of the 3' UTR of another such gene was also tested. Indeed, a GFP transgene containing the elav promoter and brat extended 3' UTR exhibited ELAV-mediated APA. These observations suggest a link between transcription initiation and ELAV-mediated APA (Oktaba, 2014).

To determine whether the promoter regions of extended genes share common sequence motifs, 252 neural-specific transcripts produced by 219 different genes exhibiting 3' UTR extensions were examined. The most significantly enriched motif is the GAGA element, which occurs in nearly half of all extended genes. To investigate the functional significance of the GAGA element in promoters of extended genes, whether 3' UTR extension is diminished in animals lacking the GAGA-binding protein, Trithorax-like (Trl) was tested. For all six genes examined, the ratio between extension sequences and coding sequences was reduced between 15% and 75% in Trl mutant flies. These observations suggest that the GAGA motifs in the promoters of extended genes are important for proper 3' UTR extension (Oktaba, 2014).

The GAGA element is a motif commonly found in the promoter regions of genes containing paused Pol II. Paused Pol II is a pervasive feature of gene regulation in metazoan development, and at least 10%-30% of all genes in Drosophila contain paused Pol II. It is thought that paused promoters are poised for rapid activation and thereby exhibit synchronous induction in the different cells of a tissue. Another function of promoter pausing might be to ensure proper recruitment of essential factors for RNA elongation and processing (Oktaba, 2014).

Most extended genes contain paused Pol II, based on whole genome Pol II ChIP-seq assays. Some extended genes express both short and long isoforms from the same promoter (for example elav), while others (e.g., brat) employ different promoters for the different isoforms. In the latter case, only the promoter driving the extended isoform contains paused Pol II (Oktaba, 2014).

To determine whether paused Pol II might be associated with the formation of 3' UTR extensions, the overall Pol II pausing index (PI) of extended genes and various control genes was examined. Extended transcripts were found to be derived from significantly more paused promoters than any of the control groups, including neural-specific (but nonextended) genes. Thus, there is a clear association between Pol II pausing and 3' UTR extension, which transcends the general pausing seen for neural-specific gene expression. Extended transcripts are also strongly paused in muscle cells, where they are not actively transcribed and where ELAV is not expressed. Thus, Pol II pausing at extended genes occurs independently of ELAV (Oktaba, 2014).

The preceding analyses raise the possibility that ELAV is selectively recruited to the promoter regions of extended genes. To test this hypothesis, ChIP-seq assays were performed using anti-ELAV antibodies. ELAV is an RNA-binding protein that directly binds and inhibits proximal poly(A) elements of target transcripts. It was therefore reasoned that it should be possible to identify the genome-wide distribution of ELAV by crosslinking ELAV/RNA complexes to associated DNA templates. ELAV ChIP-seq assays were conducted with nuclei obtained from 6-8 hr and 10-12 hr embryos. These stages were selected based on previous observations regarding the timing of 3' extensions in the nervous system (Oktaba, 2014).

6,879 genomic regions bound by ELAV were identified in 6-8 hr embryos and 8,076 regions in 10-12 hr embryos. There is a striking enrichment of ELAV in the promoter regions of extended genes. For example, argonaute1 (ago1) produces multiple APA isoforms driven from three different promoters. The two promoters that produce extended transcripts display ELAV peaks, whereas the promoter that expresses the short (ubiquitous) isoform does not. High levels of ELAV are also found at 3' poly(A) sites, consistent with previous RNA immunoprecipitation assays (Oktaba, 2014).

The ChIP-seq data were combined into a 'meta-gene' plot that provides simple visualization of key sites of ELAV binding. There is a significant enrichment of ELAV at the promoter regions of extended genes as compared with neural-specific nonextended genes. A distinct ELAV peak is seen near the TSS, although ELAV binding continually increases across the 5' UTR and peaks at ~300 bp downstream of the start site (Oktaba, 2014).

ELAV not only binds to promoter regions, but also to 3' UTRs and introns of extended genes. ELAV is strikingly depleted from coding sequences. As expected, binding markedly increases in the vicinity of proximal poly(A) sites and remains high across extended regions where there are additional poly(A) elements (Oktaba, 2014).

A meta-gene analysis was also performed of previously published Pol II ChIP-seq data. Pol II binding is highly enriched in the promoter regions of extended genes, which is consistent with earlier evidence that such genes tend to contain paused Pol II. The Pol II binding profile did not otherwise differ from nonextended neural-specific genes. It is possible that ELAV binds to both nascent transcripts and associated DNA templates, since ELAV is usually detected at distal poly(A) sites of extended genes prior to full transcriptional extension (Oktaba, 2014).

This study has presented evidence that paused Pol II fosters selective recruitment of ELAV and coordinates expression of extended 3' UTR sequences during neurogenesis. The basis for selective recruitment of ELAV is a bit of a mystery since it has been shown to interact with broadly distributed low-complexity RNA sequences (e.g., U-rich). Increased interaction between paused promoters and termination regions might help promote 3' extension, for example, by bringing ELAV to the promoter via gene looping. The observed association of ELAV with the paused promoter regions of extended genes provides a foundation for selectivity and also strengthens the link between transcription initiation and 3' cleavage. It is improbable that paused Pol II is sufficient for recruitment of ELAV, since not all paused genes exhibit APA. It is therefore likely that additional sequence elements, for example, in extended 3' UTRs, are essential for recruitment. ELAV proteins are highly conserved, and it is easy to imagine that the regulation of 3' extension in the vertebrate CNS depends on selective promoter recruitment as seen in Drosophila (Oktaba, 2014).

Reassessment of Piwi binding to the genome and Piwi impact on RNA Polymerase II distribution

Drosophila Piwi was reported by Huang (2013) to be guided by piRNAs to piRNA-complementary sites in the genome, which then recruits heterochromatin protein 1a and histone methyltransferase Su(Var)3-9 to the sites. Among additional findings, Huang (2013) also reported Piwi binding sites in the genome and the reduction of RNA polymerase II in euchromatin but its increase in pericentric regions in piwi mutants. Marinov (2015) disputed the validity of the Huang bioinformatic pipeline that led to the last two claims. This study reports an independent reanalysis of the data using current bioinformatic methods. The reanalysis agrees with Marinov (2015) that Piwi's genomic targets still remain to be identified but confirms the Huang claim that Piwi influences RNA polymerase II distribution in the genome (Lin, 2015).

RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo

It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter. Although there are well-documented examples in which Pol II is recruited to a gene but stalls, a general role for Pol II stalling in development has not been established. Comprehensive Pol II chromatin immunoprecipitation microarray (ChIP-chip) assays were carried out in Drosophila embryos and three identified distinct Pol II binding behaviors were identified: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The notable feature of the ~10% genes that are stalled is that they are highly enriched for developmental control genes, which are either repressed or poised for activation during later stages of embryogenesis. It is proposed that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development (Zeitlinger, 2007).

To determine at which genes Pol II stalling occurs during development, global Pol II occupancy was analyzed in whole Drosophila embryos. Although this is one of the few systems in which genomics approaches can easily be applied to developmental questions, interpretation is complicated by the occurrence of multiple tissues. To reduce the complexity, Toll^10b embryos (2-4 h after fertilization), a well-characterized mutant that contains a homogeneous population of mesodermal precursor cells at the expense of neuronal and ectodermal cells, was used. In Toll^10b mutants, mesodermal genes are uniformly activated, whereas genes required for the development of ectodermal and neural tissues are repressed throughout the embryo. Previous whole-genome microarray experiments have identified the transcript levels of all genes in these mutants. To distinguish between stalled and active Pol II, a mixture of antibodies that recognizes both the initiating and elongating forms of Pol II was used, and whole-genome ChIP-chip assays were carried carried out (Zeitlinger, 2007).

The results show that many genes known to be repressed in Toll^10b embryos show notably high Pol II signal near the transcription start site. In some cases, the prominent Pol II peak was tightly restricted to the promoter region (for example, at the tail-up (tup) gene, whereas at other genes Pol II was also found at low abundance throughout the transcription unit (for example, the sog and brk genes. This is consistent with previous evidence that some genes, such as sog, are transiently activated but then repressed at later stages, whereas others, such as tup, are never activated in Toll^10b mutants (Zeitlinger, 2007).

The Pol II profiles of repressed genes are clearly distinct from those of active genes. For example, the stumps (also known as Hbr) gene, which encodes a fibroblast growth factor (FGF) receptor specifically expressed in mesodermal precursors show uniformly high levels of Pol II throughout the transcription unit. Furthermore, genes that are silent in the early embryo simply lack Pol II binding altogether. Thus, there appear to be three distinct classes of genes: those with Pol II distributed throughout the transcription unit, those with preferential enrichment of Pol II at the transcription site and those that lack Pol II binding altogether (Zeitlinger, 2007).

To further characterize these three groups, a principled method was developed that classifies genes on the basis of their Pol II enrichment profiles. The ratio between Pol II enrichment at the transcription start site versus internal regions of the transcription unit was calculated. It was possible to assign 76% of the protein coding genes (10,220 of 13,448 genes) to one of the three classes. At least 27% of all genes had an active Pol II profile in which Pol II was detected uniformly throughout the transcription unit. At least 12% of all genes (1,614 of 13,448) showed disproportionate accumulation of Pol II near the transcription start site. Among this group, Pol II was tightly restricted to the transcription start site at 62% of genes. At the remaining 38% of these genes, Pol II was also detected within the transcription unit, presumably because these genes -- such as sog -- are expressed at low levels in at least a subset of cells during the time frame of the analysis (2-4 h after fertilization). Finally, 37% of all genes lacked Pol II binding altogether (Zeitlinger, 2007).

Several lines of evidence confirm that the ~1,600 genes with disproportionate enrichment of Pol II at the transcription start site have a form of stalled Pol II. First, all heat shock genes, which provide the classical example of Pol II stalling, fall into this class. Second, the Pol II peaks map an average of ~50 bp downstream of the transcription start site, consistent with the location of stalled Pol II at heat shock genes. Because this is an average profile, it is possible that a fraction of Pol II occupancy comes from inactive preinitiation complexes. However, the majority of detected Pol II signal seems to come from Pol II that is stalled downstream of the transcription start site. Third, Pol II stalling at these genes is consistent with comprehensive expression analysis using whole-genome tiling arrays. Genes with Pol II tightly restricted to the transcription start site are either silent or only weakly expressed in Toll^10b mutants. In contrast, genes with similar levels of Pol II binding but uniform distribution throughout the transcription unit are expressed at substantial levels in these mutants. Finally, permanganate footprint assays were used as an independent method to confirm stalled Pol II at selected genes. For example, the rho gene showed clear permanganate sensitivity downstream of the transcription start site (+37 bp), consistent with the Pol II stalling profile seen in Toll^10b mutants (Zeitlinger, 2007).

There are considerable differences in the expression and functions of genes in the active, stalled or no Pol II classes based on in situ expression patterns (ImaGO database) and functional annotations. The set of genes with stalled Pol II is highly enriched for developmentally regulated genes, particularly those expressed in ectodermal and neuronal precursor cells. Consistent with these results, genes with stalled Pol II are highly enriched for functions in development, including neurogenesis, ectoderm development and muscle differentiation. Many of these genes encode sequence-specific transcription factors (Hox, T-box, bHLH, zinc fingers and HMG) and components of cell signaling pathways (FGF, Wnt, Notch, EGF, TGFβ, JNK and TNF (Zeitlinger, 2007; see supplemental material to article for a full list of those genes exhibiting pausing).

In contrast, the set of genes with uniform Pol II binding is highly enriched for ubiquitously expressed genes, which function mostly in metabolism and cell proliferation. The set of genes that lacks Pol II binding is highly enriched in genes that show no staining in whole-embryo in situ hybridizations, confirming that they are not expressed during early embryogenesis. Many of these genes encode proteins that have functions in adult cells, such as cuticle proteins or proteins required for vision (Zeitlinger, 2007).

Pol II stalling could reflect two nonexclusive developmental functions. It could be indicative of active transcriptional repression, or it could prepare genes for activation at later stages of embryogenesis. The second model is particularly attractive, because Pol II stalling has already been shown to prepare heat shock genes for rapid induction. Evidence was found for both models (Zeitlinger, 2007).

Pol II stalling is particularly prevalent among genes expressed in the neuroectoderm and dorsal ectoderm, which are repressed in Toll^10b embryos. To test whether Pol II stalling is specific for repressed genes, the Pol II profile was examined of these genes in mutant embryos in which they are active. For this, two well-defined mutants, Toll^rm9/rm10 and gd⁷ (2-4 h), were used in which cells adopt neurectodermal and dorsal ectodermal fates, respectively. Indeed, at these genes, Pol II is redistributed into the transcription unit in these mutants, and some genes now show the active Pol II profile. These results indicate that Pol II stalling is associated with cell-type specific repression and is subject to dynamic changes during development (Zeitlinger, 2007).

Previous studies have shown that the repression of a large set of genes in Toll^10b embryos depends on Snail, a well-studied repressor that is constitutively expressed in Toll^10b embryos but not in Toll^rm9/rm10 and gd⁷ embryos. A statistically significant association was found between repression by Snail and Pol II stalling. For example, among the 139 genes that are occupied by Snail and show reduced expression in the Toll^10b mutant, 54% have stalled Pol II, whereas only 19% of all genes with reduced expression show Pol II stalling. This suggests that Pol II stalling in Toll^10b embryos may be regulated by Snail. A role of developmental repressors in regulating Pol II stalling is also consistent with a recent study (Wang, 2007) of Drosophila segmentation (Zeitlinger, 2007).

Multiple lines of evidence suggest that Pol II stalling also occurs at genes that are poised for activation in older embryos. Genes with stalled Pol II are highly over-represented among genes that are rapidly induced within 12 h after the time frame of the analysis. Moreover, genes with stalled Pol II are enriched for genes expressed in the derivatives of the mesoderm precursors present in Toll^10b mutants, such as the developing heart and muscle cells. These genes, such as Drop (Dr) and bap, are not yet activated at the time frame of the analysis, but they nonetheless show high levels of Pol II near the transcription start site (Zeitlinger, 2007).

To confirm that muscle genes indeed show stalled Pol II before activation, permanganate assays were carried out on wild-type Drosophila embryos at 2-4 h after fertilization. Dr and lbe showed a clear permanganate footprint downstream of transcription. These footprints were specific to the early embryo stage, since S2 cells, a cell line derived from older embryos, did not show a permanganate footprint under the same conditions. These results confirm that Pol II stalling is dynamically regulated and suggest that one of its functions is to prepare genes for activation (Zeitlinger, 2007).

This genome-wide analysis showed that genes in Drosophila embryos are found in three distinct dynamic states: active, stalled or no Pol II. Stalled Pol II is particularly associated with developmental genes that are repressed and poised for activation. It is proposed that Pol II stalling prepares genes for rapid response to developmental signals during embryogenesis and thus may represent a key regulatory step for gene transcription in development (Zeitlinger, 2007).

A global change in RNA polymerase II pausing during the Drosophila midblastula transition

Massive zygotic transcription begins in many organisms during the midblastula transition when the cell cycle of the dividing egg slows down. A few genes are transcribed before this stage but how this differential activation is accomplished is still an open question. In this study ChIP-seq experiments were performed on tightly staged Drosophila embryos, and massive recruitment of RNA polymerase II (Pol II) with widespread pausing was shown to occur de novo during the midblastula transition. However, approximately 100 genes are strongly occupied by Pol II before this timepoint and most of them do not show Pol II pausing, consistent with a requirement for rapid transcription during the fast nuclear cycles. This global change in Pol II pausing correlates with distinct core promoter elements and associates a TATA-enriched promoter with the rapid early transcription. This suggests that promoters are differentially used during the zygotic genome activation, presumably because they have distinct dynamic properties (Chen, 2013).

The results suggest that a large number of genes first recruit Pol II during the MBT with widespread Pol II pausing, while the small number of genes transcribed before that are not paused. Presumably, these pre-MBT genes are required for very early developmental events such as sex determination or cellularization, or may be involved in early patterning events that require feedback regulation over time (for example ftz. Thus, while Pol II pausing is commonly found at developmental genes and may be advantageous for their precise and synchronous expression in response to localized extracellular signals, a different mode of transcription is used during pre-MBT stages. Due to the short nuclear cycles at this stage, it is likely that transcription is optimized to achieve high levels of transcripts in a very short time period (Chen, 2013).

The fact that the TATA box enriched was found among pre-MBT genes is consistent with the known properties of TATA-containing promoters. TATA is a strong core promoter element that efficiently supports transcription in vitro, mediates efficient re-initiation in vitro, and its presence in vivo correlates with 'bursts' of transcription that produce many transcripts within a short time. Furthermore, it has been shown that TATA promotes pTEFb activity, leading to more efficient elongation rates in vitro and in vivo (Chen, 2013).

This suggests that TATA-enriched promoters and paused promoters have different transcription dynamics and serve different purposes during development. While this difference is particularly evident during the zygotic genome activation as reported in this study, it is proposed that this difference is general and also applies to later development. For example, promoters were analyzed during muscle development, and many genes were found to be induced during late stages of embryogenesis without prior Pol II pausing, and these genes were also found to be enriched in TATA. Consistent with this, statistical analyses suggest that the combination of Inr and TATA represents a separate class of promoters that is often found among genes expressed in adult tissues. Since the properties of TATA are not specific to Drosophila, it is likely that differences among promoter types and their propensity for Pol II pausing are conserved across animals (Chen, 2013).

Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation

The kinetics with which promoter-proximal paused RNA polymerase II (Pol II) undergoes premature termination versus productive elongation is central to understanding underlying mechanisms of metazoan transcription regulation. To assess the fate of Pol II quantitatively, photoactivatable GFP-tagged Pol II at uninduced Hsp70 was tracked on polytene chromosomes, and it was shown that Pol II is stably paused with a half-life of 5 min. Biochemical analysis of short nascent RNA from Hsp70 reveals that this half-life is determined by two comparable rates of productive elongation and premature termination of paused Pol II. Importantly, heat shock dramatically increases elongating Pol II without decreasing termination, indicating that regulation acts at the step of paused Pol II entry to productive elongation (Buckley, 2014).

NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila

Recent analyses of RNA polymerase II (Pol II) revealed that Pol II is concentrated at the promoters of many active and inactive genes. Negative elongation factor (NELF) causes Pol II to pause in the promoter-proximal region of the hsp70 gene in Drosophila melanogaster. In this study, genome-wide location analysis (chromatin immunoprecipitation-microarray chip [ChIP-chip] analysis) revealed that NELF is concentrated at the 5' ends of 2,111 genes in Drosophila cells. Permanganate genomic footprinting was used to determine if paused Pol II colocalized with NELF. Forty-six of 56 genes with NELF were found to have paused Pol II. Pol II pauses 30 to 50 nucleotides downstream from transcription start sites. Analysis of DNA sequences in the vicinity of paused Pol II identified a conserved DNA sequence that probably associates with TFIID but detected no evidence of RNA secondary structures or other conserved sequences that might directly control elongation. ChIP-chip experiments indicate that GAGA factor associates with 39% of the genes that have NELF. Surprisingly, NELF associates with almost one-half of the most highly expressed genes, indicating that NELF is not necessarily a repressor of gene expression. NELF-associated pausing of Pol II might be an obligatory but sometimes transient checkpoint during the transcription cycle (Lee, 2008).

This study identified 2,111 genes in the Drosophila genome that had peaks of both NELF-B and -E within 500 bp of the transcription start. The broad distribution of NELF throughout the genome revealed by ChIP-chip analysis is in excellent agreement with the broad distribution detected on Drosophila polytene chromosomes. The close association of NELF with promoter regions raises the possibility that NELF is involved in the expression of many genes (Lee, 2008).

The relationship between NELF and paused Pol II was investigated by performing permanganate genomic footprinting on the promoter regions of 58 genes and one region that exhibited an obvious peak of NELF located 9 kb downstream from the annotated start site. Of 56 NELF-associated genes, 46 had a clearly discernible footprint. The results can be extrapolated to predict the number and identity of genes that are likely to have Pol II paused in the promoter-proximal region. NELF-associated genes were ranked according to the level of NELF reflected by the Mpeak value. Forty-four of the genes analyzed are within the collection of genes with the top 1,000 Mpeak values, and 42 of these had permanganate footprints. Thus, these top 1,000 genes represent cases where detection of paused Pol II was detected with permanganate (Lee, 2008).

Comparison of these data with recent Pol II ChIP-chip data obtained for Drosophila S2 cells provides another measure of the relationship between NELF and promoter-proximal pausing. Previously 5,403 genes were associated with Pol II, and the majority of these had Pol II concentrated at the 5' end. Based on the unusually high density of Pol II found at the 5' end, 1,014 genes were judged to have stalled Pol II at the 5' end. These Pol II were described as stalled rather than paused because there is uncertainty in the conformation of the elongation complex. Nevertheless, since these conformations are related to pausing, the relationships between genes with NELF, GAGA factor, and stalled Pol II were assessed. Thirty-nine of the 46 genes with permanganate footprints were found among the genes with stalled Pol II, indicating that a significant portion of the stalled Pol II are indeed transcriptionally engaged in the promoter-proximal region. A Venn diagram shows the concordance between the current NELF-associated genes and those with stalled Pol II. Eighty percent of the genes with stalled Pol II coincide with NELF-associated genes. Collectively, the data indicate that the presence of NELF, the presence of paused Pol II, and the presence of stalled Pol II are linked to each other. The interdependence of these gene features is further supported by the finding that the distribution of Pol II was altered on 115 of 200 genes with stalled Pol II when NELF was depleted with RNA interference (Lee, 2008).

Importantly, it is also evident that pausing is not dictated solely by NELF. Ten genes that lacked a permanganate footprint were detected, yet clearly associated with NELF. A previous study observed that 85 of 200 genes with stalled Pol II displayed no significant change in the distribution of Pol II when NELF was depleted with RNA interference. It is noteworthy that NELF slows but does not stop elongation. Thus, the density of Pol II in the promoter-proximal region is probably influenced by additional factors. These could include gene-specific factors that influence initiation rates and the duration of the pause. Chromatin structure could impede elongation, as appears to be the case for hsp70 in human cells. Some cases could involve Pol II in a preinitiated state. Other cases could involve premature termination. Further analysis of individual genes is necessary to explore these possibilities (Lee, 2008).

Based on previous work, it was anticipated that NELF would repress transcription. Surprisingly, it was observed that approximately 80% of the NELF-associated genes were among the upper half of genes ranked by expression level. Almost one-half of the genes above the 90th percentile in expression associated with NELF. In addition, 35 of the 46 genes for which a permanganate footprint was detected ranked in the top 50th percentile for expression (Lee, 2008).

It has been proposed that NELF could function as a checkpoint during an early stage in elongation. The delay in elongation could allow time for both elongation factors and RNA processing factors to associate with Pol II. The checkpoint could also serve to attenuate the level of expression of some genes whose expression levels might otherwise be too high for the biological functions of the genes. This has been proposed for some estrogen-induced genes, wherein the estrogen receptor appears to recruit NELF at the same time that it is activating transcription (Lee, 2008).

A Venn diagram (see Relationships between NELF-associated genes) shows that GAGA factor associates with a significant number of genes that associate with NELF or a stalled Pol II. GAGA factor was previously implicated in pausing Pol II on hsp70. GAGA factor interacts with TFIID, the chromatin remodeling factor NURF, and the histone chaperone FACT. Thus, GAGA factor could function in initiation by recruiting TFIID and by establishing a nucleosome-free region that allows access of the transcription machinery to the promoter. It is unclear if GAGA factor directly impacts elongation. Pausing could be a default outcome of GAGA factor's inability to overcome the action of NELF, possibly because it does not recruit P-TEFb to the gene (Lee, 2008).

Approximately 40% of the GAGA factor-associated genes do not associate with NELF. More investigation is needed to determine what GAGA factor does at these genes. GAGA factor associates with polycomb response elements, which are involved in transcription repression, so GAGA factor could be involved in organizing a repressive chromatin structure that blocks transcription initiation (Lee, 2008).

Six genes for which a permanganate footprint was detected lack GAGA factor, and approximately one-half of the genes identified by previously lack GAGA factor. This raises the possibility of the existence of other sequence-specific DNA binding proteins that function similarly to GAGA factor. These could be proteins that participate in initiation but are unable to directly impact elongation. Such proteins were previously implicated in an earlier study, but a direct link to promoter-proximal pausing and NELF remains to be investigated (Lee, 2008).

DNA sequence analysis of the regions encompassing the permanganate footprints of 46 genes identified a 35-nucleotide region with three conserved patches of nucleotides, centered at positions +1, +18, and +29 relative to known or putative transcription start sites. These conserved patches align with three contacts made by TFIID in the first 30 nucleotides of the hsp70, hsp26, and histone H3 genes, strongly suggesting that the three conserved patches are recognized by TFIID. This conclusion is further supported by the similarity between the sequences of the initiator and DPE, which are recognized by TFIID, and the sequences centered at positions +1 and +29 in the conserved element (Lee, 2008).

While it is not surprising for the promoters with paused Pol II to have TFIID binding sites, it is unusual to have such a large proportion of promoters with multiple binding sites. Statistical analysis of 3,393 nonredundant Drosophila promoters revealed that only 12% of the promoters contained an Inr and a DPE, whereas this study found this combination in 61% (28/46 cases) of the cases with paused Pol II. These two regions, in combination with a third conserved patch of nucleotides near position +18, further distinguish this collection of promoters with paused Pol II from the rest of the promoters in the genome (Lee, 2008).

The combination of TFIID contacts in the regions at positions +1, +18, and +29 could contribute to pausing by increasing the affinity of TFIID for the core promoter region, thus shifting the rate-limiting step from the binding of TFIID to a subsequent step, such as promoter-proximal pausing. In addition, the contacts of TFIID downstream from the transcription start might help to establish a promoter architecture that is conducive to pausing. DNase I footprinting and UV cross-linking analyses showed that TFIID contacts DNA as far as 45 nucleotides downstream from the transcription start (Lee, 2008).

The permanganate footprinting results indicate that Pol II pauses after transcribing approximately 30 to 50 nucleotides. This agrees well with high-resolution mapping of nascent transcripts associated with paused Pol II on the hsp70 and hsp27 genes and with the locations of permanganate footprints detected on several other genes. Because the footprints at several different promoters extend further than the size of a bubble associated with a single Pol II molecule, the location of the paused Pol II must often be heterogeneous. This also agrees with previous results for the hsp70 and hsp27 genes (Lee, 2008).

The distance where Pol II begins to pause downstream from the start site is similar to the distance where NELF was found to inhibit elongation in a cell-free system. This strengthens the conclusion that NELF is involved in concentrating Pol II in the promoter-proximal region. Moreover, the results fit well with a proposed pausing mechanism in which the NELF-E subunit associates with the nascent transcript and slows elongation by restricting the extrusions of the nascent transcript from the elongation complex. This model requires that Pol II transcribe at least 18 nucleotides so that the transcript is exposed outside the elongation complex (Lee, 2008).

The location of paused Pol II provides a landmark for sequence comparisons directed at identifying sequences involved in promoter-proximal pausing. The location where Pol II pauses does not correlate with conserved sequences or conserved RNA structures. The conserved element that is likely to be recognized by TFIID is located various distances from where Pol II pauses, so it seems unlikely that this element directly influences Pol II. Mfold analyses of the portions of nascent transcripts that would be exposed from the elongation complexes failed to detect evidence of substantial secondary structure (Lee, 2008).

It is proposed that NELF's interaction with the nascent transcript associated with paused Pol II is independent of sequence and RNA secondary structure. NELF was previously shown to associate with a variety of RNAs, including some with substantial secondary structure and some without it. The proposal contrasts with studies of the human immunodeficiency virus (HIV) provirus, for which sequence-specific association of NELF-E with the TAR element has been detected and is thought to control elongation. Recent analysis of an HIV provirus, however, indicates that NELF associates with the elongation complex and causes Pol II to pause at position +45, well before the TAR element has been transcribed and well within the range where Pol II pausing is found on Drosophila genes (Lee, 2008).

Knowledge of transcriptional control is based largely on studies with yeast and cell-free systems. These studies have tended to emphasize the regulation of transcription initiation. New data provided by genome-wide analyses of Pol II significantly increase the number of genes whose transcription appears to be controlled after initiation in animal cells. Thus, a full understanding of transcriptional control in animal cells requires investigation of molecular events occurring both before and after initiation in the promoter regions. Knowledge of the genome-wide distribution of NELF and GAGA factor combined with recent data on the distribution of Pol II provides a foundation for future studies of promoter-proximal pausing in Drosophila (Lee, 2008).

Hsp90 globally targets paused RNA polymerase to regulate gene expression in response to environmental stimuli

The molecular chaperone Heat shock protein 90 (Hsp90) promotes the maturation of several important proteins and plays a key role in development, cancer progression, and evolutionary diversification. By mapping chromatin-binding sites of Hsp90 at high resolution across the Drosophila genome, an unexpected mechanism was uncovered by which Hsp90 orchestrates cellular physiology. It localizes near promoters of many coding and noncoding genes including microRNAs. Using computational and biochemical analyses, it was found that Hsp90 maintains and optimizes RNA polymerase II pausing via stabilization of the negative elongation factor complex. Inhibition of Hsp90 leads to upregulation of target genes, and Hsp90 is required for maximal activation of paused genes in Drosophila and mammalian cells in response to environmental stimuli. These findings add a molecular dimension to the chaperone's functionality with wide ramifications into its roles in health, disease, and evolution (Sawarkar, 2012).

Since its discovery, most of the cellular functions of Hsp90 have been attributed to stabilization of client proteins involved in signaling. The results outlined in this study argue for an additional, and hitherto ignored, role of this chaperone at a different level of the information-processing pathway - gene transcription. Although this activity may be a result of the function of Hsp90 to stabilize a protein-forming paused pol II complex, it ultimately results in a much wider control of cellular behavior (Sawarkar, 2012).

These studies linking Hsp90 with pol II pausing were suggested solely by genomic comparisons of several digital data sets arising from a single homogeneous cell line. Use of inhibitors, rather than slow-acting RNAi, with rapid and genome-wide follow-up of downstream gene activity led to a mechanism with minimal complications arising from secondary effects of Hsp90 inhibition, such as cell-cycle arrest and cell death. The upregulation of Hsp90 targets within minutes of radicicol treatment seen on a global scale confirms the conjecture that the immediate transcriptional effect of Hsp90 inhibition is via its chromatin function and not as an effect on cytosolic function (Sawarkar, 2012).

The list of Hsp90 targets includes several genes important for growth homeostasis. c-myc, a potent protooncogene, exhibits pol II pausing in mammalian cells and Hsp90 binding in Drosophila cells. Given the conservation of the process described in this study, it is highly possible that human Hsp90 also targets the paused c-myc promoter. Hsp90 depletion, thus, may also alter expression of c-Myc, p53, and other signaling components in human cells via pol II pausing, a possibility that is significant in light of anticancer activities of Hsp90 inhibitors (Sawarkar, 2012).

In addition to c-myc and p53, other Hsp90 targets such as bantam and mir-278 are implicated in apoptosis and growth control. Their direct regulation by the chaperone caused by release of paused pol II in the gene body adds yet another aspect to Hsp90 biology. In addition to the chaperone's role in Argonaute function, its involvement in miRNA-gene expression may represent a major pathway by which Hsp90 orchestrates cellular physiology and pathology (Sawarkar, 2012).

Recently, Hsp90 was shown to bind HIV promoter and regulate its expression and eventually viral infectivity. The viral genome exhibits pol II pausing at its promoter. Moreover NELF-E depletion results in increased virion production. Taken together with the current results, it is most likely that Hsp90 targets the paused pol II located on HIV promoter via NELF-E and, similar to several genes reported in this study, affects release of paused pol II from the viral locus. Thus, the fundamental observation with Drosophila cells may help explain several findings of biomedical importance, paving a way for a more rational drug design (Sawarkar, 2012).

Most heat shock loci are targeted by heat shock factor (HSF) at high temperature, leading to their activation. A comparison between binding sites of HSF mapped in heat-shocked S2 cells and those of Hsp90 in normal cells reported in this study showed that three-quarters of a total 437 HSF targets were occupied by the chaperone before heat shock, implying a collaboration between HSF and Hsp90 at chromatin. Given that HSF itself is a client of Hsp90, the promoter-bound Hsp90 may assist HSF binding to nearby heat shock elements or its activity thereafter. Notably, not all loci bound by HSF show elongation factor recruitment on polytene chromosomes. It is possible that HSF binds to many sites but activates genes only in the context of bound Hsp90 and paused polymerase. It should be noted that HSF binds very strongly to only one locus prior to heat shock, Hsp83 locus, the promoter of which is also targeted by Hsp90. The control of this locus by promoter-bound Hsp90 may be assisted by HSF and shows an important motif in the chaperone circuitry (Sawarkar, 2012).

What targets Hsp90 to specific sites across chromatin? Given the association of Hsp90 with pausing factors, it's likely that paused complex acts as a docking site for Hsp90. NELF complex may be only one of the several pausing factors required by Hsp90 to get recruited to chromatin as Hsp90 occupancy only mildly changes upon NELF knockdown. No pausing of pol II is reported in yeast S. cerevisiae, and it lacks NELF complex, suggesting that TSS association of Hsp90 may be metazoan specific. It will be highly interesting to see whether Hsp90 is associated with distinct chromatin regions even in the yeast (Sawarkar, 2012).

This study provides several lines of evidence for a functional association between Hsp90 and pol II pausing. From genome-wide comparison of Hsp90 ChIP and pol pausing data sets and biochemical interaction between Hsp90 and pol pause factors to functional links in culture cells and flies, the results buttress the model of Hsp90's action through NELF-mediated regulation of pol II pausing. Hsp90 inhibition does not cause upregulation of all target paused genes, for example CecC and CecA1. This is visible in the nonuniform changes in gene expression of radicicol-treated cells. Knockdown of the best characterized pol pause factor, NELF complex, has previously been shown to upregulate only a small fraction of paused genes but downregulate many more genes. This may be attributed to a competition between nucleosomal occupancy and pol II binding or another pause-associated regulatory factor like Polycomb. The results with Hsp90 inhibition thus reiterate that there are different layers of regulation, and a final picture is an integrated outcome of a variety of these processes in a gene-specific way (Sawarkar, 2012).

The molecular details of how Hsp90 is involved in pol II pausing may share similarities with how Hsp90 poises steroid receptors for activation. The chaperone may keep the paused pol II components such as NELF and pol II itself in a conformation receptive for signal-mediated phosphorylation by P-TEFb. Radicicol causes paused pol II to be instantaneously but inefficiently released in elongation mode owing to NELF malfunction, causing a transient increase in expression of many genes. On the other hand, in absence of Hsp90, P-TEFb may not be able to phosphorylate and activate pol II efficiently following an extracellular signal such as LPS. It should be noted that P-TEFb is thought to be a client of Hsp90. Additionally, activators of transcription such as Trx are also degraded after a few hours of radicicol treatment, suggesting a possible feedback regulation of increased transcription upon Hsp90 inhibition (Sawarkar, 2012).

Past studies have linked Hsp90 depletion with cellular effects via its cytosolic function of protein stabilization. In light of the current results, earlier observations need to be reinterpreted to accommodate the direct effects of Hsp90 inhibition on gene expression. Hsp90 is thought to dampen phenotypic manifestation of genetic variants) in protein-coding as well as cis-regulatory regions. The relative contribution of cytosolic and chromatin-bound Hsp90 to different categories of genetic variants needs to be assessed. The recent demonstration of Hsp90's function in signal-mediated transactivation of the inducible nitric oxide synthase (iNOS) gene in addition to stabilization of iNOS protein underlines the need for reanalysis. In this regard, it is important to devise methods to distinguish cytosolic and nuclear functions of Hsp90. Either a nucleus-specific cochaperone network or nuclear entry of Hsp90 could be targeted by small-molecule regulators. Given the biomedical importance of Hsp90 in cancer and infectious diseases, development of such tools would be the next significant milestone. Thus the work presented here unites two rather disparate branches of biology -- molecular chaperones and gene regulation -- and it should open a new avenue of therapeutic importance for integrating vast amounts of data in both fields (Sawarkar, 2012).

SAYP and Brahma are important for 'repressive' and 'transient' Pol II pausing

Drosophila SAYP, a homologue of human PHF10/BAF45a, is a metazoan coactivator associated with Brahma and essential for its recruitment on the promoter. The role of SAYP in DHR3 activator-driven transcription of the ftz-f1 gene, a member of the ecdysone cascade was studied. In the repressed state of ftz-f1 in the presence of DHR3, the Pol II complex is pre-recruited on the promoter; Pol II starts transcription but is paused 1.5 kb downstream of the promoter, with SAYP and Brahma forming a 'nucleosomal barrier' (a region of high nucleosome density) ahead of paused Pol II. SAYP depletion leads to the removal of Brahma, thereby eliminating the nucleosomal barrier. During active transcription, Pol II pausing at the same point correlates with Pol II CTD Ser2 phosphorylation. SAYP is essential for Ser2 phosphorylation and transcription elongation. Thus, SAYP as part of the Brahma complex participates in both 'repressive' and 'transient' Pol II pausing (Vorobyeva, 2012).

The mechanism of ftz-f1 transcription activation has been analyzed in S2 cells. Sequential addition and removal of ecdysone allows the DHR3 and ftz-f1 genes in these cells to be activated in accordance with their expression pattern in vivo. This system is of considerable interest, since only a few Drosophila models of activated transcription are available. It also provides the possibility of studying the mechanism of pausing in the active and repressed transcription states of the same gene, whereas previous such studies have been performed with different genes (Vorobyeva, 2012).

Pol II pausing on ftz-f1 occurs at about 1.5 kb downstream of the promoter, i.e. at a much greater distance than that described for other genes (from +30 to +100 nt). Future studies will show how widespread is this mode of pausing. It is of interest in this context that a case of Pol II pausing at 800 bp downstream of the promoter was described for the β-actin gene (Vorobyeva, 2012).

The ftz-f1 activation at the molecular level is a several-stage process. At the first stage, when the ecdysone titer and DHR3 expression are high, DHR3, SAYP, TFIID, Brahma and Pol II accumulate at the promoter. Transcription is initiated, but Pol II is paused 1.5 kb downstream of the promoter; DHR3, SAYP and Brahma are also present at this site, where a nucleosomal barrier is formed. At the next stage, ~1 h after ecdysone removal, promoter-bound factors remain at the same levels, except for SAYP (its level on the promoter decreases). Pol II and associated factors disappear from the site of pausing, and the nucleosomal barrier is eliminated, but the transcription level does not increase. The following stage is characterized by rapid intensification of transcription, which reaches a maximum within several hours; the level of Pol II increases in the body of the gene, and its pausing is observed again, with SAYP and Brahma being present at the corresponding position. In addition, the level of SAYP on the promoter is recovered, indicating that it is highly regulated at different transcription stages. The DHR3 activator is present at the site of pausing, and its level does not change upon SAYP knockdown. This is evidence that DHR3 may participate in SAYP recruitment for subsequent nucleosomal barrier formation and Pol II pausing (Vorobyeva, 2012).

The region of high nucleosome density (nucleosomal barrier) is specific for the repression stage, at which the DHR3 activator induces the assembly of the Pol II preinitiation complex on the promoter and makes paused Pol II competent for transcription initiation. Nucleosomal barrier disruption by SAYP knockdown leads to the full-length transcript synthesis, indicating that the nucleosomal barrier contributes to preventing the entry of Pol II to the transcribed region. The data show that SAYP and Brahma play the crucial role in organization of the nucleosomal barrier: this barrier coincides in location with the peak of these coactivators and disappears after SAYP knockdown, which leads to elimination of Brahma from the gene. Thus, SAYP and Brahma at the stage of repressed transcription have an important role in blocking the synthesis of full-length transcripts. Although the transcription increases upon SAYP depletion and elimination of the nucleosomal barrier, its level remains low, compared with that in the permissive state. This is evidence for the existence of different mechanisms of Pol II pausing regulation, which also correlates with the fact that the depletion of NELF, an important factor of Pol II pausing, causes a 2.5-fold increase in the transcription of hsp70 or hsp26 gene in the repressed state, which, however, does not reaches the level characteristic of a fully activated gene (Vorobyeva, 2012).

The question arises as to the structure of the nucleosomal barrier. As shown previously, the human SWI/SNF complex can not only erase nucleosomes from the template but also produce a stable remodeled dimer of mononucleosome core, with this complex being also needed for converting this product back to the cores. One may suggest that the Drosophila Brahma complex operates in the same way. In the current experiments, the level of histone H3 increased ~2-fold in the region of the nucleosomal barrier, compared with its general level on the gene, which agrees with the assumption concerning the presence of a nucleosome dimer. The fact that the region of nucleosomal barrier is significantly enriched in sequences with a high nucleosome-positioning probability indicates that DNA sequences probably contribute to organization of this barrier (Vorobyeva, 2012).

Previous experiments have revealed a relationship between Pol II pausing and the nucleosomal structure of the template. It has been shown that Pol II stops at the site where the nucleosome density is restored to the average level characteristic of the gene. However, no specific nucleosome-dense regions preventing Pol II transcription have been described as yet (Vorobyeva, 2012).

The transition to the transcription-permissive state correlates with significant rearrangements in the promoter-distal region (disappearance of Brahma, SAYP, Pol II and nucleosomal barrier at the site of Pol II pausing). However, no increase in the ftz-f1 transcription level has been observed within the first 30 min after this transition. As shown in the study on estradiol (ER)-mediated gene expression, productive transcription is preceded by an unproductive cycle (~40 min) that is necessary for promoter preparation to this process. This may be the case for ftz-f1, with a certain period of time being required for rearrangements preceding its active transcription (Vorobyeva, 2012).

At the (+;-) stage, the level of SAYP on the promoter is recovered within 2-3 h after the onset of transcription, with SAYP RNAi influencing the Brahma and TFIID levels on the promoter. Pol II pausing correlating with its Ser2 modification is again observed as the transcription level increases. Although SAYP and Brahma occur again together with paused Pol II, their function appears to be different from that at the repression stage. The nucleosomal barrier is not restored, and SAYP depletion has only a slight effect on chromatin structure (Vorobyeva, 2012).

However, SAYP depletion severely disturbs transient pausing, interfering with Ser2 phosphorylation. This impairs proper transition to productive elongation and leads to a decrease in Pol II level on the body of the gene. Thus, SAYP knockdown not only affects the level of ftz-f1 activation but also shifts the timing of its expression. The slower kinetics of transcription induction together with the slight decrease in the Pol II level on the promoter upon SAYP knockdown are evidence for the retarded Pol II passage in the coding region of the gene and, hence, for disturbances in the elongation mechanisms. Similar consequences are observed for other genes regulating on pausing mechanisms (Vorobyeva, 2012).

The results of this study show that SAYP is important for proper timing of ftz-f1 transcription during Drosophila metamorphosis. The ftz-f1 gene is a major regulator of metamorphosis, that is why its precise activation in time is crucial during development. On the whole, the data provide evidence for the important role of pausing in sequential activation of genes in cascades and indicate that this mechanism may have a general role in development (Vorobyeva, 2012).

In addition, these results also support the idea that Pol II pausing may require not only NELF and DSIF but also other factors, such as nucleosome-remodeling complexes. Interestingly, the depletion of NELF proved to result in an increased nucleosome occupancy at the promoters of some genes (Vorobyeva, 2012).

In summary, this study has found that Pol II pausing is dependent on the interplay of several molecular mechanisms, including the formation of a specific chromatin structure via the action of coactivators. These results indicate that, although Pol II pausing is a genome-wide phenomenon, the specific molecular mechanism controlling paused Pol II activity on individual genes may vary significantly (Vorobyeva, 2012).

Structural analysis of nucleosomal barrier to transcription

Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. This study shows that both DNA-histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone-histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA-protein and protein-protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed (Gaykalova, 2015).

The data identify the transition from the +48 to +49 position as the key step during transcription through a nucleosome where Pol II makes a choice between arrest and further transcription. In the minimal system containing only Pol II and a nucleosome, this choice is dictated primarily by the sequence of nucleosomal DNA that determines the affinity of DNA-histone interactions and the probability of Pol II backtracking. In particular, R3 DNA sequence at the +(99-102) region is critical for escape from the +48 position (Gaykolova, 2015).

The arrest at position +48 becomes nearly irreversible on the 601 nucleosome due to Pol II backtracking along DNA and partial recoiling of nucleosomal DNA on the octamer after backtracking. The ability of Pol II to backtrack most likely depends on the DNA sequence immediately upstream of the active center of the enzyme and is typically reversible on DNA. However, recoiling of nucleosomal DNA on the octamer 'locks' Pol II and makes backtracking irreversible; a similar mechanism has been proposed previously (Gaykolova, 2015).

The choice between the arrest and further transcription through the nucleosome can be affected by transcription factors and histone chaperones. Thus, elongation factor TFIIS that facilitates transcription through chromatin in vitro can facilitate escape from the arrested state. Histone chaperone FACT facilitates transcription through chromatin via transient interaction with the open surface of histone octamer. Thus, depending on the sequence of nucleosomal DNA and presence of dedicated proteins, various steps during transcription through a nucleosome can be rate limiting and possibly regulated in vivo (Gaykolova, 2015).

After overcoming the nucleosomal barrier, further Pol II transcription is typically accompanied by nucleosome survival mediated by formation of a small intranucleosomal DNA loop on the surface of the histone octamer (Ø loop). The high efficiency of nucleosome survival has been puzzling because transcription by various RNA polymerases is accompanied by uncoiling of an extended DNA region (up to 80 bp) from the octamer, which is expected to induce immediate loss of an H2A/H2B dimer. The observed stability of the +42 complex containing a H2A/H2B dimer that is exposed into the solution suggests that binding of the H2A/H2B dimer to the H3/H4 tetramer could be allosterically stabilized and provides an explanation for the remarkable nucleosome stability of nucleosomal structure during various processes including transcription, replication, and ATP-dependent chromatin remodeling. Although one dimer is constitutively displaced from nucleosomes during transcription in vitro, this displacement likely occurs after transcription through the position +49 because the EC +49 contains both H2A/H2B dimers (Gaykolova, 2015).

The +42/48 pausing described in this study is related to nucleosomal pausing observed in Drosophila and yeast. In yeast, the primary nucleosomal barrier during transcript elongation by Pol II is encountered when the active center of the enzyme is +(40-55) bp in the nucleosome. A similar barrier is encountered by Pol II in Drosophila during transcription through nucleosomes localized more than ~400 bp from the transcription start site. However, there are additional nucleosomal barriers at the positions -7 and +20 bp in Drosophila, characteristic for nucleosomes localized immediately downstream of the transcription start site (Gaykolova, 2015).

Recent studies suggest that the density of Pol II complexes along the transcribed genes likely dictates the critical outcomes of transcription through chromatin-the height of nucleosomal barrier and the extents of histone displacement and exchange. At the same time, multiple factors including histone modifications, histone variants, histone chaperones, and chromatin remodelers further modify the histone dynamics on transcribed genes. Some of these factors interact with tumor suppressors and present important targets for the development of anticancer drugs (Gaykolova, 2015).

Genome-wide mapping of matrix attachment regions in Drosophila melanogaster

Eukaryotic genome acquires functionality upon proper packaging within the nucleus. This process is facilitated by the structural framework of Nuclear Matrix, a nucleo-proteinaceous meshwork. Matrix Attachment Regions (MARs) in the genome serve as anchoring sites to this framework. This study reports direct sequencing of the MAR preparation from Drosophila melanogaster embryos and identifies >7350 MARs. This amounts to ~2.5% of the fly genome and often coincides with AT rich non-coding regions. Significant association of MARs with the origins of replication, transcription start sites, paused RNA Polymerase II sites and exons, but not introns, of highly expressed genes. Sequence motifs and repeats were identified that constitute MARs. These data reveal the contact points of genome to the nuclear architecture and provide a link between nuclear functions and genomic packaging (Pathak, 2014).

Drosophila dosage compensation involves enhanced Pol II recruitment to male X-linked promoters

Through hyperacetylation of histone H4 lysine 16 (H4K16), the male-specific lethal (MSL) complex in Drosophila approximately doubles transcription from the single male X chromosome in order to match X-linked expression in females and expression from diploid autosomes. By obtaining accurate measurements of RNA polymerase II (Pol II) occupancies and short promoter-proximal RNA production, a consistent, genome-scale increase in Pol II activity was detected at the promoters of male X-linked genes. Moreover, enhanced Pol II recruitment to male X-linked promoters is largely dependent on the MSL complex. These observations provide insights into how global modulation of chromatin structure by histone acetylation contributes to the precise control of Pol II function (Conrad, 2012).

These data suggest that dosage compensation mainly arises from a near two-fold increase in Pol II activity at the promoters of male X-linked genes, most likely reflecting enhanced transcription initiation on the male X chromosome. In addition, release of Pol II into gene bodies, the entire gene from the transcription start site to the end of the transcript, is also slightly enhanced, suggesting that the transition into elongation might be facilitated. An initiation-based model readily explains the recent observation that the promoter sequence and associated transcription factor-binding sites influence the susceptibility of a reporter to dosage compensation. It is also consistent with the global hyperacetylation of male X-linked promoters and intergenic regions, which elevates the general accessibility of regulatory sequences to transcription factors. At the same time, hyperacetylation within gene bodies is a prerequisite for MSL complex binding to the transcribed regions of X-linked genes. Restricting the MSL complex to these sites may prevent the physical obstruction of promoters in males and so help balance the transcriptional output between sexes (Conrad, 2012).

X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila

The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. This study used global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome (Larschan, 2011).

In addition to increasing the transcription of X-linked genes for dosage compensation, MSL complex also positively regulates the roX noncoding RNA components of the complex, to promote their male-specificity. roX1 expression is low in the SL2 cell line, but GRO-seq data indicate that active transcription of roX2 is highly dependent on MSL2 as predicted. Interestingly, there is a strong GRO-seq peak at the 3′ roX2 DHS which contains sequences important for targeting MSL complex to the X chromosome. Sites of roX gene transcription are thought to be critical for MSL complex assembly. Therefore, it is possible that paused RNAP II at the roX2 DHS could promote an open chromatin structure that facilitates MSL complex targeting or incorporation of noncoding roX2 RNA into the complex (Larschan, 2011).

In summary, it is hypothesized that MSL complex functions on the male X chromosome to promote progression and processivity of RNAP II through the nucleosomal template. Improving transcriptional output downstream of typical gene-specific regulation makes biological sense when compensating the diverse set of genes found along an entire chromosome (Larschan, 2011).

'Jump Start and Gain' model for dosage compensation in Drosophila based on direct sequencing of nascent transcripts

Dosage compensation in Drosophila is mediated by the MSL complex, which increases male X-linked gene expression approximately 2-fold. The MSL complex preferentially binds the bodies of active genes on the male X, depositing H4K16ac with a 3' bias. Two models have been proposed for the influence of the MSL complex on transcription: one based on promoter recruitment of RNA polymerase II (Pol II), and a second featuring enhanced transcriptional elongation. This study utilize nascent RNA sequencing to document dosage compensation during transcriptional elongation. Also, comparison was made of X and autosomes from published data on paused and elongating polymerase in order to assess the role of Pol II recruitment. The results support a model for differentially regulated elongation, starting with release from 5' pausing and increasing through X-linked gene bodies. The results highlight facilitated transcriptional elongation as a key mechanism for the coordinated regulation of a diverse set of genes (Ferrari, 2013).

This study has systematically dissected the mechanism of DC during distinct steps of transcription. Multiple high-resolution, genome-wide approaches converge on the following model: paused Pol II is not augmented in general on the male X, but Pol II release from pausing ('jump-start' in the model) appears to be a key rate-limiting step that is facilitated by X-specific enrichment of H4K16ac in gene bodies. The increasing MSL and H4K16ac levels over the bodies of genes further reduce steric hindrance, leading to a 'gain' of Pol II density. Currently, it cannot be determined whether this gain is the result of (1) increased processivity (reduced termination) or (2) positive feedback to 5' Pol II, to further increase pausing release. In either case, it is believed that facilitated elongation through an acetylated chromatin template enables coordinate control of X-linked genes with widely differing mechanisms of individual, gene-specific regulation (Ferrari, 2013).

PNUTS/PP1 regulates RNAPII-mediated gene expression and is necessary for developmental growth

In multicellular organisms, tight regulation of gene expression ensures appropriate tissue and organismal growth throughout development. Reversible phosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) is critical for the regulation of gene expression states, but how phosphorylation is actively modified in a developmental context remains poorly understood. Protein phosphatase 1 (PP1) is one of several enzymes that has been reported to dephosphorylate the RNAPII CTD. However, PP1's contribution to transcriptional regulation during animal development and the mechanisms by which its activity is targeted to RNAPII have not been fully elucidated. This study shows that the Drosophila orthologue of the PP1 Nuclear Targeting Subunit (dPNUTS) is essential for organismal development and is cell autonomously required for growth of developing tissues. The function of dPNUTS in tissue development depends on its binding to PP1, which is targeted by dPNUTS to RNAPII at many active sites of transcription on chromosomes. Loss of dPNUTS function or specific disruption of its ability to bind PP1 results in hyperphosphorylation of the RNAPII CTD in whole animal extracts and on chromosomes. Consistent with dPNUTS being a global transcriptional regulator, loss of dPNUTS function was found to affect the expression of the majority of genes in developing 1st instar larvae, including those that promote proliferative growth. Together, these findings shed light on the in vivo role of the PNUTS-PP1 holoenzyme and its contribution to the control of gene expression during early Drosophila development (Ciurciu, 2013).

Reversible phosphorylation plays important roles in the regulation of transcriptional networks and in coordinating spatial and temporal patterns of gene expression. Phosphorylation of RNA polymerase II (RNAPII) at multiple sites on its C-terminal domain (CTD) is critical for gene expression and its regulation. Different phospho-forms of the CTD appear at different stages of the transcription cycle, and these are thought to facilitate initiation, elongation and termination by recruiting specific histone and RNA modifiers. The consensus view from studies of RNAPII occupancy in budding yeast is that there is a stereotypical pattern of phosphorylation at most gene loci during the transcription cycle. However, numerous lines of evidence suggest that there is active control of CTD phosphorylation in response to environmental cues and during developmental transitions, e.g. in which restriction of CTD phosphorylation to particular lineages is used to control cell fate. Furthermore, studies of the enzymes responsible for regulating CTD phosphorylation indicate that phosphorylation may be modified at specific loci to determine gene-specific patterns of expression (Ciurciu, 2013).

Serine/threonine protein phosphatase type 1 (PP1) is one of four protein phosphatases known to contribute to the regulation of CTD phosphorylation (Washington, 2002), the others being FCP1, SCP1 and Ssu72. In Drosophila, PP1 is found at multiple sites on chromosomes where it has been postulated to play important roles in regulating developmentally controlled gene expression (Rudenko, 2003; Rudenko, 2004). However, analysing the role of PP1 in transcriptional regulation has been complicated by its pleiotropic roles (Kirchner, 2007) and broad in vitro substrate specificity. In vivo, PP1 has been shown to associate with different targeting subunits that restrict its activity towards particular substrates (Bollen, 2010). Therefore, a full understanding of PP1 function requires the identification and characterisation of these regulatory proteins (Ciurciu, 2013).

In mammalian cells, the PP1 Nuclear Targeting Subunit (PNUTS) is one of the two most abundant PP1-interacting proteins in the nucleus and is known to be chromatin-associated during interphase and not during mitosis. Its reassociation with chromatin during telophase and its ability to augment chromosome decondensation in vitro and in vivo have indicated a possible role in cell cycle progression. Several lines of evidence also indicate that PNUTS is required for cell survival and contributes to cellular responses to environmental stress, including hypoxia and DNA damage. These roles may be especially important during ageing since loss of PNUTS expression is associated with an age-dependent increase in cardiomyocyte apoptosis and decline in cardiac function. Targeting of PNUTS to chromatin is likely to be in part through association with the DNA-binding factor Tox4/Lcp1, which is capable of recognising DNA adducts generated by platinum anticancer drugs. PNUTS and Tox4 have also been reported to form a stable multimeric complex with Wdr82, which was previously identified as an integral component of a distinct complex containing Set histone H3-Lys4 methyltransferases. Although the role of Wdr82 bound to PNUTS is not known, Wdr82 may mediate interactions with initiating and early elongating RNAPII by recognising Ser5-phosphorylated CTD, as it does when it is associated with the Set1 complex. A role for PNUTS in transcription has been further suggested by recent reports that it associates with RNAPII complexes. Despite these insights, an understanding of the physiological roles of PNUTS remains incomplete (Ciurciu, 2013).

This study shows that null mutants in the D. melanogaster orthologue of PNUTS (dPNUTS), display a larval growth defect and are larval lethal. Mutant clones show a cell autonomous growth defect and are eliminated from wild type epithelia due to cell competition. RNA-Sequencing (RNA-Seq) analysis indicates that dPNUTS affects the expression of the majority of genes in 1st instar larvae, including those that are highly expressed and are involved in cellular metabolism and larval development. The function of dPNUTS in tissue development is dependent on binding to the catalytic subunit of Protein phosphatase 1 (PP1), which is targeted by dPNUTS to RNA polymerase II in cell extracts and at many active sites of transcription on polytene chromosomes. Loss of dPNUTS function, or displacement of dPNUTS-PP1 using a non-PP1 binding mutant of dPNUTS, results in hyperphosphorylation of the C-terminal domain of RNA Polymerase II in whole animal extracts and on chromosomes. Taken together, these data suggest that dPNUTS-PP1 is a global regulator of gene expression via effects on RNAPII phosphorylation and is required in larvae to promote normal developmental growth (Ciurciu, 2013).

This study reports the functional analysis of Drosophila PNUTS, a regulatory subunit of PP1 that is highly conserved between flies and humans. dPNUTS is essential for organismal growth, with mutant animals arresting early in larval development. Survival of the null zygotic mutants until the early larval stage is most likely due to perdurance of maternal dPNUTS gene products, raising the possibility of additional roles for dPNUTS during embryological development that were not uncovered in this study. Clonal analysis indicates that dPNUTS has a cell autonomous effect on growth, with mutant clones failing to survive unless given a growth advantage. Transcriptomics characterisation of dPNUTS mutant animals indicates that the larval arrest phenotype is associated with the underexpression of many RNAPII-dependent genes, including those that normally support developmental growth. Of particular interest in this regard is the significant enrichment of genes involved in cellular metabolism. The underexpression of these genes suggests that an important role of dPNUTS during larval growth might be to ensure transcription of highly expressed metabolic pathways responsible for fuelling energy production and generating the macromolecular precursors for RNA and protein synthesis. Metabolic state is monitored in developing epithelia, ensuring that the fittest cells are selected as organ precursors. The failure to compete with wild type neighbours is consistent with an altered metabolic state that is recognised by cell competition, triggering cells to be outcompeted by their neighbours and lost by caspase-dependent apoptosis (Ciurciu, 2013).

Is the effect on RNAPII-dependent transcription the cause of growth defects? It is conceivable that roles that have been assigned to hPNUTS, e.g. in the DNA damage response and chromatin condensation, are conserved in dPNUTS and these might contribute to the larval lethality exhibited by dPNUTS mutants. Indeed the non-identical distribution of dPNUTS and RNAPII on chromosomes suggests that dPNUTS is present in chromatin-associated complexes lacking RNAPII. Notably no detectable condensation defects were detected in dPNUTS mutant clones but the possibility cannot be excluded that dPNUTS may also contribute to other processes that underlie tissue growth, such as transcription-independent cell cycle control, as has been reported for other enzymes that regulate CTD phosphorylation, such as FCP1 (Visconti, 2012). Nevertheless, loss of expression of any one of the cell metabolism pathways affected by dPNUTS is sufficient to cause larval growth arrest and is likely to explain the failure of dPNUTS larvae to grow in size prior to their eventual demise (Ciurciu, 2013).

Like its mammalian counterpart, dPNUTS is shown to be a nuclear protein that localises to chromatin during interphase. By utilising larval polytene chromosomes, which are readily visible by light microscopy, it was possible to extend this analysis by determining the distribution of dPNUTS on interphase chromosomes in situ. These analyses show co-localisation of dPNUTS with many transcriptionally active sites marked with RNAPII, suggesting that the widespread changes in gene expression that were observe upon loss of dPNUTS function are likely to be due to the direct involvement of dPNUTS in RNAPII-mediated transcriptional regulation. Correspondingly, it was found that dPNUTS is complexed to the large subunit of RNAPII in cell extracts. However, it is important to note that not all RNAPII sites stain for dPNUTS (and vice versa) and the relative amounts of the two proteins vary widely amongst these sites. This suggests that the association of dPNUTS with RNAPII, or with associated factors, which may affect the availability of the dPNUTS epitope for detection by the antibody used in this study, may be differentially regulated. PNUTS contains a number of conserved macromolecular-interaction domains, which have led to the suggestion it might serve as a multivalent adapter protein. However, it has not yet been established to what extent the known interactors, Tox4 and Wdr82 aid in the recruitment of PNUTS to chromosomal loci. These issues will require investigation of the genome-wide sites of dPNUTS binding, as well as identification and comprehensive characterisation of dPNUTS-interacting proteins and their role in dPNUTS recruitment (Ciurciu, 2013).

Since PP1-binding was found to be necessary for dPNUTS function, it was reasoned that dPNUTS affects transcription by targeting PP1 to specific substrates on chromosomes. Several lines of evidence indicate that one important target of dPNUTS-PP1 in this context is the CTD of RNAPII: 1) dPNUTS is complexed with RNAPII in nuclear extracts and regulates RNAPII CTD phosphorylation in a PP1-dependent manner; 2) RNAPII CTD Ser5-P levels are elevated in dPNUTS mutant larval extracts and tissues; 3) dPNUTS colocalises with PP1 and RNAPII on chromosomes; 4) ectopic expression of a mutant version of dPNUTS that displaces PP1 from polytene chromosomes results in elevated RNAPII CTD Ser5-P levels on chromosomes. dPNUTS-PP1 appears to preferentially target Ser5-P of the CTD as only a modest effect on Ser2-P levels wasobserved and no effect on phosphorylation of other RNAPII-CTD residues in dPNUTS mutant larval extracts was revealed by Western blotting. However, PNUTS/PP1 is not the only PP1 holoenzyme that has been implicated in regulation of RNAPII phosphorylation (Jerebtsova, 2011), raising the possibility that different PP1 holoenzymes possess different RNAPII CTD specificities (Ciurciu, 2013).

Changes in the pattern of gene expression that were observed in dPNUTS mutant animals are correlated with the normal expression level of the affected transcripts; these changes may also reflect the spatial distribution of dPNUTS expression during development. During embryogenesis it was observed that the levels of dPNUTS expression in the gut and the ventral nerve cord correlates with stages in which these tissues are undergoing periods of rapid expansion and development. In an analogous fashion to SCP1, which restricts RNAPII dephosphorylation of neuronal genes to non-neuronal cells by virtue of its expression pattern (Yeo, 2005), the enrichment of dPNUTS in proliferating tissues may function to promote expression of highly expressed transcripts, such as those involved in cellular metabolism, in these tissues, to support their development. In mammals, the gradual decrease from a high level of PNUTS during embryogenesis to a relatively low level in adults has been taken to imply that PNUTS could play a role in cortical development, but could equally reflect a requirement during growth of developing tissues. Notably, PNUTS is not found in some metazoans such as C.elegans, where strictly controlled cell lineage determines tissue architecture. An evolved function of PNUTS might therefore be to support proliferative states in organisms where compensatory mechanisms such as cell competition are at play (Ciurciu, 2013).

How do dPNUTS and RNAPII hyperphosphorylation regulate gene expression? Studies of other enzymes that control CTD phosphorylation state indicate that maintaining correct levels of CTD phosphorylation is critical for normal levels of transcription and that hyperphosphorylation of RNAPII can increase or reduce gene expression depending on what stage of the transcriptional cycle phosphorylation is affected. For instance, FCP1 targets Ser2-P in vivo and is thought to recycle RNAPII after the complex has dissociated from the transcribed region. Correspondingly, conditional knockout of FCP1 in yeast results in a global defect in transcription affecting 77% of genes. SCP1 and Ssu72 both target Ser5-P, but have contrasting roles in transcriptional regulation: knockdown of SCP1 unmasks neuronal gene expression, indicating it normally acts as a transcriptional repressor, whilst Ssu72 facilitates transcription by promoting the elongation stage of the transcription cycle. ChIP experiments from larvae expressing dPNUTS^W726A suggest that displacement of PP1 binding to dPNUTS does not result in accumulation of RNAPII on the coding region of affected loci. The precise mechanisms of how loss of dPNUTS function and RNAPII hyperphosphorylation disrupt gene expression require further investigation. However, it might be expected that processes dependent on normal CTD phosphorylation, including RNA processing, transcription-coupled chromatin modification and transcription-associated homologous recombination, are affected. In this regard, it is notable that inhibition of TFIIH kinase activity, which phosphorylates promoter-bound RNAPII at Ser5, predominantly affects mRNA capping and stability rather than transcription per se (Ciurciu, 2013).

In summary, the analysis of dPNUTS reveals an important function for this evolutionarily conserved chromatin-associated protein, via association with PP1, in the regulation of RNAPII phosphorylation and the appropriate expression of genes during larval development, which support organismal growth. These findings provide insight into the role of PNUTS and RNAPII phosphorylation during normal development, and may also be of relevance to the understanding of aberrant gene expression patterns observed in disease processes and ageing (Ciurciu, 2013).

P-TEFb kinase promotes transcription at heat shock loci

P-TEFb kinase recruitment to heat shock loci during the heat shock response and functions to stimulate promoter-paused RNA polymerase II (Pol II) to enter into productive elongation. P-TEFb is located at >200 distinct sites on Drosophila polytene chromosomes. Upon heat shock, P-TEFb, like the regulatory factor heat shock factor (HSF), is rapidly recruited to heat shock loci, and this recruitment is blocked in an HSF mutant. Yet, HSF binding to DNA is not sufficient to recruit P-TEFb in vivo, and HSF and P-TEFb immunostainings within a heat shock locus are not coincident. Insight to the function of P-TEFb is offered by experiments showing that the direct recruitment of a Gal4-binding domain P-TEFb hybrid to an hsp70 promoter in Drosophila cells is sufficient to activate transcription in the absence of heat shock. Analyses of point mutants show this P-TEFb stimulation is dependent on Cdk9 kinase activity and on Cdk9's interaction with cyclin T. These results, coupled with the frequent colocalization of P-TEFb and the hypophosphorylated form of Pol II found at promoter-pause sites, support a model in which P-TEFb acts to stimulate promoter-paused Pol II to enter into productive elongation (Lis, 2000).

P-TEFb is required to produce full-length transcripts from a variety of cellular DNA templates in an in vitro transcription system that accurately recapitulates the normal DRB-sensitive transcription seen in cells (Marshall, 1995). These results suggest that P-TEFb may have a role in transcription of many cellular genes. If so, this kinase may localize to chromosomal loci that possess genes that are the target of its activity. The chromosomal distribution of P-TEFb was examined by staining salivary gland polytene chromosomes with a highly specific antibody to the cyclin T regulatory subunit. This cyclin T subunit binds tightly to Cdk9 and is a critical component of the P-TEFb activity (Peng, 1998). Moreover, immunodepletion experiments show that the vast majority of Cdk9 is associated with a cyclin T subunit (Peng, 1998), and probing of phosphocellulose fractions from Drosophila Kc cell nuclear extracts indicates that cyclin T is present only where P-TEFb activity is found. Therefore, the cyclin T antibody provides a good means of tracking the P-TEFb complex (Lis, 2000).

Heat shock causes a rapid and dramatic activation of transcription of heat shock genes and a concomitant reduction in transcription of many normally expressed genes. Immunofluorescence analysis of polytene chromosomes reveals that Pol II relocates to heat shock loci after a brief heat shock. P-TEFb distribution also changes dramatically following heat shock. In uninduced larvae, P-TEFb is undetectable at major heat shock loci 87A and 87C, which contain the five hsp70 genes, or at 59B, which, in this strain, contains an hsp70-lacZ transgene. After a 20-min heat shock, these and all the other major heat shock loci at 63B, 67B, 93D, and 95D are the prominent sites of labeling. Loci that had high levels of P-TEFb before heat shock now have a reduced level. Therefore, P-TEFb redistributes to heat shock loci following heat shock (Lis, 2000).

The Pol II level on the 5' end of the hsp70 gene begins to be elevated in as little as 70 sec following a very rapid heat shock induction (mixing cells with warm medium), and Pol II is detected beyond the pause region and in the middle of the gene in as little as 2 min. This rapid transcriptional activation leads to a very high density of hyperphosphorylated Pol II on these genes. Could P-TEFb be playing a role in the transition of Pol II to its hyperphosphorylated, elongationally competent mode? If so, then one might expect P-TEFb to be recruited as rapidly as Pol II to these newly activated heat shock sites (Lis, 2000).

The kinetics of localization to heat shock loci at 87A and 87C and to 59B, which in this strain contains an Hsp70-lacZ transgene, were examined. No P-TEFb is detected at the native or the transgenic sites before heat shock. However, within 2 min, staining is apparent at 87A and 87C, each of which contain multiple copies of hsp70. Some staining is also detectable at the transgenic copy of Hsp70-lacZ. By 5 min of heat shock, staining at all heat shock loci is strong and this high level persists and may even increase in the 10- and 15-min time points. The level remains high during heat shock measured out to 60 min. A shift back to normal fly culture temperature (e.g., a 60-min recovery) reduces heat shock gene transcription and the normal pattern transcription is largely re-established (Lis, 2000).

The recruitment of P-TEFb to heat shock loci is completely dependent on HSF. A Drosophila temperature-sensitive mutant HSF strain, hsf4, shows a much reduced induction of heat shock gene transcription and chromosome puffing. In this strain, heat shock fails to concentrate P-TEFb at heat shock loci. Additionally, heat shock does not lead to a dramatic loss of P-TEFb at the normally active chromosomal sites in the HSF mutant strain as exemplified at 88D (Lis, 2000).

Heat shock rapidly stimulates the trimerization and binding of HSF to the heat shock elements (HSEs) located upstream of every heat shock gene. HSF acquires strong DNA-binding activity and localizes to heat shock loci on polytene chromosomes within 2 min following heat shock. Therefore, the rapid induction of HSF binding is similar to the rapid recruitment of P-TEFb seen here. Could HSF itself be sufficient to recruit P-TEFb through a stable interaction? This hypothesis was tested in vivo using a transgenic line containing a polymer of native HSF-binding sites that are unlinked to the rest of the hsp70 promoter. Following heat shock, HSF is known to localize to sites on polytene chromosomes containing this polymer. This anti-HSF staining is more than an order of magnitude stronger than that seen at the regulatory region of a single hsp70 gene, and can be compared with the 87A and 87C loci that contain two and three copies of native hsp70, respectively. The 87C signal is considerably stronger than 87A (Lis, 2000).

If HSF is sufficient to recruit P-TEFb to heat shock loci in vivo, then one would expect to see high levels of P-TEFb at the polymer site. There is detectable P-TEFb at the polymer site, but the level is less than at the native heat shock loci 87A and 87C. Moreover, the ratio of P-TEFb to HSF staining is much higher at heat shock genes than at the polymer site. These results indicate that HSF does not on its own recruit P-TEFb, and other features of the heat shock promoters are required to provide P-TEFb's strong recruitment to heat shock genes (Lis, 2000).

P-TEFb appears to resolve from HSF at the 87A locus. In most extended chromosomes examined, it is observed that the P-TEFb label separates into a doublet with HSF overlapping and falling between the peaks of the P-TEFb doublet. This can be interpreted in terms of the known arrangement of hsp70 genes at 87A. The hsp70 genes are divergently transcribed and the regulatory DNA containing the binding sites for HSEs resides in this region between the genes. HSF binds these regulatory regions as was seen from a band of fluorescence in the middle of the puff. In contrast, the centers of P-TEFb staining appear to reside downstream of the HSEs on both copies of the hsp70 gene. The partial separation of P-TEFb and HSF is also consistent with the idea that P-TEFb does not derive its stable association with heat shock genes solely through interaction with HSF (Lis, 2000).

A biochemical assay of the interaction of HSF and P-TEFb adds further support to the conclusion that these proteins do not interact strongly. Plasmids that express HSF, Cdk9-Flag, and cyclin T-6His were cotransfected into Drosophila cells. Following a standard heat shock treatment, cleared lysates were prepared from these cells, and the lysates were then chromatographed over nickel-NTA beads, which bind the 6His-tag. Portions of the lysates and nickel-bound fractions were then examined by Western blotting using HSF or Flag antibodies. Whereas Cdk9 is efficiently recovered in the Ni-bound fraction, HSF is not recovered at levels exceeding the background from cells lacking cyclin T-6His. These results and the in vivo results indicate that the high levels of P-TEFb association with heat shock loci cannot be explained by an interaction of HSF with P-TEFb (Lis, 2000).

Does the redistribution of P-TEFb to heat shock loci influence transcription of the heat shock genes? The effects of directly recruiting P-TEFb subunits, Cdk9 or cyclin T, to the hsp70 promoter were tested. A pair of Gal4-binding sites (UASgal) was introduced upstream of a Drosophila hsp70-M reporter gene. The expression of this hybrid reporter gene can be distinguished from native hsp70 genes since it is marked by fusion to a bacterial DNA sequence. This reporter construct and copper-inducible expression vectors, which express the Gal4 DNA-binding alone (G4) or G4 fused to Cdk9, cyclin T and a variety of controls, were cotransfected into Drosophila cells. The inserted UASgal sequences are upstream of the regions critical for heat shock expression, so, as anticipated, transcription of this reporter gene is heat inducible, albeit at about a twofold lower level than the control containing no UASgal insert. The reporter gene containing UASgal sites is strongly activated without heat shock when cells are cotransfected with G4 fused to the activation domain of HSF (G4-HSF). The reporter gene carrying the UASgal sites is also strongly activated without heat shock when cells are cotransfected with plasmids expressing G4-Cdk9 or G4-cyclin T. A point mutation that disrupts the activity of the kinase subunit, Cdk9/D199N (Peng, 1998), also disrupts the ability of the G4-Cdk9 hybrid protein to activate transcription from the hsp70 reporter. The levels of expression of wild-type and mutant G4-Cdk9 are similar. Also, a pair of mutations in cyclin T that disrupt its ability to interact with Cdk9, the double point mutant CycT/2XMut (Bieniasz, 1999), greatly impairs the ability of G4-cyclin T to activate transcription. These results demonstrate that artificially recruiting P-TEFb to the promoter by directly recruiting either of its two subunits is sufficient to strongly activate an hsp70 gene. A similar activation by G4-HSF, G4-Cdk9, and G4-cyclin T was observed with UASgal sequences inserted further upstream at -256, although the level of activation was reduced two to threefold (Lis, 2000).

A model is proposed in which P-TEFb acts on promoter-paused Pol II complexes to stimulate their escape into productive elongation. If P-TEFb is a major kinase that acts on the promoter-paused Pol II complex, its distribution should overlap at least some of the chromosomal sites that accumulate unphosphorylated RNA polymerase II (Pol IIa). However, the correlation need not be perfect, since the rate of formation of a promoter-paused Pol IIa is likely to be governed by mechanisms distinct from those that are responsible for recruiting P-TEFb. These mechanisms appear to be quite independent in an extreme case of heat shock genes, in which Pol IIa is present at full occupancy on the uninduced hsp70 promoter, and heat shock is needed to trigger both high levels of transcription and recruitment of P-TEFb. However, when a gene is active, Pol IIa is being continuously recruited to the promoter and maturing into a productive hyperphosphorylated polymerase II (Pol IIo) elongation complex. In the case of heat shock genes, the entry is fast enough to keep the pause region fully occupied with Pol II even when the gene is fully induced. Therefore, both the kinase responsible for phosphorylation and the Pol IIa would be expected to be present on active promoters, and their respective levels would be dictated by the relative rates of Pol entry and its maturation into a productive elongation complex (Lis, 2000 and references therein).

Chromosome were stained with antibodies to P-TEFb and Pol IIa. Most chromosomal sites in unstressed larvae that are labeled strongly by the P-TEFb (cyclin T) antibody are also labeled to various extents by the Pol IIa antibody; however, the ratio of labeling by these two antibodies varies at different sites. Therefore, the level of Pol IIa must be governed by factors that act at least somewhat independently from factors that govern the level of P-TEFb at specific sites. Nonetheless, the strong tendency of these proteins to colocalize is consistent with a model in which Pol IIa is a substrate for P-TEFb, and this phosphorylation serves to convert Pol II into a productive elongation complex (Lis, 2000).

The hyperphosphorylated form, PoI IIo, labels many more sites than does P-TEFb. Numerous sites are strongly labeled with antibody to Pol IIo, but not detectably labeled with antibody to P-TEFb. This pattern does not easily fit a model in which P-TEFb has a universal role in all Pol II transcription elongation. Presumably, there are distinct mechanisms (and other kinases) for producing Pol IIo that do not require the stable and continuous association of P-TEFb with a locus (Lis, 2000).

In contrast, there are few chromosomal sites that have P-TEFb, but no Pol IIo. A simple interpretation of this result, which is consistent with the known properties of P-TEFb, is that the recruitment of P-TEFb to a locus generally leads to efficient formation of transcription elongation complexes. These results also indicate that there is little recruitment of P-TEFb to sites that are not transcriptionally active (Lis, 2000).

P-TEFb is a kinase/cyclin heterodimer that was critical for overcoming an early block to transcriptional elongation (Marshall, 1995). Interestingly, the short transcripts of 20-40 nucleotides that are produced in the absence of P-TEFb are remarkably similar in size to those measured in vivo at genes that show promoter-associated pausing. Such pausing has been observed at a variety of genes; however, the heat shock promoters of Drosophila are perhaps the most thoroughly studied in eukaryotes. P-TEFb stimulates production of full-length transcripts in vitro (Marshall, 1995), and also from HIV templates in vivo (Mancebo, 1997). P-TEFb is normally located at >200 loci, but upon heat shock, it redistributes to native and transgenic heat shock loci with a robustness and rapidity that make it a good candidate for playing a critical role in the activation of heat shock gene transcription. The normally broad distribution of P-TEFb is simplified during heat shock, where the bulk of P-TEFb concentrates at all the major heat shock genes. The resolution of HSF and P-TEFb staining within the 87A locus is consistent with the long-held view that the DNA within this activated heat shock locus is in a very extended configuration. The two divergently transcribed hsp70 genes at this locus are separated by only 2 kb, and yet the P-TEFb staining resolves as two distinct bands. The major HSF staining resides between the two P-TEFb bands. HSF binding sites are known to reside in the region between the start sites of these genes. If the DNA in a highly decondensed puff approximates B-form DNA, that is, it has a chromatin packing ratio similar to that of highly transcribed ribosomal DNA, then the distance between the start sites of the two hsp70 genes would be ~0.7 µm. The centers of the two bands of staining are approximately twice that distance, implying that P-TEFb may be distributed over a region that extends downstream of the hsp70 start sites. Higher resolution biochemical methods will be required to precisely define the limits of the P-TEFb distribution. Nonetheless, the partial resolution of HSF and P-TEFb staining supports a view that these two components act at distinct points in the process of activating heat shock genes (Lis, 2000).

P-TEFb is not simply recruited by the hypophosphorylated Pol IIa. Pol IIa is the form of Pol II that is at the promoter pause region of hsp70 and other heat shock genes. Yet very little or no P-TEFb is detected at these sites prior the heat shock. It is speculated that a separate event must occur at these promoters to cause the association of P-TEFb, for example, another protein or proteins could recruit P-TEFb to these promoters. In the case of HIV, the Tat protein interacts with cyclin T to recruit the P-TEFb complex (Garber, 1998). For normal cellular genes, other host transcription factors may also play such a role; P-TEFb has been shown recently to be functionally recruited to MHC class II gene promoters by the CIITA activator (Kanazawa, 2000). Alternatively, transcription activation may normally allow a paused Pol IIa, a likely in vivo substrate of P-TEFb, to undergo a change or unmasking that now allows its association with P-TEFb (Lis, 2000).

P-TEFb is normally located at many chromosomal sites that are transcriptionally active. The chromosomal loci scored as positive with the cyclin T antibody may represent only a fraction of the genes that could be regulated by P-TEFb, owing to the dynamic developmental regulation of the Drosophila genome. Also, the existence of additional cyclin subunits that can couple with Cdk9 may produce a P-TEFb activity lacking cyclin T. Although whether P-TEFb activates transcription at all of the loci containing cyclin T cannot be evaluated, in the case of heat shock genes, the direct recruitment (via a Gal4 DNA-binding domain) of P-TEFb to an hsp70 promoter leads to an activation of this gene in the absence of heat shock. Although this activation is less than the very high level of activation caused by directly recruiting the activation domain of HSF, it is nonetheless clearly dependent on P-TEFb kinase activity. Interestingly, related kinases, Cdk2 and Cdk7, fail to activate this promoter and critical point mutations in the P-TEFb kinase or cyclin T disrupt the activation. The fact that Cdk7, the kinase of the TFIIH complex, fails to activate is worth noting, because it, like P-TEFb, can phosphorylate efficiently the CTD of Pol II. Perhaps these kinases have specificity for discrete steps in transcription. For example, P-TEFb may be capable of stimulating the elongation of the paused Pol II, whereas TFIIH kinase fulfills another role such as providing activity for an earlier step in transcription that does not necessarily lead to the full phosphorylation and maturation of elongationally competent Pol II. This specificity issue requires further, more focused investigation (Lis, 2000 and references therein).

Direct recruitment of P-TEFb to an HIV promoter has been shown to activate HIV transcription fully and bypasses the need for Tat (Bieniasz, 1999). Although the activation of hsp70 by directly recruiting P-TEFb that is observed in uninduced cells is strong, it is still less than that seen when the HSF activation domain is directly recruited. This fact suggests that HSF may be providing a function beyond triggering the events that lead to P-TEFb recruitment. The HSF activation domain is large enough to accommodate multiple interactions and functions (Lis, 2000 and references therein).

The colocalization of the hypophosphorylated Pol IIa with P-TEFb is intriguing, because the promoter-paused Pol II associated with all genes examined in Drosophila is hypophosphorylated. If the Pol IIa distribution is a general indicator of sites in which promoter-pausing is a part of the transcription mechanism, then P-TEFb may be stimulating the maturation of Pol II and its entry into productive elongation at a significant subset of active genes. Three of the four constitutively active genes that have been reported to have promoter-paused Pol II are at chromosomal sites that show significant P-TEFb. The fourth, Gapdh-2, is at 13F, a region that shows light P-TEFb staining. A higher resolution analysis will be required for a rigorous assignment of the P-TEFb signals to these specific genes (Lis, 2000 and references therein).

The failure to see a quantitative correlation of the intensity of staining of anti-Pol IIa and anti-P-TEFb at specific sites on polytene chromosomes is consistent with models in which the mechanism of generating paused Pol IIa is distinct from the mechanism that recruits P-TEFb. The extreme case of this is hsp70, in which, before heat shock, the promoter is fully occupied with Pol IIa, but has very little P-TEFb. Heat shock triggers the dramatic recruitment of P-TEFb, and the accumulation of Pol IIo on heat shock puffs. During heat shock, the paused Pol IIa still forms, but it escapes into productive elongation faster, once every 4 sec as compared with the uninduced level of once every 10 min. It is hypothesized that P-TEFb participates in this escape at heat shock genes and the subset of other genes that have promoter-paused Pol II (Lis, 2000).

Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation

The RNA polymerase II (Pol II) C-terminal domain (CTD) serves as a docking site for numerous proteins, bridging various nuclear processes to transcription. The recruitment of these proteins is mediated by CTD phospho-epitopes generated during transcription. The mechanisms regulating the kinases that establish these phosphorylation patterns on the CTD are not known. This study reports that three CTD kinases, CDK7, CDK9, and BRD4, engage in cross-talk, modulating their subsequent CTD phosphorylation. BRD4 phosphorylates PTEFb/CDK9 at either Thr-29 or Thr-186, depending on its relative abundance, which represses or activates CDK9 CTD kinase activity, respectively. Conversely, CDK9 phosphorylates BRD4 enhancing its CTD kinase activity. The CTD Ser-5 kinase CDK7 also interacts with and phosphorylates BRD4, potently inhibiting BRD4 kinase activity. Additionally, the three kinases regulate each other indirectly through the general transcription factor TAF7. An inhibitor of CDK9 and CDK7 CTD kinase activities, TAF7 also binds to BRD4 and inhibits its kinase activity. Each of these kinases phosphorylates TAF7, affecting its subsequent ability to inhibit the other two. Thus, a complex regulatory network governs Pol II CTD kinases (Devaiah, 2012).

Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs

Erk1/2 activation contributes to mouse ES cell pluripotency. This study found a direct role of Erk1/2 in modulating chromatin features required for regulated developmental gene expression. Erk2 binds to specific DNA sequence motifs typically accessed by Jarid2 and PRC2. Negating Erk1/2 activation leads to increased nucleosome occupancy and decreased occupancy of PRC2 and poised RNAPII at Erk2-PRC2-targeted developmental genes. Surprisingly, Erk2-PRC2-targeted genes are specifically devoid of TFIIH, known to phosphorylate RNA polymerase II (RNAPII) at serine-5, giving rise to its initiated form. Erk2 interacts with and phosphorylates RNAPII at its serine 5 residue, which is consistent with the presence of poised RNAPII as a function of Erk1/2 activation. These findings underscore a key role for Erk1/2 activation in promoting the primed status of developmental genes in mouse ES cells and suggest that the transcription complex at developmental genes is different than the complexes formed at other genes, offering alternative pathways of regulation (Tee, 2014).

Cdk9 is an essential kinase in Drosophila that is required for heat shock gene expression, histone methylation and elongation factor recruitment

Phosphorylation of the large RNA Polymerase II subunit C-terminal domain (CTD) is believed to be important in promoter clearance and for recruiting protein factors that function in messenger RNA synthesis and processing. P-TEFb is a protein kinase that targets the (CTD). The goal of this study was to identify chromatin modifications and associations that require P-TEFb activity in vivo. The catalytic subunit of P-TEFb, Cdk9, was knocked down in Drosophila using RNA interference. Cdk9 knockdown flies die during metamorphosis. Phosphorylation at serine 2 and serine 5 of the CTD heptad repeat were both dramatically reduced in knockdown larvae. Hsp 70 mRNA induction by heat shock was attenuated in Cdk9 knockdown larvae. Both mono- and trimethylation of histone H3 at lysine 4 were dramatically reduced, suggesting a link between CTD phosphorylation and histone methylation in transcribed chromatin in vivo. Levels of the chromo helicase protein CHD1 were reduced in Cdk9 knockdown chromosomes, suggesting that CHD1 is targeted to chromosomes through P-TEFb-dependent histone methylation. Dimethylation of histone H3 at lysine 36 was significantly reduced in knockdown larvae, implicating CTD phosphorylation in the regulation of this chromatin modification. Binding of the RNA Polymerase II elongation factor ELL was reduced in knockdown chromosomes, suggesting that ELL is recruited to active polymerase via CTD phosphorylation (Eissenberg, 2007).

Cdk9, the catalytic subunit of P-TEFb, is highly conserved among eukaryotes. The yeast kinases Ctk1 and Bur1 are both homologs of Cdk9, and both are CTD kinases in Drosophila, although loss of Bur1 has no effect on CTD phosphorylation yeast. Bur1 is essential but Ctk1 is not (Eissenberg, 2007).

RNAi knockdown of Cdk9 in transgenic flies results in lethality at the pupal stage. This is considerably later than the embryonic lethality reported for C. elegans RNAi knockdown of Cdk9. While this difference could reflect differences in the requirements for Cdk9 in these organisms, it is more likely that differences in timing or efficiency of RNAi, Cdk9 protein turnover and/or maternal Cdk9 loading accounts for the much later lethality in knockdown flies. Nevertheless, these results confirm and extend the finding that P-TEFb is essential in metazoan development (Eissenberg, 2007).

In contrast, Cdk9 homologs in fission yeast and Neurospora are not essential. Since CTD phosphorylation has been linked to promoter clearance, pre-mRNA processing and chromatin modification, it is not possible to say what aspect of P-TEFb activity is essential in metazoa. RNAi knockdown of the Drosophila Cdk9 in cultured cells causes arrest of the cell cycle at the G1-S transition, implicating this kinase in cell cycle control. It is unlikely that cell cycle arrest is causing the lethality in knockdown flies, since cell cycle mutations in Drosophila generally are associated with reduced or missing imaginal discs, and the discs in Cdk9 knockdown larvae appear overtly normal. The finding that Hsp70 transcripts are reduced in Cdk9 knockdown larvae is consistent with the reduced Hsp70 transcription previously reported in Cdk9 RNAi cultured cells. Hsp 70 is not essential in Drosophila, but the effects on Hsp70 suggest that defects in gene expression could underlie the essential requirement for Cdk9 in Drosophila development (Eissenberg, 2007).

Cdk9 knock-down flies show dramatic reductions in both serine 2 and serine 5 phosphorylation. In contrast, flavopiridol treatment of cultured cells has been found to selectively reduce serine 2 phosphorylation. The significance of this difference is unclear, but could reflect differences in experimental protocol. For example, flavopiridol treatments were limited to 15-20 min, while RNAi knockdown third instar larvae are subject to knockdown conditions for several days before assay. Longer periods of Cdk9 inactivation may be required for reduction in serine 5 phosphorylation. Alternatively, it is possible that knockdown of Cdk9 protein levels results in inhibition of TFIIH, the other known CTD kinase. Regardless of the mechanism, the RNAi knockdown clearly results in reduced phosphorylation of the CTD, enabling a test of the consequences of loss of CTD phosphorylation on chromatin modification and recruitment of RNA Polymerase II-associated factors (Eissenberg, 2007).

Loss of CTD phosphorylation in Cdk9 knockdown larvae is associated with reduced binding of the RNA Polymerase II elongation factor ELL genome-wide. ELL is broadly co-localized with phosphorylated RNA Polymerase II on polytene chromosomes, and is rapidly recruited to heat shock loci after a brief heat shock. These results suggest that the efficient recruitment of ELL to transcribed loci requires CTD phosphorylation. Whether this reflects a direct interaction of ELL with the CTD is unknown (Eissenberg, 2007).

Despite the fact that Elongin A affects the same kinetic parameter in RNA Polymerase II catalysis as ELL, Elongin A binding is not reduced by loss of CTD phosphorylation. As with ELL, the nature of Elongin A binding to RNA Polymerase II is unknown, but these observations suggest their binding can be distinguished by sensitivity to the phosphorylation state of the CTD. Since no increase of Elongin A was observed under conditions of reduced ELL binding, it seems unlikely that ELL and Elongin A compete for RNA Polymerase II binding (Eissenberg, 2007).

Spt4 and Spt5 are subunits of DSIF, which is implicated in the regulation of RNA Polymerase II elongation. Previous work suggested that reduced serine 2 phosphorylation of the RNA Polymerase II CTD has no effect on Spt5 recruitment to a heat shock gene in cultured cells (Ni, 2004). In Cdk9 knockdown flies, in which both serine 2 and 5 phosphorylation are reduced, the chromosomal distribution of Spt5 is unchanged genome-wide. This is consistent with previous reports that Spt5 interacts with both phosphorylated and unphosphorylated RNA Polymerase II (Wen, 1999; Lindstrom, 2001; Lindstrom, 2003; Eissenberg, 2007 and references therein).

The chromo domain motif is a binding site for methylated histone tails. The role of the CHD1 chromo domain in methylated histone binding is controversial. However, recent structural data determined that the double chromo domain of mammalian CHD1 binds methylated H3K4 in vitro (Flanagan, 2005). This study shows that Cdk9 knockdown leads to a loss of chromosomal CHD1. This observation is most easily interpreted as the result of loss of H3K4 methylation that also occurs in Cdk9 knockdown chromosomes. Thus, the finding reported in this study lends support to the in vitro binding data and strongly suggests that the chromo domain-methylated histone interaction plays a dominant role in targeting CHD1 to active chromatin in vivo (Eissenberg, 2007).

The observation that both H3K4 and H3K36 methylation are significantly reduced in Cdk9 knockdown chromosomes suggests a linkage between phosphorylation of the CTD and histone methylation at transcribed genes. In this respect, Cdk9 subsumes activities found in yeast Bur1/Bur2 and yeast Ctk1. Since no significant difference was observed in ASH1 protein levels on Cdk9 knockdown chromosomes, a model is favored in which Cdk9-dependent RNA Polymerase II elongation plays a mechanistic role in H3 tail methylation. In this model, RNA Polymerase II passage destabilizes histone-DNA contacts, making the histones better substrates for efficient methylation. Reduced CTD phosphorylation would lead to reduced rates of RNA Polymerase II transcription genome-wide, resulting in reduced efficiency of histone tail methylation. While the mechanism connecting CTD phosphorylation to RNA Polymerase II elongation rate is likely to be complex in vivo, the observation that reduced CTD phosphorylation is associated with reduced dELL binding suggests that loss of dELL association could be a contributing factor (Eissenberg, 2007).

Mutation in Ash1 in Drosophila results in loss of all detectable H3K4 methylation, but has no effect on H3K36 methylation. This is consistent with independent mechanisms for these two chromatin modifications. A Polymerase II passage model provides a simple mechanism to account for similar effects on both modifications based on substrate availability (Eissenberg, 2007).

P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo

Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. This study demonstrates that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered (Ni, 2008).

This study has found that P-TEFb inhibition leads to a rapid depletion of elongating Pol II from the Hsp70 gene. Furthermore, the majority of polymerases remaining on the gene are restricted to the 5' end and are transcriptionally engaged. Together, these data indicate that P-TEFb is critical for the escape of Pol II into productive elongation in vivo. It was also found that upon P-TEFb inhibition, levels of all elongation and RNA-processing factors so far tested (P-TEFb, Spt5, TFIIS, Spt6, and CstF50) were dramatically reduced, with kinetics indistinguishable from the depletion of elongating Pol II. Therefore, at least a subset of transcription factors appear to depend on the continual presence of elongating Pol II for their association with chromatin (Ni, 2008).

The dependence of P-TEFb, Spt5, TFIIS, and CstF50 on Pol II for association with the Hsp70 gene was not surprising. P-TEFb, Spt5, and TFIIS all interact with Pol II and track with Pol II during activated transcription. CstF50 physically interacts with the CTD of Pol II, and CTD Ser2 phosphorylation is required for the association of cleavage and polyadenylation factors in S. cerevisiae. However, Spt6 was not necessarily expected to require Pol II elongation for its association with the gene. Spt6 directly interacts with histones and separates somewhat from Pol II on Hsp70: Spt6 does not colocalize with promoter-proximal Pol II but does colocalize with Pol II on the body of the gene. It is possible that Spt6 interacts cooperatively with Pol II and nucleosomes or that Spt6 interacts only with the productively elongating, Ser2-phosphorylated form of Pol II. Consistent with the latter possibility, Spt6 was recently shown to interact directly with Ser2-phosphorylated Pol II (Yoh, 2006). Interestingly, however, the interaction of Spt6 with Pol II is not required for the positive elongation activity of Spt6 in vivo. Future studies using live-cell-imaging techniques should shed light on the dynamics and mechanism of Spt6 recruitment to chromatin during transcription (Ni, 2008).

Evidence has also been obtained for the stable association of promoter-proximally stalled polymerases with the Hsp70 gene. Following a short treatment with Flavopiridol (FP), a potent and highly specific inhibitor of P-TEFb kinase activity treatment, the only transcriptionally engaged polymerases remaining on the gene were promoter proximal and the usual rapid recovery of fluorescence after photobleaching of GFP-tagged polymerase was abolished. This observation may also apply to the promoter-proximally paused polymerase on Hsp70 in uninduced cells and in the absence of FP and argues against rapid cycles of initiation, pausing, and premature termination. In further support of a stable association, the two main protein complexes believed to promote promoter-proximal pausing, DSIF and negative elongation factor, repress Pol II elongation in vitro but do not induce premature termination. Furthermore, the Spt5 subunit of DSIF was shown to cooperate with the Tat activator in preventing premature RNA dissociation from Pol II in an in vitro transcription assay. Both Spt5 and low levels of the activator HSF are present at the Hsp70 gene under noninducing conditions. It is considered unlikely, but it cannot be ruled out, that the FP prevents polymerases from prematurely terminating. It is extremely unlikely that FP prevents new polymerases from initiating, since FP is a kinase inhibitor and initiating polymerases are most favorably unphosphorylated. The definitive test of the status of paused Pol II in untreated cells awaits additional technological advances that allow rapid mapping of the paused Pol II associated with uninduced, unpuffed loci (Ni, 2008).

Consistent with previous analyses, this study has found that active P-TEFb kinase activity is not required for Pol II that is already productively elongating to continue to do so: polymerases already elongating at the time of P-TEFb inhibition cleared from the middle and downstream regions of the gene. Furthermore, these elongating polymerases still had substantial levels of Ser2 phosphorylation after FP addition, indicating that there was minimal Ser2 phosphatase activity upstream of the polyadenylation site (Ni, 2008).

In the course of this study, it was also found that Pol II levels across Hsp70 recovered with time after FP treatment. This discovery explains why only a small reduction in Pol II density on heat shock genes was detected after 20 min of FP treatment in previous work (Ni, 2004). In that work, it was concluded that the major defect in Hsp70 expression following P-TEFb inhibition was at the level of 3'-end processing of RNA. This study now shows that there is a Pol II elongation defect immediately following P-TEFb inhibition. While the persistent mRNA-processing defects likely account for a significant reduction in Hsp70 mRNA levels following a long FP treatment, the initial elongation defect demonstrated in this study may also contribute to the low mRNA levels. Previous work, in addition to demonstrating that severe mRNA-processing defects exist following P-TEFb inhibition, also showed that in the recovered phase, Ser2 phosphorylation levels are still low. Therefore, elongation and RNA processing may require different extents or patterns of phosphorylation of the CTD or other targets of P-TEFb (Ni, 2008).

There are several possible mechanisms for the recovery of Pol II levels after FP treatment. The FP may become inactivated with time, metabolized, or expelled from the cell. Alternatively, an FP-resistant kinase may compensate for reduced P-TEFb kinase activity. It is also possible that a very small residual activity of P-TEFb persists in the presence of FP and eventually enables recovery. Another intriguing possibility is that cellular levels of active P-TEFb are increased in response to FP treatment. In mammalian cells, two P-TEFb complexes exist: a large, inactive complex and a small, active complex. The treatment of mammalian cells with P-TEFb inhibitors promotes the release of P-TEFb from the large complex to increase the pool of active P-TEFb. If this same mechanism exists in Drosophila, following FP treatment, P-TEFb may be released from the complex that sequesters it in an inactive form to create a larger population of active P-TEFb molecules. Consistent with this possibility, P-TEFb levels on the 5' end of Hsp70 recovered after 3 min of FP treatment. The recovery in Pol II elongation is not yet seen at a 10-min time point, and so the recovery of P-TEFb levels at this time may indicate the beginning of the rescue of Pol II elongation ability, which then occurs gradually over the course of the next 20 min. While support for this explanation exists, a combination of the above-described possibilities contributing to the recovery of Pol II elongation ability with time after FP treatment cannot be ruled out (Ni, 2008).

The data presented in this study also show that, at least for some drugs, it is important to examine cells during very early periods of treatment to observe the immediate effect of the drug. The short FP treatments used in this study now provide evidence that P-TEFb is indeed required for the escape of Pol II into productive elongation at the Hsp70 gene. Other cases in which an elongation defect is not apparent in the presence of P-TEFb inhibitors may be so explained, or promoter-proximal pausing may not be a regulatory feature of the genes in question. In budding yeast, in which regulated promoter-proximal pausing is absent, the deletion of the Pol II CTD Ser2 kinase Ctk1 does not affect transcription elongation (Ni, 2008).

Regulation of the transcriptional activity of poised RNA polymerase II by the elongation factor ELL

Many developmentally regulated genes contain a poised RNA polymerase II (Pol II) at their promoters under conditions where full-length transcripts are undetectable. It has been proposed that the transcriptional activity of such promoters is regulated at the elongation stage of Pol II transcription. In Drosophila, the heat-shock loci expressing the Hsp70 genes have been used as a model for the regulation of the transcriptional activity of poised Pol II. Drosophila ELL (dELL) is a Pol II elongation factor capable of stimulating the rate of transcription both in vivo and in vitro. Although ELL and the elongation factor Elongin A have indistinguishable effects on RNA polymerase in vitro, the loss-of-function studies indicate that these proteins are not redundant in vivo. This study used RNAi to investigate the physiological properties of dELL and a dELL-associated factor (dEaf) in a living organism. Both ELL and Eaf are essential for fly development. dELL is recruited to heat shock loci upon induction, and its presence with Pol II at such loci is required for proper heat-shock gene expression. Consistent with a role in elongation, dELL knockdown reduces the levels of phosphorylated Pol II at heat-shock loci. This study implicates dELL in the expression of loci regulated by Pol II elongation (Smith, 2008).

Efficient transcription by RNA polymerase II (Pol II) is an intricate process that requires multiple contacts with the DNA template and nascent RNA that inevitably leads to frequent stalling during the transcription of a gene. The average rate of transcription by Pol II in vivo is an order of magnitude higher than that obtained in vitro despite additional impediments, such as traversing through nucleosomes. Using biochemical approaches, two Pol II elongation factors, Eleven nineteen lysine-rich leukemia (ELL) and Elongin A, were isolated from cell extracts as factors capable of stimulating Pol II activity by suppressing transient pausing. Despite similar in vitro activities, the Drosophila orthologs of ELL and Elongin A are each essential for development. This observation indicates that their in vivo activity is not redundant (Smith, 2008).

Recent genome-wide studies have found a large number of developmentally regulated genes that contain a paused Pol II at their promoters. Therefore, it has been proposed that the transcriptional activity of such poised Pol IIs is regulated at the level of transcription elongation. The classic model for studying genes regulated by promoter-proximal paused polymerase is Hsp70 gene induction in Drosophila. Previous studies have shown that several Pol II elongation factors are rapidly recruited to the Hsp70 genes after heat shock. Although much work has been done on the role of these factors in gene regulation in cultured cells, less is known about the role of these factors in the regulation of heat-shock gene expression in the whole organism. Although there are several mutants in the gene encoding Drosophila ELL (dELL), all of these alleles are embryonic lethal. Therefore, it was possible to use these alleles to further characterize the role of the elongation factor ELL in the regulation of the transcriptional activity of poised Pol II and Hsp70 loci. To test the role for dELL in gene expression, RNAi was used to reduce expression levels of both dELL and dELL-associated factor (dEaf) expression levels during development, and the in vivo effect of their reduction on transcription and development was examined. It was found that knockdown of dELL and dEaf results in lethality. Furthermore, knockdown of these elongation factors results in reduced Hsp70 transcript accumulation after heat shock. Immunolocalization of phosphorylated Pol II in heat-shocked dELL knockdown salivary glands demonstrates reduced levels of the elongating form of Pol II at the Hsp70 loci in the absence of dELL. These studies demonstrate that dELL is essential for full induction of heat-shock gene expression and are consistent with a role for dELL in Pol II elongation. These findings provide a role for an RNA Pol II elongation factor in the transcriptional regulation of poised Pol II (Smith, 2008).

dELL has been shown to be essential; homozygous mutant clones do not survive in the eye and homozygotes for loss-of-function alleles die at the end of embryogenesis or in early first instar. To investigate the role of dELL in transcription in flies, dELL was knocked down by RNAi, which typically reduces, but does not eliminate, the targeted gene products. A 600-bp portion of the dELL coding region was inserted into a P-element vector that drives the expression of dsRNA through two convergent Gal4 UAS promoters that flank the insert. Several transgenic lines were generated and tested for effects on viability by crossing to an Actin5C-Gal4 driver line that expresses yeast Gal4 under the cytoplasmic actin promoter. All eight dELL RNAi lines show significant loss of viability when expressed under this driver. When adult escapers were obtained, very few males were observed, indicating that males are more susceptible to loss of dELL. Greater numbers of females than males were observed at the third instar larval stage, indicating that males are dying earlier than females. A significant genome-wide reduction of dELL protein is observed by immunofluorescence analysis of dELL RNAi larval polytene chromosomes (Smith, 2008).

Through two-hybrid analysis, two interacting partners of ELL have been characterized in humans, Eaf1 and Eaf2. Eaf1 and Eaf2 are highly related and can stimulate the elongation activity of ELL in vitro. Recently, the association of Eaf with ELL was shown to be evolutionarily conserved, with the finding that Schizosaccharomyces pombe homologs SpEaf and SpELL directly interact with each other. Additionally, SpEaf enhances the stimulation by SpELL of Pol II transcription in vitro. Because Drosophila also has a single Eaf homolog, RNAi was used to knock down dEaf levels and assessed the viability of dEaf-knockdown flies in six different transgenic RNAi lines. In all lines, significant reductions were observed in the number of adult progeny of RNAi-expressing flies compared with control siblings. In addition, a consistent reduction in the male-female sex ratio was observed for dEaf RNAi, suggesting that the male-enhanced lethal phenotype (not observed for other elongation factors) is due to loss of a dELL-dEaf complex (Smith, 2008).

To test for the effectiveness of the RNAi knockdowns, dELL and dEaf mRNA levels were measured in knockdown larvae and their control siblings. Significant reductions in dELL transcripts are observed in the dELL RNAi larvae. dELL transcripts, as measured by RT-PCR, are not reduced by RNAi to the same level as dELL protein, as assessed by immunofluorescence on polytene chromosomes. Previously, it was observed that knockdown of dRTF1 by RNAi was more effective at the protein than the RNA levels presumably because the long dsRNAs produced are processed as miRNAs and interfere with translation. Because dELL is nested in an intron of the gene encoding the chromatin remodeling enzyme dMi-2, transcript levels for this gene were measured and no reduction was found of dMi-2 RNA in dELL RNAi larvae. Additionally, it was found that dEaf RNA levels are reduced in dEaf RNAi larvae. Interestingly, a significant increase in dELL levels is observed in dEaf RNAi larvae, possibly compensating for the lower dEaf levels (Smith, 2008).

dELL was previously shown to be recruited to heat-shock genes upon heat shock. To determine whether dELL is required for heat-shock gene expression, the levels of Hsp70 transcripts after heat shock were compared in dELL knockdown larvae and their control siblings. By immunofluorescence analysis, little or no dELL is seen at the Hsp70 gene after heat shock in dELL knockdown larvae, whereas the control siblings without the Gal4 driver showed the expected recruitment of dELL to the Hsp70 gene. Northern blot analysis showed reduced levels of Hsp70 mRNA levels in the dELL RNAi larvae. A similar analysis was done with dEaf RNAi larvae, and reduced Hsp70 mRNA also occurs after heat shock, although the deficit was less than observed for the dELL RNAi larvae. Similar results were observed when Hsp70 levels were measured by RT-PCR, showing greater reductions in Hsp70 RNA levels in dELL RNAi than dEaf RNAi larvae (Smith, 2008).

Chromosomal levels of dELL are markedly reduced in the absence of Cdk9, the catalytic subunit of the Pol II C-terminal domain (CTD) kinase PTEF-B. To determine whether dELL knockdown affects the recruitment of Pol II to the Hsp70 genes, dELL knockdown and control polytene chromosomes were probed with antibodies to the Ser-2-phosphorylated, elongating form of Pol II. Lower levels of Ser-2-phosphorylated Pol II were consistently observed at the Hsp70 heat-shock loci in dELL-knockdown larvae, suggesting a close link between dELL function and phosphorylation of the Pol II CTD (Smith, 2008).

ELL belongs to a class of transcription elongation factors that have been shown to stimulate the K_m and/or V_max of RNA Pol II in vitro by alleviating pausing on a purified DNA template. Another member of this class is Elongin A and its Drosophila ortholog dEloA. From the present and previous studies, it is clear that both dELL and dEloA localize to the Hsp70 gene upon heat shock, and each is required for full levels of heat-shock gene expression, suggesting that the in vivo roles of these elongation factors in Hsp70 gene transcription are not redundant. Similarly, it was observed that the knockdown phenotypes of these two proteins can be unique, such as the enhanced male lethality in dELL RNAi larvae. How could both elongation factors be redundant in vitro, yet nonredundant in vivo? The in vitro studies were performed on naked DNA templates, whereas the chromatin environment of RNA Pol II-transcribed genes can provide additional challenges to the polymerase. Each of these elongation factors has its own interaction partners and may be recruited to distinct states of the polymerase, such as initiating, elongating, or stalled polymerase. Consistent with this view, knockdown of dELL, but not dEloA, results in decreased levels of Ser-2-phosphorylated Pol II at the Hsp70 and other loci. Interestingly, the chromosomal targeting of dELL, but not dEloA, is dramatically reduced by the knockdown of CDK9, the Pol II CTD kinase, suggesting that dELL and dEloA are recruited to genes by distinct mechanisms. Fine mapping of dELL and dEloA on the well characterized Hsp70 gene at different time points after activation could clarify the distinct roles for these enzymes (Smith, 2008).

The lesser effect of dEaf knockdown on Hsp70 gene induction could be indicative of a requirement of dEaf for optimal function of dELL, whereas dELL can partially function without dEaf. Indeed, in vitro transcription studies have demonstrated that human Eaf proteins, in combination with ELL, stimulate transcription elongation by Pol II above the levels obtained with ELL alone. In dEaf RNAi larvae, it was observed that dELL levels are increased, conceivably as a cellular response to increased pausing resulting from lower dEaf levels (Smith, 2008).

Previous work on the function of dELL made use of alleles of the Su(Tpl) locus, which encodes dELL. All known Su(Tpl) alleles are embryonic lethal. In contrast, RNAi of dELL allows survival to the larval or adult stages depending on the insertion line of the dsRNA construct. Interestingly, the few 'escaper' dELL RNAi adults are overwhelmingly female. As seen with the heat-shock defect, the difference in male and female viability is less in dEaf RNAi flies than in dELL RNAi flies, consistent with dEaf enhancing, but not being absolutely required for, dELL function. A previous study showed that males express much higher levels of a dELL transcript than females, although the functional significance of this difference has not been investigated. One hypothesis is that dELL is needed in males as part of the process of X chromosome dosage compensation; Drosophila dosage compensation factors are thought to enhance transcription elongation of X-linked genes in males, and loss of any of these factors leads to male-specific lethality. In addition, reduced levels of several global chromatin regulators, including the supercoiling factor, Jil-1 H3 kinase, heterochromatin protein HP1, and the chromatin remodeler ISWI, have been reported to differentially affect the survival of males and/or the morphology of the X chromosome. However, in dELL knockdowns, MSL localization and the male polytene X chromosome morphology appears similar in dELL knockdown male larvae and their control brothers. Whether there are specific defects in dosage compensation of X-linked genes may be an interesting avenue for future investigations. Alternative explanations for a male-enhanced lethality also should be considered. For example, Drosophila males differ from females not just in having one less X chromosome, but also in carrying a Y chromosome, which comprises ~12% of the male genome. A number of genes are male-lethal due to the presence of the mostly heterochromatic Y chromosome, including modulators of position effect variegation, such as the Su(var)3-3 gene that encodes the histone demethylase LSD1, the uncharacterized Su(var)2-1, as well as the HP1-interacting protein Bonus (dTIF1), an enhancer and suppressor of position-effect variegation. For Su(var)2-1 and Bonus, the Y-lethal effect is not Y-specific but can be phenocopied by other sources of heterochromatin. A role for dELL in the regulation of heterochromatin is unknown but could conceivably be required for the expression of heterochromatin components (Smith, 2008).

CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription

CTCF is a versatile transcription factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of elongation by RNA polymerase (pol) II. This study shows that CTCF knockdown abrogates pol II pausing at the early elongation checkpoint of c-myc by affecting recruitment of DRB-sensitivity-inducing factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of negative elongation factor (NELF). In addition, CTCF is required for recruitment of positive elongation factor b (P-TEFb), which phosphorylates NELF, DSIF and Ser2 of the pol II CTD to activate elongation of transcription of c-myc and recognition of the snRNA gene-specific 3' box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling pol II transcription through the elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes (Laitem, 2015).

Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II

Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L) is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II). Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, this study characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although significant overlap was observed between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L does not bind methylated histone tails in vitro, and loss of TRX or ASH1 function does not alter the association of KIS-L with chromatin. By contrast, loss of kis function leads to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and is accompanied by increased histone H3 lysine 27 methylation - a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. These findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin (Srinivasan, 2008).

NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila

Transcriptional elongation regulators NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF (FlyBase term: Spt5) from a Drosophila nuclear extract reduces the level of polymerase that pauses in the promoter proximal region of hsp70. Depletion of one Negative elongation factor E (NELF) subunit in salivary glands using RNA interference also reduces the level of paused polymerase. In vivo protein-DNA cross-linking shows that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborates the cross-linking result and shows that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF are strongly recruited to chromosomal puffs harboring the hsp70 genes. It is proposed that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation (Wu, 2003).

It is proposed that promoter proximal pausing occurs when the nascent transcript emerges from the RNA exit channel of the Pol II and is grabbed by the NELF-E subunit. Tethering of the NELF-E to the elongation complex would generate a rigid body that could restrict the movement of the Pol IIa. This model is supported by several observations. The paused polymerase is in the Pol IIa state, and NELF and DSIF only inhibit elongation by Pol IIa. In vitro transcription analysis indicates that the elongation complex is not receptive to inhibition by NELF and DSIF until the nascent transcript is ~30 nucleotides long. This length coincides approximately to the distance polymerase elongates on hsp70 before it pauses. In vitro transcription analyses indicate that DSIF and NELF associate with polymerase shortly after initiation but probably before the polymerase reaches the region of pausing. Finally, NELF-E has an RNA-binding motif that is essential for its inhibitory action (Wu, 2003 and references therein).

Although NELF and DSIF are sufficient to slow the elongation rate of purified Pol IIa, it is suspected that additional proteins are involved in stably pausing Pol II on the hsp70 promoter. In cell-free transcription reactions done with other promoters, the pausing caused by DSIF and NELF appears to be transient -- the polymerase eventually moves forward if given enough time. In contrast, several observations indicate that the Pol II on hsp70 is stably paused. The paused Pol II remains associated with the hsp70 promoter when nuclei are isolated from uninduced cells, and sarkosyl or high salt must accompany addition of nucleotides to cause the Pol II to resume elongation. In a cell-free system, Pol II remains stably paused on the hsp70 promoter for at least 30 min. GAGA factor might be involved in stabilizing the pause because mutations in the GAGA element result in a loss of paused Pol II (Wu, 2003).

Heat shock rapidly induces transcription as a result of the association of HSF with sites located upstream from the TATA element. The data suggest that HSF may activate transcription in part by causing NELF to dissociate from the Pol II. How HSF might cause the release of NELF is unclear. Phosphorylation of Pol IIa is likely to be an important step because the Pol II found in the body of the gene during heat shock is hyperphosphorylated. Phosphorylation of DSIF is another possibility as this has been observed to occur early in elongation in vitro. It is also unclear which kinase might be responsible for phosphorylating the Pol II. P-TEFb (see Cdk9) is a candidate because it associates with the hsp70 gene during heat shock induction, and HSF can be bypassed by directing a Gal4/P-TEFb fusion protein to the hsp70 promoter. No interaction, however, has been detected between P-TEFb and HSF. Recent results show that HSF associates with the mediator. Drosophila mediator contains a kinase that phosphorylates the CTD, and phosphorylation can occur synergistically with the TFIIH kinase. Perhaps HSF recruits the mediator and in turn the mediator releases the paused polymerase by phosphorylating the CTD (Wu, 2003).

The strong immunofluorescence staining observed for DSIF at heat shock loci during heat shock indicates that DSIF is associated with many of the polymerase molecules transcribing the gene. RNA polymerase initiates at a rate of once every few seconds during heat shock resulting in a train of elongation complexes traversing the gene. In the absence of NELF, DSIF might act as a positive elongation factor. Shortly after DSIF was discovered, another investigation identified DSIF as a cofactor required for reconstituting tat-dependent transcription. In this situation, DSIF appears to be stimulating elongation. DSIF has been found in a complex with another positive elongation factor called Tat-SF1. Tat-SF1 was first identified as a stimulatory factor for Tat, but subsequent results indicate that Tat-SF1 may promote elongation on cellular genes. In yeast, DSIF appears to act as either a positive or negative regulator of elongation depending on circumstances. A hypothesis that unites the positive and negative activities of DSIF considers this factor an adaptor that connects other modulators to the elongation complex. In this regard, DSIF has been shown to bind on its own to Pol II, whereas the stable association of NELF with Pol II requires the presence of DSIF (Wu, 2003 and references therein).

NELF and DSIF appear to associate with several hundred interbands in polytene chromosomes. Each interband could contain many genes. The weak staining of interbands by Hoecsht suggests that the DNA in the interbands is in a decondensed state. Residing in these decondensed regions could be genes whose primary control mechanism does not involve a disruption of chromatin structure or even assembly of the initiation complex. Instead, alleviating repression by NELF and DSIF could underlie the mechanism of activation (Wu, 2003).

How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes

Interactions between RNA guanylyltransferase (GTase) and the C-terminal domain (CTD) repeats of RNA polymerase II and elongation factor Spt5 are thought to orchestrate cotranscriptional capping of nascent mRNAs. The crystal structure of a fission yeast GTase*Pol2 CTD complex reveals a unique docking site on the nucleotidyl transferase domain for an 8-amino-acid Pol2 CTD segment, S5PPSYSPTS5P, bracketed by two Ser5-PO4 marks. Analysis of GTase mutations that disrupt the Pol2 CTD interface shows that at least one of the two Ser5-PO4-binding sites is required for cell viability and that each site is important for cell growth at 37 degrees C. Fission yeast GTase binds the Spt5 CTD at a separate docking site in the OB-fold domain that captures the Trp4 residue of the Spt5 nonapeptide repeat T(1)PAW(4)NSGSK. A disruptive mutation in the Spt5 CTD-binding site of GTase is synthetically lethal with mutations in the Pol2 CTD-binding site, signifying that the Spt5 and Pol2 CTDs cooperate to recruit capping enzyme in vivo. CTD phosphorylation has opposite effects on the interaction of GTase with Pol2 (Ser5-PO4 is required for binding) versus Spt5 (Thr1-PO4 inhibits binding). It is proposed that the state of Thr1 phosphorylation comprises a binary 'Spt5 CTD code' that is read by capping enzyme independent of and parallel to its response to the state of the Pol2 CTD (Doamekpor, 2014).

Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II

The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is an essential component of transcriptional regulation and RNA processing of protein-coding genes. A large body of data also implicates the CTD in the transcription and processing of RNAPII-mediated small nuclear RNAs (snRNAs). However, the identity of the complex (or complexes) that associates with the CTD and mediates the processing of snRNAs has remained elusive. Here, we describe an RNA polymerase II complex that contains at least 12 novel subunits, termed the Integrator, in addition to core RNAPII subunits. Two of the Integrator subunits display similarities to the subunits of the cleavage and polyadenylation specificity factor (CPSF) complex. Integrator is shown to be recruited to the U1 and U2 snRNA genes and mediates the snRNAs' 3' end processing. The Integrator complex is evolutionarily conserved in metazoans and directly interacts with the C-terminal domain of the RNA polymerase II largest subunit (Baillat, 2005).

Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS

Uninduced heat shock genes are poised for rapid activation, with RNA polymerase II (Pol II) transcriptionally engaged, but paused or stalled, within the promoter-proximal region. Upon heat shock, this Pol II is promptly released from the promoter region and additional Pol II and transcription factors are robustly recruited to the gene. Regulation of the heat shock response relies upon factors that modify the efficiency of elongation through the initially transcribed sequence. This study reports that Pol II is susceptible to transcription arrest within the promoter-proximal region of Drosophila hsp70 and that transcript cleavage factor TFIIS is essential for rapid induction of hsp70 RNA. Moreover, using a tandem RNAi-ChIP assay, it was discovered that TFIIS is not required to establish the stalled Pol II, but that TFIIS is critical for efficient release of Pol II from the hsp70 promoter region and the subsequent recruitment of additional Pol II upon heat induction (Adelman, 2005; full text of article).

In a search for elongation factors that directly affect the heat shock response, a role for the transcript cleavage factor TFIIS was investigated. Like the bacterial Gre factors, TFIIS rescues RNA polymerase that has undergone reverse translocation, or 'backtracking' along the DNA template. Backward movement misaligns the 3' end of the nascent RNA with the RNA polymerase active site, thereby prohibiting continued RNA synthesis. Transcript cleavage factors restart the arrested RNA polymerase by inducing internal cleavage of the RNA by the polymerase active site, creating a new 3' end that is properly aligned for catalysis. The activity of transcript cleavage factors has been reported to stimulate promoter escape and transcription elongation and to decrease pausing. Recently published structural and functional analyses of transcript cleavage factors GreB and TFIIS complexed with their respective RNA polymerases elucidate the mechanism of this activity: TFIIS inserts a long coiled-coil domain into the RNA polymerase secondary channel, helping to coordinate a Mg+2 ion required for the reverse-catalytic reaction. However, although the detailed mechanism of TFIIS activity is known, the in vivo roles for this activity remain poorly defined (Adelman, 2005 and references therein).

To test whether Pol II complexes stalled within the promoter-proximal region were inactive due to transcription arrest, whether they could be rescued by transcript cleavage factor TFIIS was investigated. Stalled early elongation complexes (EEC) formed in a partially fractionated embryo extract lacking TFIIS were isolated and washed before restarting transcription in the presence or absence of purified TFIIS. The data show that the addition of NTPs leads to little or no transcription elongation in the absence of TFIIS. The presence of purified TFIIS alone induced efficient cleavage of RNA products associated with stalled Pol II. The sensitivity of specific RNAs to TFIIS-dependent cleavage signifies that these RNA species are associated with backtracked, arrested Pol II complexes. Cleavage of these RNAs in the presence of TFIIS reactivates the stalled complexes, allowing the labeled RNA species to be elongated upon addition of NTPs. It is concluded that TFIIS-induced cleavage rescues the promoter-proximal stalled, arrested Pol II (Adelman, 2005).

Taken together, these data suggest that intrinsic pause sites within the promoter-proximal region of hsp70 are recognized in vitro, perhaps with the aid of regulatory elongation factors, and that Pol II at these locations rapidly become inactive. However, the experiments demonstrating transcription arrest involve EEC that were artificially stalled and stringently washed prior to analysis, which does not accurately reflect the dynamics of hsp70 transcription. Thus, to investigate whether Pol II actively transcribing through the promoter-proximal region is susceptible to arrest and to determine the role of TFIIS in this process, a transcription assay was performed in a fractionated Drosophila embryo extract that lacked TFIIS (Adelman, 2005).

EEC were radiolabeled during elongation to position +16 nt and washed thoroughly with transcription buffer plus heparin to remove unincorporated NTPs and unbound extract proteins and to prevent reinitiation. The resulting EEC were split into two equivalent reactions, one of which was supplemented with purified TFIIS. Unlabeled NTPs were added to restart transcription, and aliquots were removed various time points. In the absence of TFIIS, Pol II accumulated in the promoter-proximal region and was not able to escape from sites of stalling during the time course. In contrast, inactive Pol II complexes were barely detectable in the presence of TFIIS. Instead, TFIIS stimulated rapid and efficient elongation of the labeled +16 nt RNA through the promoter-proximal region, leading to the formation of increased levels of full-length transcript. TFIIS activity also generated cleavage products that were released from Pol II. These data indicate that the initially transcribed sequence of hsp70 contains intrinsic sites at which Pol II pauses or stalls during active transcription, and that TFIIS is critical for efficient elongation through this region (Adelman, 2005).

To verify the functional relevance of TFIIS in the heat shock response in vivo, TFIIS levels were depleted in Drosophila S2 cells using RNAi. S2 cells that were untreated or treated with dsRNA targeting TFIIS were heat shocked to induce production of hsp70 RNA before harvesting cells and isolating total RNA. The depletion of TFIIS was not complete, perhaps due to the abundance or low turnover of the TFIIS protein; nonetheless, TFIIS-depleted cells were estimated to contain only ~10% of normal levels of TFIIS (Adelman, 2005).

Analysis of hsp70 RNA levels by quantitative RT-PCR reveals that TFIIS-depleted cells are indeed deficient in the heat shock response. In particular, there is a dramatic delay in hsp70 production in TFIIS-depleted cells, with hsp70 levels barely increasing above background after 2.5 min of heat shock. The significant kinetic block in hsp70 RNA production in TFIIS-depleted cells observed after a short heat shock, begins to be overcome at later time points, leading to an overall heat shock response of approximately 50%-60% normal hsp70 levels. These data demonstrate that TFIIS is required in vivo for maximal expression of hsp70 and suggest that TFIIS may serve to regulate the kinetics of the heat shock response by maintaining Pol II in a readily inducible conformation (Adelman, 2005).

These results suggest that TFIIS is involved in mediating the magnitude and efficiency of the heat shock response; additionally, TFIIS has been proposed to function broadly in transcription elongation by Pol II. To view the distribution of TFIIS both over the entire genome and at heat shock loci, Drosophila polytene chromosomes were stained with an antibody that is highly specific for TFIIS. Over 150 specific loci are stained by anti-TFIIS, including several interbands and chromosomal puffs, which contain the Pol II-transcribed developmental genes, the native and transgenic heat shock genes, and the nucleolus organizer, which contains the Pol I-transcribed rRNA genes. The consistent, prominent labeling of the nucleolus organizer suggests that TFIIS plays a role in Pol I elongation. A functional interaction between TFIIS and Pol I has been reported previously; however, conflicting reports have indicated that Pol I transcription is stimulated by a distinct transcript cleavage factor (Adelman, 2005).

Perhaps most surprising is the strong staining of many condensed chromosomal bands. These are sites that are not actively transcribed by RNA Pol I, II, or III. These transcriptionally inactive regions of TFIIS accumulation may be indicative of an as yet uncharacterized function of TFIIS, or may represent storage or proposed transcriptosome assembly loci akin to the TFIIS-containing Cajal Bodies in Xenopus oocytes (Adelman, 2005).

Upon stimulation of the heat shock response, TFIIS accumulates at heat shock loci. However, in contrast to many other transcription factors, TFIIS can still be observed at many other loci on the chromosomes, and in particular, the strong colocalization with condensed DNA bands persists. This result is consistent with recent data on the localization of TFIIS in yeast, where it was noted that TFIIS was not generally required for Pol II transcription but appeared to be specifically recruited to actively transcribed genes during times of cellular stress and when transcription was compromised (i.e., 6-AU treatment or temperature shift). In agreement with these results, Drosophila TFIIS is recruited to heat shock loci rapidly after heat induction and TFIIS appears to travel into the body of the gene along with Pol II, since it can be seen to colocalize throughout the puff with active Pol II (Adelman, 2005).

To analyze the localization of TFIIS at hsp70 at higher resolution, chromatin immunoprecipitation (ChIP) assays were performed followed by real-time PCR. This method allows for quantitative analysis of both the spatial and temporal distribution of TFIIS on the hsp70 gene. Pol II (detected using an antibody that recognizes the Pol II Rpb3 subunit) is associated specifically with the promoter region of hsp70 prior to heat shock (Boehm, 2003). Upon heat induction, Pol II is rapidly detected in the body of the gene and a robust recruitment of additional Pol II is observed (Adelman, 2005).

Strikingly, TFIIS is also present at the uninduced hsp70 promoter. This result is consistent with the idea that TFIIS associates with the promoter-proximal stalled Pol II to rescue it from arrest, thereby maintaining the Pol II in a rapidly responsive, active state. During heat shock, TFIIS is further recruited to the promoter region of hsp70 and TFIIS is seen to track along with the elongating Pol II into the body of the gene, in agreement with its role as an accessory factor for transcription elongation (Adelman, 2005).

All of the above data are consistent with the hypothesis that the promoter-proximal stalled Pol II has a tendency to fall into transcription arrest and that TFIIS serves to rescue the arrested Pol II so that it can be induced to elongate upon heat shock. Thus, one would predict that, in the absence of TFIIS, Pol II that becomes inactive in the promoter-proximal region would remain inactive, thereby presenting a steric obstacle to the rapid recruitment of additional Pol II molecules upon heat shock. The Pol II density at the hsp70 promoter before heat shock would thus remain unchanged (i.e., one Pol II present within each hsp70 promoter region), but the movement of Pol II into the body of the gene and the recruitment of additional Pol II upon heat shock should be diminished or delayed (Adelman, 2005).

To test this idea, a protocol was developed to perform ChIP on S2 cells that had been depleted of TFIIS by RNAi. TFIIS and LacZ RNAi-treated cells were crosslinked directly, or after a short, 2.5 min, heat shock. Depletion of TFIIS has no effect on the level of Pol II detected in the hsp70 promoter region before heat shock. This result indicates that TFIIS is not required for Pol II to stall within the promoter-proximal region. However, depletion of TFIIS leads to a significant reduction in the heat shock-induced recruitment of Pol II to the promoter. In fact, the Pol II density remains equivalent to that observed before heat induction. Moreover, the reduction in recruitment of Pol II is accompanied by a decrease in the Pol II signal throughout the body of the gene. This result indicates that the stalled Pol II is not efficiently released into the gene in the TFIIS-depleted cells, and that this “stuck” Pol II blocks recruitment of additional Pol II (Adelman, 2005).

As a control for the level of depletion, the presence of TFIIS at hsp70 was assayed in LacZ and TFIIS-treated cells. The 10-fold depletion of TFIIS observed by Western analysis leads to a similar reduction in TFIIS detectable on the hsp70 gene under both NHS and HS conditions. Importantly, depletion of TFIIS has no effect on the levels of HSF recruited to hsp70 upon heat shock, indicating that TFIIS-depleted cells did not display a general, nonspecific loss of factor recruitment. These results demonstrate that, while TFIIS is not required to establish the stalled Pol II at hsp70, depletion of TFIIS interferes with efficient release of Pol II from the promoter region and the rapid recruitment of additional Pol II (Adelman, 2005).

Pol II and/or general transcription factors have been found to occupy a growing number of promoters of preactivated genes. These varied promoters may utilize similar mechanisms for selectively recruiting certain components of the transcription machinery and for regulating transcription initiation and elongation through the promoter-proximal region. The efficiency of synthesis through the initially transcribed sequence is particularly sensitive to perturbation and is thus a prime target for gene regulation. Factors that impede the progress of the RNA polymerase within the first 10–40 nt, which often include both protein components and the nucleic acid sequence, have been shown to influence transcriptional pausing, arrest, and termination efficiency. Identification and characterization of the factors that modulate the regulatory pausing and/or stalling of Pol II within the promoter-proximal region is essential to understanding the regulation of genes like hsp70, wherein this step is rate limiting for gene expression (Adelman, 2005).

Transcription of hsp70 in vitro revealed positions of pausing that corresponded faithfully with locations that had been identified in vivo as harboring Pol II complexes that were not efficiently elongated. Likewise, Pol II artificially halted at these positions in vitro rapidly lost the capacity to resume transcription, even after removal of negatively acting elongation factors through stringent washing with sarkosyl. These results demonstrate that Pol II can become inactive within the promoter-proximal region. This work expands upon these observations by establishing that the inactive Pol II can be rescued by transcript cleavage factor TFIIS and thus represent arrested species. It is interesting to note that the predominant sites at which Pol II is found on the uninduced hsp70 gene are positions to which Pol II stably backtracks in vitro (Adelman, 2005).

These data suggest the following model for the role of TFIIS in hsp70 gene expression. Under uninduced conditions, Pol II is recruited to the hsp70 promoter and begins to transcribe through the promoter-proximal region. Intrinsic pause sites within the initially transcribed sequence induce transient stops in elongation, giving the regulatory negative-elongation factors time to bind and impede further movement into the gene. However, Pol II stalled for an extended time at the pausing sites have a tendency to backtrack along the template, displacing the 3′ end of the RNA from the catalytic site and prohibiting further elongation. In the absence of TFIIS, the arrested, inactive Pol II are unable to resume transcription rapidly upon heat induction, even after the negatively acting factors have been removed. However, in the presence of TFIIS, TFIIS-dependent cleavage returns inactive Pol II to a transcriptionally active conformation so that, upon heat shock and the removal of negatively acting factors, Pol II can be rapidly released from the promoter region. The movement of the first Pol II away from the promoter region allows for the recruitment of subsequent Pol II molecules. It is noted that this model is supported by a recent study of factors that interact genetically with Dst1 (the yeast gene encoding TFIIS), which suggested a general role for TFIIS in the transition from initiation to elongation (Adelman, 2005 and references therein).

These results are reminiscent of the role of bacterial Gre factors in mediating transcription efficiency through a regulatory pause in the late gene operon of λ bacteriophage. In the λ system, interactions between the RNA polymerase σ subunit and the promoter-proximal DNA sequence induce a transient pause in transcription, during which the λ Q protein binds and modifies the RNA polymerase, rendering it termination resistant. The Gre proteins modulate the kinetics of transcription through the pause site and are required for efficient function of the λ Q protein. Similarly, the activity of transcript cleavage factor TFIIS is necessary for efficient induction of hsp70 through its activation of promoter-proximally stalled Pol II. Thus, the current results indicate that, in addition to structural and mechanistic similarity between the Gre and TFIIS proteins, these factors may perform similar roles in vivo, serving to mediate the expression of genes that undergo pausing within the initially transcribed sequence (Adelman, 2005).

NELF potentiates gene transcription in the Drosophila embryo

A hallmark of genes that are subject to developmental regulation of transcriptional elongation is association of the negative elongation factor NELF with the paused RNA polymerase complex. This study used a combination of biochemical and genetic experiments to investigate the in vivo function of NELF in the Drosophila embryo. NELF associates with different gene promoter regions in correlation with the association of RNA polymerase II (Pol II) and the initial activation of gene expression during the early stages of embryogenesis. Genetic experiments reveal that maternally provided NELF is required for the activation, rather than the repression of reporter genes that emulate the expression of key developmental control genes. Furthermore, the relative requirement for NELF is dictated by attributes of the flanking cis-regulatory information. It is proposed that NELF-associated paused Pol II complexes provide a platform for high fidelity integration of the combinatorial spatial and temporal information that is central to the regulation of gene expression during animal development (Wang, 2010).

A principle conclusion that emerges from this study is that NELF can play a positive role in supporting transcription in the Drosophila embryo. This finding is somewhat surprising based on NELF's well-characterized properties as a transcriptional inhibitor in vitro and the current view of its role in regulating the hsp70 gene in vivo. So how does a factor that antagonizes transcription elongation play a positive role in promoting gene expression? Depletion of NELF in Drosophila S2 cells leads to reduced expression of a number of genes, and this drop in expression levels correlates with the re-positioning of nucleosomes around the promoter. The idea that NELF stabilizes the local architecture at the promoter that supports transcription is attractive, but the current results indicate these presumptive architectural contributions are not essential for transcription of several endogenous loci in the early embryo (Wang, 2010).

Central to understanding the requirement for NELF in promoting transcription is defining the key differences between the endogenous eve, ftz, rho and slp1 loci and the NELF-dependent reporter genes containing different specific cis-regulatory enhancers from these genes. The results strongly suggest that it is not the basal promoter per se that dictates the requirement for NELF. The basal promoter region contained in the composite slp1[DESE+PESE]-lacZ reporter extends from 260 bp upstream to 121 bp downstream of the transcription start site and includes the entire 5′ untranslated region of the slp1 mRNA. Even more telling is the observation that expression of the ftz-lacC reporter is lost in NELF-A GLC embryos. This reporter contains 6.5 kb of contiguous upstream cis-regulatory DNA extending to 120 bp downstream of the transcription start site. This stands in contrast to the NELF-independent expression of the P{PZ}slp1[05965] enhancer trap inserted 44 bp upstream of the slp1 transcription start site. Transcription of lacZ mRNA from this enhancer trap presumably initiates at the P-element promoter located at the 5′ terminus of this transgene insertion (Wang, 2010).

Although the requirement for NELF appears not to be dictated by the basal promoter, the observations that NELF is specifically localized to promoter regions and does not travel with elongating Pol II complexes strongly suggest the requirement involves NELF-associated Pol II complexes paused downstream of the promoter. The differential effect of NELF-E depletion on expression of the different slp1-lacZ reporters further indicates that the relative requirement for NELF is a function of the extent of flanking cis-regulatory information. Taken together these observations suggest that the relative requirement for NELF depends on interactions involving these flanking cis-regulatory DNA regions and NELF-associated paused Pol II complexes. It is proposed that NELF interacts with Pol II complexes that have initiated transcription but that are not fully competent to enter productive elongation and helps to stabilize these complexes in a form that is competent for responding to activating (or repressing) cues from enhancer-bound transcription factors. In this model the relative requirement for NELF in allowing for active transcription would depend on the strength of the interaction between a promoter and an enhancer and the relative efficiency of generating productive elongation complexes. Although the enhancers contained in the different lacZ reporters used in these experiments are all clearly capable of communicating with the promoter it would certainly be expected that this communication would be less efficient than for endogenous loci that contain the full complement of flanking DNA that has evolved to optimize the regulation of gene expression at this stage. Importantly, the NELF-dependent expression of these reporters strongly suggests that the generation of a productive Pol II elongation complex is the key step in the transcription cycle that is targeted for integrating the regulatory cues that drive the patterned expression of these genes in the early embryo (Wang, 2010).

NELF clearly has a pleiotropic role during Drosophila development. Perturbations in maternally provided NELF lead to two distinct embryonic lethal phenotypes. The observation that both phenotypes, albeit with different penetrance are produced either by elimination of maternal NELF-A or by reduction of maternal NELF-E strongly suggests both phenotypes are due to decreased activity of the NELF complex. The early arrest phenotype occurs prior to the onset of transcription in the embryo and thus is most likely due to defects that occur during oogenesis. The maternally provided histone mRNAs are one likely candidate as a prospective target of NELF activity during oogenesis. NELF is required for the proper processing of the 3′ termini of replication-dependent histone mRNAs in HeLa cells, and Drosophila embryos with defects in the processing of maternally provided histone mRNAs arrest during the nuclear division cycles that precede the blastoderm stage. Further studies should reveal whether the early arrest of NELF-A and NELF-E GLC embryos reflects a conserved role for NELF in the 3′-end processing of histone mRNAs (Wang, 2010).

The finding that many genes have paused Pol II complexes at their 5′ end strongly suggests that the regulation of transcription elongation is a widespread phenomenon in higher eukaryotes. Recent studies indicate that more than one third of all genes in Drosophila S2 cells generate short, 5′-capped RNAs similar to those produced by stalling of Pol II. The results of Pol II chromatin immunoprecipitation whole genome microarray assays suggest that paused Pol II complexes are formed on approximately 10% of genes in the blastoderm stage Drosophila embryo. This is almost certainly an underestimate as five of the seven genes for which NELF association has been demonstrated were not identified as having paused Pol II complexes at this stage. Indeed, the stringent cut-off used in this study led to the assignment of slp1 as a member of the 27% of genes that have uniform Pol II association in the blastoderm embryo (Wang, 2010).

It is furthermore clear that NELF association is developmentally regulated as neither srya nor any of the five segmentation genes for which NELF association in the early embryo was demonstrated in this study are also associated with NELF in S2 cells. Amongst these six genes with early embryonic association of NELF there are differences in the level of association at different developmental stages. The two genes with the most rapid loss of NELF, ftz and srya show little to no expression after four hours of development. Thus NELF is not involved in the stable maintenance of repression at these later stages, which involves instead other mechanisms such as epigenetic maintenance by the Polycomb group proteins and specific histone methylation marks. The observation that NELF association is also reduced on genes such as en and wg that have increased expression levels at later stages may suggest that NELF is not involved in the ongoing expression of these genes at later stages. However, as the embryo is comprised of a mixture of expressing and non-expressing cells it will be important to examine NELF association specifically in cells expressing these genes before coming to this conclusion (Wang, 2010).

The high levels of NELF association with the promoter regions of a number of genes involved in segmentation and other early developmental processes serves to emphasize the unique and pivotal aspects of this critical stage of Drosophila embryogenesis. Pre-blastoderm nuclei are totipotent and come to be specified in response to maternally-provided positional information and the action of the genetic systems that respond to this information. The regulation of gene transcription is central to the initial specification of cell fates along both the anterior-posterior and dorsal-ventral axes of the early embryo, and it is clear that regulation of transcription elongation is central to this process. Similar to Drosophila blastoderm nuclei, the pluripotent properties of human embryonic stem cells are reflected by the presence of paused Pol II complexes on a wide number of genes, including many key developmental regulators. Further studies on the mechanisms of developmentally regulated transcription elongation are clearly of great importance for understanding the initial programming of cell fates expression during animal embryogenesis (Wang, 2010).

Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex

Negative elongation factor (NELF) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) are involved in pausing RNA Polymerase II (Pol II) in the promoter-proximal region of the hsp70 gene in Drosophila, before heat shock induction. Such blocks in elongation are widespread in the Drosophila genome. However, the mechanism by which DSIF and NELF participate in setting up the paused Pol II remains unclear. The interactions were analyzed among DSIF, NELF, and a reconstituted Drosophila Pol II elongation complex to gain insight into the mechanism of pausing. The results show that DSIF and NELF require a nascent transcript longer than 18 nt to stably associate with the Pol II elongation complex. Protein-RNA cross-linking reveals that Spt5, the largest subunit of DSIF, contacts the nascent RNA as the RNA emerges from the elongation complex. Taken together, these results provide a possible model by which DSIF binds the elongation complex via association with the nascent transcript and subsequently recruits NELF. Although DSIF and NELF were both required for inhibition of transcription, no NELF-RNA contact is detected when the nascent transcript was between 22 and 31 nt long, which encompasses the region where promoter-proximal pausing occurs on many genes in Drosophila. This raises the possibility that RNA binding by NELF is not necessary in promoter-proximal pausing (Missra, 2010).

DSIF and NELF are key factors in pausing Pol II in the promoter-proximal region of genes in Drosophila and human cells. To gain insight into the mechanism by which DSIF and NELF contribute to promoter-proximal pausing, a system was developed in which the physical interaction of DSIF and NELF with a Pol II elongation complex could be monitored using a native gel electrophoresis assay. Previously, it was demonstrated that DSIF alone could associate with the Pol II elongation complex. In this study a method was developed to purify Drosophila NELF, thus allowing exploration of the interplay of DSIF and NELF with the elongation complex (Missra, 2010).

The results show that the association of NELF with the elongation complex is dependent on the presence of DSIF. Previous work provided evidence that NELF associated with preformed complexes of DSIF and Pol II in nuclear extracts but the interaction of DSIF and Pol II was not dependent on NELF. These interactions were likely occurring outside the context of an elongation complex and were relatively weak because the bulk of DSIF, NELF, and Pol II exist independent of each other in nuclear extracts. In contrast, the current results show that NELF can significantly influence the binding of DSIF to Pol II within the context of an elongation complex when limiting amounts of DSIF are present. Since Pol II, DSIF, and NELF have been shown to interact individually with each other, it is likely that this network of interactions contributes to stable association of these proteins in the context of the elongation complex (Missra, 2010).

The binding assays show that the length of the nascent transcript affects the association of DSIF and NELF with the elongation complex. While binding of DSIF alone or in combination with NELF to the elongation complex was evident for an elongation complex with a nascent transcript of 22 nt, no binding was detected when the nascent transcript was 18 nt long. These results are consistent with the finding that human DSIF and NELF require transcripts ≥18 nt long to inhibit transcription, and also a recent study showed human DSIF preferentially bound elongation complexes containing transcripts that were at least 25 nt long. The 5′ end of an 18-nt-long nascent transcript just begins to emerge from the surface of Pol II. Exposure of four additional nucleotides appears to be sufficient for binding of DSIF alone or with NELF. Notably, the association of DSIF with the elongation complex is not simply due to nonspecific interaction with the RNA or DNA because previous experiments show that binding of DSIF to the elongation complex requires specific contacts with Pol II (Missra, 2010).

One way in which nascent transcript length could affect the association of DSIF and NELF is by providing an additional binding site in the elongation complex. Previous results have directed attention at an RRM in NELF-E. Mutations in this RRM impair the capacity of NELF to inhibit elongation in the presence of DSIF. However, these experiments focused on elongation over distances greater than 100 nt. The finding that DSIF associates with elongation complex containing a 22-nt-long radioactive transcript (EC22) but not EC18 suggests that DSIF rather than NELF might be interacting with the nascent transcript, and RNA-protein cross-linking data support this hypothesis. The Spt5 subunit of Drosophila and human DSIF contains five Kyprides, Ouzounis, Woese (KOW) domains. An isolated KOW domain from Aquifex aeolicus NusG has been shown to associate with RNA, so it is possible that one of these domains in Spt5 is contacting the nascent transcript as it emerges from the elongation complex (Missra, 2010).

Cross-linking analysis detected contact between NELF-E and the nascent transcript in EC70 but not in EC31. The 5′ end of the nascent transcript contacts Rpb7 when its length is between 26 to 32 nt. Therefore it is possible that a longer nascent transcript is required to allow contact with NELF. Given that promoter-proximal pausing can occur before Pol II transcribes 30 nt, it is proposed that the RRM of NELF-E is not involved in promoter-proximal pausing. Its role could be limited to processes involving longer nascent transcripts such as regulation that appears to involve the transactivation response element of HIV or 3′ end formation of histone mRNAs (Missra, 2010).

The finding that DSIF and NELF associate with EC22 but not with EC18 is very relevant to the process of promoter-proximal pausing. Permanganate genomic footprinting of over 60 different promoters reveals that Pol II pauses in the promoter-proximal region 20 to 50 nt downstream from the transcription start site. Those cases where the Pol II appeared to be pausing closer to a transcription start site were found to have the start sites inaccurately mapped. Thus, the promoter-proximal limit for the range where Pol II pauses is likely to be dictated by the minimum length of RNA required for DSIF to associate with the elongation complex (Missra, 2010).

From the results presented in this study, it is proposed that the first step in promoter-proximal pausing involves binding of DSIF to the nascent transcript. NELF subsequently associates to form a stable complex. Importantly, this complex alone is not sufficient to stably pause the Pol II as the results show that elongation is slowed but not halted in reactions involving only these three proteins. Hence other factors that remain to be identified are likely to act in concert with this core complex of DSIF, NELF, and Pol II to stably pause Pol II in the promoter-proximal region of genes. Since transcription in vivo occurs on chromatin, nucleosomes may cooperate with DSIF and NELF in setting up the paused polymerase. The experimental approach described in this study could serve as a way to identify additional factors involved in pausing (Missra, 2010).

Negative elongation factor accelerates the rate at which heat shock genes are shut off by facilitating dissociation of heat shock factor

Promoter-proximal pausing of RNA polymerase II (Pol II) occurs on thousands of genes in animal cells. This pausing often correlates with the rapid induction of genes, but direct tests of the relationship between pausing and induction rates are lacking. hsp70 and hsp26 in Drosophila are rapidly induced by heat shock. Contrary to current expectations, depletion of negative elongation factor (NELF), a key factor in setting up paused Pol II, reduced pausing but did not interfere with rapid induction. Instead, depletion of NELF delayed the time taken for these genes to shut off during recovery from heat shock. NELF depletion also delayed the dissociation of HSF from hsp70 and hsp26, and a similar delay was observed when cells were depleted of the histone acetyltransferase CBP. CBP has been reported to associate with Pol II, and acetylation of HSF by CBP has been implicated in inhibiting the DNA-binding activity of HSF. It is proposed that NELF-mediated pausing allows Pol II to direct CBP-mediated acetylation of HSF, thus causing HSF to dissociate from the gene. Activators are typically viewed as controlling Pol II. These results reveal a possible reciprocal relationship in which paused Pol II influences the activator (Ghosh, 2011).

Reduction of promoter proximal pausing on hsp70 does not alter the rate of heat shock induction. Correlations between the presence of paused Pol II and rapid induction of gene expression have led to the hypothesis that promoter proximal pausing provides the basis for rapid induction. However, none of these studies have actually tested if disruption of promoter proximal pausing delays expression of genes. The current analysis reveals that reduction of promoter proximal pausing at hsp70 does not detectably alter the rate of heat shock induction. This was evident by monitoring the association of Pol II at both the 5’ and 3’ ends of hsp70 using permanganate genomic footprinting and also by measuring synthesis of hsp70 RNA. The two types of assays are in good agreement with each other in that a substantial increase in the level of transcript and in the presence of Pol II at the 3’ end of the gene occurs between 2 and 4 minutes after heat shock. Also, the elongation rate of approximately 1.25 kb/min for transcription of hsp70 that can be deduced from permanganate data is in excellent agreement with other types of measurements, thus validating the use of permanganate footprinting as a means for analyzing the kinetics of transcription induction and elongation. It has been proposed that the establishment of paused Pol II at a promoter allows for rapid induction because steps that precede initiation such as chromatin remodeling and Pol II recruitment are bypassed. In the case of the heat shock genes, these steps are still likely to occur prior to heat shock induction even when promoter proximal pausing is impaired. TFIID, which provides the foundation for assembling a preinitiation complex, makes multiple sequence-specific contacts with hsp70 including the TATA box, the initiator, and downstream sequences. In addition, another DNA binding protein called GAGA factor maintains the promoter region in an accessible state even when TFIID binding is impaired. Since HSF associates with a transgenic hsp70 promoter deleted of its TATA box, the accessible state of the promoter in lieu of TFIID appears to be sufficient for HSF binding. Thus access of DNA by the general transcriptional machinery and HSF may not be limiting for hsp70 even when promoter proximal pausing is disrupted. The extent to which promoters retain an accessible state upon loss of paused Pol II is likely to vary. The Drosophila heat shock genes could represent one end of a spectrum where accessibility is retained without sustaining full occupancy of the promoter with paused Pol II because of the binding of GAGA factor and TFIID. At the other end of the spectrum may be genes that require the paused Pol II to maintain a potentially inducible state. Cases have been identified where the paused Pol II appears to contribute to induction by preventing a nucleosome from assembling over the promoter (Ghosh, 2011).

Depletion of NELF impacts the ability of hsp70 to shut-off during recovery from heat shock. Permanganate genomic footprinting provides a high-resolution view of the behavior of Pol II. Permanganate reactivity on hsp70 at positions +7 and +8 are indicative of newly initiated Pol II whereas reactivity downstream from +34 corresponds to Pol II that has read through the pause. Similarly for hsp26, permanganate reactivity at +9 and +10 are indicative of newly initiated Pol II whereas reactivity downstream from +45 corresponds to Pol II that has read through the pause. The patterns of permanganate reactivity that were observed indicate that both initiation and read-through persist for a longer time in NELF-depleted glands than in control glands during recovery from heat shock for both hsp70 and hsp26. Corroborating this conclusion is the demonstration that a brief heat shock causes these two genes to be more highly expressed in NELF-depleted glands than in control glands. Limiting the production of hsp70 protein to a level appropriate to the degree of stress could be critical for proper cell development, since ectopic expression of hsp70 in salivary glands under non-heat shock conditions inhibits growth of the cells in this tissue (Ghosh, 2011).

These results now expand the types of scenarios in which NELF and promoter proximal pausing serve to attenuate gene expression. In the case of the estrogen-mediated activation of the pS2 gene, direct interaction between the NELF-B subunit and the estrogen receptor causes estrogen-mediated association of NELF with the target gene. Depletion of NELF results in hyper-activation of the pS2 gene, indicating that the estrogen receptor is mediating both activation and repression to achieve a particular level of expression in the presence of stimuli. In the case of junB, depletion of NELF increased the expression of junB both before and after serum-mediated activation. The current results now reveal a role for NELF in shutting off gene expression upon withdrawal of the stimulus - this case being heat shock (Ghosh, 2011).

To investigate why the shut off of the heat shock genes was delayed by depletion of NELF, the association of HSF with hsp70 and hsp26 in salivary glands was measured using chromatin immunoprecipitation. Significantly more HSF was detected on the heat shock genes after 45 minutes of recovery in NELF-depleted than control glands indicating that the delay in shutting off the heat shock genes could be due to a delay in the dissociation of HSF and that NELF is somehow involved in this dissociation. It is unlikely that the dissociation of HSF is due to stress induced by depletion of NELF, since there is no evidence of this depletion inducing a stress response in salivary glands. Prior to heat shock, both control and NELF-depleted glands exhibit low levels of heat shock gene expression, low levels of HSF at heat shock gene promoters, and undetectable levels of HSF DNA-binding activity in amount of HSF detected in glands nor did it alter a heat shock-dependent shift in the mobility of HSF on SDS-PAGE. Approximately 2-fold higher HSF binding activity was detected in extracts from NELF-depleted glands than control glands after recovery from heat shock, but the standard deviations in the measurements indicate that this difference is not significant. Since the results showed that depletion of NELF resulted in a delay in the dissociation of HSF from heat shock genes during the recovery from heat shock, recent indirect evidence in human cells that acetylation of HSF by CBP regulates HSF binding activity was intriguing. The finding that depletion of CBP delays the dissociation of HSF from the heat shock genes provides the first direct evidence that CBP is involved in regulating HSF’s interaction with heat shock genes in vivo. Since depletion of CBP and NELF each delays the dissociation of HSF from the heat shock genes during recovery, it is proposed that their actions are linked. It is posited that NELF-mediated pausing of Pol II could position CBP to acetylate HSF, thus causing HSF to dissociate from the heat shock gene. Several observations provide support for this model. CBP associates with Pol IIa and not Pol IIo and the Pol II paused in the promoter proximal region is in the IIa state. Also, CBP associates with the heat shock genes in Drosophila during heat shock induction. Further investigation of this proposal will be aided by biochemical analyses of interactions between CBP and reconstituted paused elongation complexes. Multiple mechanisms can attenuate the activity of HSF during heat shock, so the possibility cannot be ruled out that the actions of NELF and CBP are independent of each other or that these proteins are acting indirectly on HSF by influencing other regulators of HSF. The heat shock proteins such as hsp70 produced during heat shock repress the activation domain of HSF thus providing a negative feedback loop that limits heat shock gene transcription. These chaperons have also been implicated in attenuating the DNA binding activity of HSF in Drosophila during extended periods of a moderate heat shock. Changes in phosphorylation of HSF have also been implicated in regulating its activity, but such changes do not appear to effect the DNA binding activity of HSF in Drosophila. The mechanisms by which heat shock genes are returned to their uninduced state during recovery from heat shock remain to be fully elucidated. The use of permanganate footprinting to directly monitor the behavior of Pol II at heat shock genes during recovery from heat shock in cells depleted of specific proteins provides a way to identify candidates involved in shutting off transcription of the heat shock genes. A significant advantage of this approach is that it allows one to directly monitor the reestablishment of the paused state (Ghosh, 2011).

Paused Pol II could function as a regulator of gene regulatory factors. Many stimulus-responsive and developmental genes are found to have paused Pol II, and attenuation of transcription of these genes is likely to be critical for establishing appropriate physiological or developmental programs of gene expression. The finding that paused Pol II influences dissociation of an activator from the promoter provides a mechanism for rapidly shutting off gene expression. The pausing of Pol II could function more broadly by allowing the Pol II to remain stationary at a promoter while it serves to recruit modulators of chromatin structure and gene expression (Ghosh, 2011).

Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation

Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter or regulated pausing of RNA polymerase II (Pol II) in early elongation. This study reports that a striking difference between genes that use these distinct regulatory strategies lies in the 'default' chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly regulated genes whose sequences inherently disfavor nucleosome formation within the gene but favor occlusion of the promoter by nucleosomes. In contrast, housekeeping genes that lack pronounced Pol II pausing show higher nucleosome occupancy downstream, but their promoters are deprived of nucleosomes regardless of polymerase binding. These results indicate that a key role of paused Pol II is to compete with nucleosomes for occupancy of highly regulated promoters, thereby preventing the formation of repressive chromatin architecture to facilitate further or future gene activation (Gilchrist, 2011).

The data support a general model for gene regulation wherein the underlying DNA sequence around promoters directly influences both chromatin architecture and the step in the transcription cycle that is rate-limiting for gene expression. Genes with high levels of Pol II pausing inherently favor the formation of nucleosomes over the promoter, establishing an active competition between Pol II and nucleosomes for promoter occupancy. It is proposed that this intrinsically repressive chromatin structure prevents aberrant expression of paused genes, which are often components of highly-regulated pathways. Nucleosome remodeling, likely initiated by proteins such as GAGA factor, would be required to disassemble nucleosomes at these promoters and allow for gene activity. Nucleosome removal would uncover strong promoter motifs that facilitate efficient, stable recruitment of the transcription machinery. Extended NELF-mediated pausing of polymerase at these promoters makes the transition to productive elongation slow. However, upon pause release, low levels of downstream nucleosomes would minimize barriers to transcription elongation and additional Pol II molecules would be rapidly recruited to maintain high Pol II occupancy and prevent nucleosome formation (Gilchrist, 2011).

In contrast, genes that lack extended pausing appear to disfavor promoter nucleosome assembly and instead harbor nucleosomes flanking the nucleosome-deprived promoter region. Localized DNA accessibility near TSSs could both help target the transcription machinery to the promoter region and diminish the requirement for nucleosome remodeling to allow gene activity. The dearth of core promoter elements could make these genes more reliant on activator binding for recruitment of the transcription machinery, and Pol II recruitment would be the rate-limiting step for expression of these genes. Pausing would be short-lived at these genes, and despite higher downstream nucleosome occupancy, polymerase escapes efficiently into productive synthesis (Gilchrist, 2011).

Importantly, these two strategies present different opportunities for gene regulation. Highly paused genes present two distinct steps at which they can be regulated: promoter accessibility and release of Pol II from pausing. It is proposed that this two-step mechanism facilitates precise control of gene expression. It is envisioned that the first step, nucleosome remodeling, functions as a molecular switch that relieves repression by chromatin to permit expression. This step can be temporally uncoupled from gene activation and could potentiate genes for future activation rather than prompting their immediate expression. The second step, release of paused Pol II, might be analogous to a volume dial, which permits fine-tuning of expression levels in response to changing conditions. Transcription levels could be rapidly regulated solely by manipulating the efficiency of P-TEFb recruitment through its interactions with DNA-binding transcription activators and histone modifications. This idea is supported by observations that activation of highly paused genes is both fast and synchronous. In contrast, genes that lack promoter-proximal pausing and nucleosome occupancy rely chiefly on a single-step mechanism to alter gene expression: regulated, step-wise recruitment of the transcription machinery. This mode of regulation has been suggested to be inherently more stochastic and prone to transcriptional noise, which may explain why many genes regulated by recruitment are constitutively active housekeeping genes (Gilchrist, 2011).

This study proviedes evidence that NELF-mediated pausing during early elongation is a general feature of the transcription cycle that is exploited at some genes to regulate transcription output. It is proposed that each round of transcription entails pausing, perhaps serving as an early 'checkpoint' to ensure proper maturation of the elongation complex before release into productive elongation. At some genes, this halt in elongation may be transient, whereas at others it may involve a long-lived paused complex that becomes rate-limiting for gene expression. Importantly, these results imply that the release from pausing through P-TEFb recruitment is an important, regulated step that broadly impacts gene expression, in agreement with recent work. It is noted that general recruitment of NELF during early elongation likely explains the seemingly paradoxical observation made in several systems that NELF levels increase at activated genes that experience robust recruitment of additional Pol II (Gilchrist, 2011).

The data also reveal that the inherent preference towards repression of highly-regulated promoters by nucleosome occlusion is an evolutionarily conserved phenomenon. Moreover, the results are in agreement with recent work in yeast which reveals that Pol II plays a role in displacing nucleosomes from promoter regions. However, in yeast, nucleosome disassembly is coupled directly to gene activation, whereas in Drosophila nucleosome disassembly is coupled to Pol II pausing. Perhaps Drosophila and other metazoans have evolved promoter-proximal pausing as an additional layer of regulation to accommodate increased demands for precise and rapid gene regulation during development and organismal responses to stress. In addition, it might be beneficial to maintain highly-regulated promoters poised in an open chromatin state, to prevent their incorporation into the more inaccessible, condensed heterochromatin that exists in metazoans (Gilchrist, 2011).

In summary, this paper reports that a primary function of paused Pol II is to prevent promoter-proximal nucleosome formation. This represents a fundamental shift in thinking about the role of Pol II pausing, which has long been thought to simply repress gene expression. Instead, it is argued that pausing should be viewed as a mechanism to fine-tune gene expression, and to potentiate genes for further or future activation. In addition, it was shown that sequence-specified 'default' nucleosome architecture instructs the regulatory properties of Drosophila promoters. It is proposed that metazoans have evolved a gene regulatory strategy in which nucleosomes and paused Pol II compete for promoter occupancy, affording multiple opportunities for regulation of gene expression (Gilchrist, 2011).

Regulation of Hox gene activity by transcriptional elongation in Drosophila

Hox genes control the anterior-posterior patterning of most metazoan embryos. Their sequential expression is initially established by the segmentation gene cascade in the early Drosophila embryo. The maintenance of these patterns depends on the Polycomb group (PcG) and trithorax group (trxG) complexes during the remainder of the life cycle. This study provides both genetic and molecular evidence that the Hox genes are subject to an additional tier of regulation, i.e., at the level of transcription elongation. Both Ultrabithorax (Ubx) and Abdominal-B (Abd-B) genes contain stalled or paused RNA polymerase II (Pol II) even when silent. The Pol II elongation factors Elongin-A and Cdk9 are essential for optimal Ubx and Abd-B expression. Mitotic recombination assays suggest that these elongation factors are also important for the regulation of Notch-, EGF-, and Dpp-signaling genes. Stalled Pol II persists in tissues where Ubx and Abd-B are silenced by the PcG complex. It is proposed that stalling fosters both the rapid induction and precise silencing of Hox gene expression during development (Chopra, 2009).

Recent studies suggest that the regulation of polymerase II (Pol II) elongation might be a common feature of developmental gene control in the Drosophila embryo. Chromatin immunoprecipitation (ChIP)-chip assays in cultured cell lines suggest that a significant fraction of all protein-coding genes contain stalled Pol II. As many as 10% of all protein-coding genes in the early Drosophila embryo contain Pol II prior to their expression. Many of these genes are developmental control genes, such as those encoding components of cell-signaling pathways, including Wnt, FGF, and Dpp (TGFβ). Moreover, four of the eight Hox genes in Drosophila appear to contain stalled Pol II (lab, Antp, Ubx, and Abd-B) in the early embryo. This study investigated the role of Pol II elongation factors in Hox gene expression (Chopra, 2009).

To confirm the preliminary evidence for stalled Pol II at the Ubx and Abd-B loci, conventional ChIP assays were performed with different antibodies against Pol II -- namely, 8WG16, which recognizes the CTD of Pol II, and H14, which recognizes the initiating form (Ser-5 phosphorylation) of Pol II. Both of these antibodies have been used in earlier ChIP as well as in ChIP-chip assays to elucidate and map distinct functions of the Pol II complex. Chromatin crosslinking was performed on 0-2 hr wild-type embryos prior to the onset of Hox gene expression. The chromatin was sonicated and precipitated with anti-Pol II antibodies, and then the extracted DNA was used as a template for PCR amplification. Hsp70 was used as a control because it represents the prototypic example of paused Pol II. As expected, the hsp70 promoter region contains strong Pol II signals with both the 8WG16 and H14 antibodies, indicating that an initiated Pol II is bound to the hsp70 promoter prior to heat shock induction. The Ubx and Abd-B promoter regions also exhibit strong signals, whereas PCR amplification performed with exonic probes failed to detect Pol II binding within the main body of the transcription unit. The presence of the H14 signal at these promoters suggests that Ser5 of the Pol II CTD is phosphorylated (initiated Pol II) prior to the activation of Ubx and Abd-B expression. As predicted from the previous ChIP-chip assays, the abd-A promoter region lacks Pol II (Chopra, 2009).

The preceding studies suggest that Ubx and Abd-B contain a stalled form of Pol II in early embryos. Additional assays were done to investigate Pol II binding in wing and haltere imaginal discs. The hsp70 promoter region contains strong Pol II signals in both wing and haltere discs, consistent with previous studies suggesting that the gene is stably paused in most or all tissues prior to induction by heat shoc. The ChIP assays also identify strong Pol II signals in the Ubx promoter region of wing discs, where the gene is silenced by the PcG complex. In contrast, a probe directed against exon 1 failed to detect significant levels of Pol II within the main body of the transcription unit (Chopra, 2009).

Very different results were obtained with haltere discs, in which Ubx is strongly expressed and the resulting Ubx repressor inhibits wing development. In this case, strong Pol II signals are detected in both the promoter region and exon, as would be expected for an actively expressed gene. These findings were strengthened by the use of qPCR assays. For these experiments, ChIP assays were done with a cocktail of Pol II antibodies (both 8WG16 and H14). Pol II signals are detected in both the promoter region and exon of the Ubx locus in haltere discs, where the gene is active. In contrast, there are substantially higher levels of Pol II in the promoter region than exon in wing discs where Ubx is silent. Permanganate protection assays are consistent with the occurrence of paused Pol II located between +18 and +35 bp downstream of the Ubx transcription start site (Chopra, 2009).

Abd-B also exhibits higher levels of Pol II binding in the promoter region as compared with exon 1. However, unlike Ubx, Abd-B is silent in both the wing and haltere discs, so it is not surprising that Pol II is not significantly detected in exon 1 in either tissue. As seen in early embryos, the promoter region of abd-A lacks significant binding of Pol II in wing discs (Chopra, 2009).

Pol II stalling raises the possibility that Ubx might be regulated at the level of transcriptional elongation. A number of elongation factors have been identified in cell culture assays, including negative elongation factors (NELF) A-E, ELONGIN-A (Elo-A), suppressor of termination (SPT) 4 and 5, and cyclin-dependent kinase 9 (CDK9). Reduced levels of Ubx⁺ activity cause a slight transformation of halteres into wings because Ubx functions as a repressor of wing development in the halteres. It was reasoned that, if Ubx is regulated at the level of Pol II elongation, then reduced levels of critical elongation factors should enhance the patterning defects observed in weak Ubx mutants (Chopra, 2009).

mutations in four different elongation factors were specifically examined: Elo-A, Cdk9, Spt4, and Spt5. Cdk9 has been shown to be a critical activator of paused Pol II at the hsp70 promoter. Heterozygotes for each mutation were examined in a Ubx¹/+ background, which displays a weak expansion of the halteres. Elo-A/+; Ubx¹/+ double heterozygotes display an enhanced transformation of halteres into wings. In particular, several wing-like bristles appear at the leading margin of the halteres. A similar phenotype was observed for Cdk9/+; Ubx¹/+ double heterozygotes. Spt4 mutations cause a slight suppression of the Ubx¹/+ phenotype, consistent with their dual activities in both attenuating and augmenting Pol II elongation (Chopra, 2009).

Cdk9 and Elo-A are thought to regulate distinct aspects of Pol II elongation. The Cdk9 kinase phosphorylates Ser-2 of the Pol II CTD, which is critical for the release of Pol II from the pause site in the hsp70 promoter. Inhibition of Cdk9 activity causes a global reduction in Ser-2 phosphorylation. In contrast, Elo-A appears to act at a later point of Pol II elongation after release from the pause site. Mutations in Cdk9 and Elo-A cause an additive enhancement in the Ubx¹/+ phenotype. Triple heterozygotes display an expansion in the overall size of the haltere, and the anterior margin contains a series of bristles like those seen in wings. This phenotype suggests that diminished levels of Cdk9 and Elo-A cause significant reductions in Ubx⁺ activity (Chopra, 2009).

ChIP-chip and conventional ChIP assays suggest that the Abd-B promoter region might also contain a stalled form of Pol II. As seen for Ubx, reduced levels of Cdk9 and Elo-A cause significant enhancements in the Abd-B^M1/+ mutant phenotype. In particular, Abd-B^M1/+ heterozygotes display a weak transformation of posterior abdominal segments into anterior segments, particularly the seventh abdominal segment (A7) into A6 (ectopic partial pigmentation) and A6 to A5 (ectopic bristles in A6 sternite). These phenotypes are augmented by reductions in either Cdk9 or Elo-A activity. Double heterozygotes display a more complete A7-to-A6 transformation, as well as an increase in the number of bristles in A6, suggesting a more severe A6-to-A5 transformation. These segmental transformations are weakly enhanced (not suppressed) by lower levels of Spt4 and Spt5. In contrast, mutations in the negative elongation factor Nelf-E strongly suppress the Abd-B^M1 phenotype, which is consistent with enhanced transcription of Abd-B. Triple heterozygotes, Abd-B^M1/+; Cdk9/+; Elo-A/+, display an even more dramatic transformation of A7 to A6 and A6 to A5. Thus, as seen for Ubx, reduced levels of Cdk9 and Elo-A cause a significant diminishment in Abd-B+ gene activity (Chopra, 2009).

Stalled Pol II appears to be disproportionately associated with developmental control genes as compared with 'housekeeping' genes that control cell metabolism and proliferation. A substantial fraction of stalled genes exhibit localized patterns of expression during embryogenesis, such as Hox genes and genes encoding components of signaling pathways (e.g., Dpp, FGF, Notch, etc.). Therefore, the possibility was explored that elimination of Cdk9 and Elo-A activity via the production of mitotic clones might produce specific developmental defects in adult appendages. In these experiments, there is no perturbation of Ubx or Abd-B activity. Cdk9 and Elo-A activities are disrupted in an otherwise wild-type background (Chopra, 2009).

The localized loss of Cdk9 or Elo-A activity in the haltere discs leads to weak wing transformation phenotypes, similar to those seen for reductions in Ubx. In particular, there is an expansion in the size of the halteres, and wing-like bristles appear at the margins. At least some of these phenotypes appear to arise from the specific loss of Ubx expression. Haltere discs containing clonal patches of Cdk9⁻/Cdk9⁻ tissue (identified by the loss of GFP expression) display localized reductions in Ubx activity, as judged by the use of an anti-Ubx antibody. This observation suggests that Ubx transcription is particularly sensitive to diminished activities of Pol II elongation factors, which is consistent with the evidence that the Ubx promoter region contains stalled Pol II (Chopra, 2009).

Cdk9 and Elo-A mitotic clones produce a variety of patterning defects in the wing and notum. Most notably, there is notching of the wing margins, ectopic wing veins, short crossveins, and both losses and duplications of macrochaete in the notum. These phenotypes might arise from perturbations in Notch, EGF, and Dpp (TGFβ) signaling. Genes encoding components of each of these pathways appear to contain stalled Pol II in early embryos (Chopra, 2009).

This study has presented evidence that the elongation factors Cdk9 and Elo-A are essential for optimal expression of at least a subset of Drosophila Hox genes, particularly Ubx⁺ activity in the developing halteres. Small patches of Elo-A⁻/Elo-A⁻ or Cdk9⁻/Cdk9⁻ mutant tissue also cause specific patterning defects in the wings and notum. Both Pol II elongation factors are probably required for normal expression of a great number of genes in the Drosophila genome. Indeed, both elongation genes are essential, and every attempt to create large mitotic clones resulted in larval lethality. Such lethality presumably reflects the general role of Elo-A and Cdk9 in gene expression. Previous studies have documented the general importance of the elongation factors ELL and Elo-A in Drosophila larval development and metamorphosis. Nonetheless, it would appear that a small number of patterning genes, including Ubx, are particularly sensitive to the loss of Elo-A and Cdk9 activity (Chopra, 2009).

It has been extensively argued that Polycomb might mediate repression by propagating an inactive form of chromatin, for example, by methylation of H3K27 followed by recruitment of HP1 or other proteins that package chromatin in an inactive state. However, the demonstration that TBP and Pol II are present in the Ubx proximal promoter in wing imaginal discs suggests that PcG silencing does not render the chromatin inaccessible for the binding of even large protein complexes. Instead, it is proposed that paused Pol II could contribute to PcG silencing by excluding the binding of additional Pol II complexes. Such occlusion by steric hindrance might help reduce transcriptional noise and thereby maintain Ubx repression. Mutations in the elongation factor, ELL [Su(Tpl)], suppress Scr phenotypes caused by the Pc⁴ Polycomb mutant, raising the possibility that Pol II elongation factors somehow communicate with the PcG-silencing complex. It is proposed that stalling might serve the dual role of fostering both silencing and rapid induction and thereby provide a sharp on/off switch in Hox regulation (Chopra, 2009).

The Snail repressor inhibits release, not elongation, of paused Pol II in the Drosophila embryo

The development of the precellular Drosophila embryo is characterized by exceptionally rapid transitions in gene activity, with broadly distributed maternal regulatory gradients giving way to precise on/off patterns of gene expression within a one-hour window, between two and three hours after fertilization. Transcriptional repression plays a pivotal role in this process, delineating sharp expression patterns (e.g., pair-rule stripes) within broad domains of gene activation. As many as 20 different sequence-specific repressors have been implicated in this process, yet the mechanisms by which they silence gene expression have remained elusive. This study reports the development of a method for the quantitative visualization of transcriptional repression. The focus of this study was the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm. Elongating Pol II complexes were found to complete transcription after the onset of Snail repression. As a result, moderately sized genes (e.g., the 22 kb sog locus) are fully silenced only after tens of minutes of repression. It is proposed that this 'repression lag' imposes a severe constraint on the regulatory dynamics of embryonic patterning and further suggest that posttranscriptional regulators, like microRNAs, are required to inhibit unwanted transcripts produced during protracted periods of gene silencing (Bothma, 2011).

Snail typically binds to repressor sites located near upstream activation elements within distal enhancers. Repression might result from the passive inhibition of upstream activators, such as the failure of the activators to mediate looping to the core promoter. Alternatively, Snail might alter the chromatin state of the promoter region, resulting in diminished access of the Pol II transcription complex. Such repression mechanisms might cause a lag in gene silencing due to the continued elongation of Pol II complexes that were released from the promoter prior to the onset of repression. As in the case of the delay in the production of mature mRNAs after initiation, the lag in repression would be commensurate with the size of the gene, with large genes taking longer to silence than small genes. This can take a significant amount of time due to the surprisingly slow rate of Pol II elongation, only ∼1 kb/min (Ardehali, 2009; Bothma, 2011 and references therein).

Alternatively, elongating Pol II complexes might be arrested or released from the DNA template due to changes in chromatin structure and/or attenuation of Pol II processivity. Such mechanisms could lead to the immediate silencing of all genes regardless of size. Recent studies have documented rapid changes in the chromatin structure across the entire length of genes, exceeding the rate of Pol II processivity (Petesch, 2008). Certain corepressors in the Drosophila embryo (e.g., Groucho) are thought to mediate repression by a 'spreading' mechanism that modifies chromatin over extensive regions. Indeed, this type of mechanism has been invoked to account for the repression of the pair-rule gene even-skipped (eve) by the gap repressor Knirps. The attenuation of Pol II elongation has been implicated in a variety of processes. For example, Pol II attenuation has been documented for the transcriptional repression of MYC. Moreover, the activation of the HIV genome is regulated by Pol II processivity. In an effort to distinguish these potential mechanisms, the repression dynamics of several Snail target genes were visualized, because they are silenced in the presumptive mesoderm of precellular embryos (Bothma, 2011).

short gastrulation (sog) encodes an inhibitor of BMP/Dpp signaling that restricts peak Dpp signaling to the dorsal midline of cellularizing embryos. The sog locus is ∼22 kb in length and contains three large introns, including a 5′ intron that is ∼10 kb in length and a 3′ intron that is ∼5 kb in length. The use of separate intronic hybridization probes permits independent detection of 5′ and 3′ sequences within nascent sog transcripts. Individual nuclei are then false colored according to the probe combination they contain (Bothma, 2011).

sog exhibits synchronous activation at the onset of cell cycle 13 (cc13), ∼2 hr after fertilization. There is a lag between the time when nascent transcripts are first detected with the 5′ probe and subsequently cross-hybridize with both the 5′ and 3′ intronic probes. This lag is consistent with the established rates of Pol II elongation in flies, ∼1.1–1.5 kb/min. cc13 persists for ∼20 min, and by the completion of this time window, most of the nuclei in ventral and lateral regions exhibit yellow staining, indicating the presence of multiple nascent transcripts containing 5′ and 3′ intronic sequences within each nucleus. There is little or no repression in ventral regions, presumably due to insufficient levels of the Snail repressor prior to cc14 (Bothma, 2011).

As shown previously, nascent transcripts are aborted during mitosis. Consequently, only the 5′ hybridization probe detects nascent sog transcripts at the onset of cc14. Moreover, a small number of nuclei (at the ventral midline) fail to exhibit nascent transcripts with either the 5′ or 3′ probe, suggesting repression by Snail. This repression becomes progressively more pronounced during cc14 (Bothma, 2011).

Within about 10 min of the first detection of nascent sog transcripts at the onset of cc14, most of the nuclei exhibiting sog expression stain yellow, indicating expression of both 5′ (green) and 3′ (red) intronic sequences. During the next several minutes, progressively more nuclei exhibit only 3′ (red) hybridization signals in ventral regions. This transition from yellow to red continues and culminates in a 'red flash' where the majority of the ventral nuclei that contain nascent transcripts express only the 3′ (red) probe. As cc14 continues, there is a progressive loss of staining in the presumptive mesoderm, and eventually, nascent sog transcripts are lost entirely in the presumptive mesoderm (Bothma, 2011).

These results suggest that after its release from the promoter, Pol II continues to elongate along the length of the sog transcription unit, even as Snail actively represses its expression in the mesoderm. The red flash observed during mid-cc14 represents partially processed nascent sog transcripts that have lost the 5′ intron (hence no green signals with the 5′ hybridization probe) but retain 3′ sequences. Previous studies are consistent with sequential processing of nascent transcripts, beginning with the removal of 5′ intronic sequences and concluding with the removal of 3′ introns. As a control, two separate hybridization probes were used to label opposite ends of sog intron 1. As expected, there was no red flash, because both hybridization signals were simultaneously lost when intron 1 was spliced (Bothma, 2011).

There is an ∼20 min lag between the onset of repression at early cc14 and the complete silencing of sog expression in the presumptive mesoderm during mid- to late cc14. To determine whether this repression lag is a common feature of Snail-mediated gene silencing, additional target genes, including ASPP, Delta, canoe, and scabrous (sca) were examined. ASPP encodes a putative inhibitor of apoptosis, whereas Delta encodes the canonical ligand that induces Notch signaling. All four of these genes exhibit repression lag as they are silenced in the presumptive mesoderm of cc14 embryos (Bothma, 2011).

With the notable exception of Delta, the genes examined in this study contain promoter-proximal paused Pol II, as do most developmental patterning genes active in the precellular embryo. Moreover, results from whole-genome Pol II binding assays indicate that these genes maintain promoter-proximal paused Pol II in the presumptive mesoderm as they are actively repressed by Snail. These findings are consistent with the observation that the segmentation gene sloppy paired 1 retains promoter-proximal paused Pol II even after being silenced by the ectopic expression of Runt and Ftz. Thus, the Snail repressor does not appear to affect Pol II recruitment but rather inhibits the release of Pol II from the promoter-proximal regions of paused genes. At every round of de novo transcription, each Pol II complex at the pause site must receive an activation signal for its release into the transcription unit. It is proposed that the Snail repressor interferes with this signal, resulting in the retention of Pol II at the pause site (Bothma, 2011).

It is currently unclear whether repression lag is a general feature of transcriptional silencing. A recent study suggests that the gap repressor Knirps reduces the processivity of Pol II complexes across the eve transcription unit (Li, 2011). Snail and Knirps might employ distinctive modes of transcriptional repression. Snail recruits the short-range corepressor CtBP, whereas Knirps recruits either CtBP or the long-range corepressor Groucho. When bound to certain cis-regulatory elements within the eve locus, Knirps recruits Groucho, which might propagate a repressive chromatin structure. In contrast, Snail-CtBP might interfere with the release of Pol II from the proximal promoter, as discussed above. There is a considerable difference in the lengths of the genes examined in the two studies. The eve transcription unit is only 1.5 kb in length, less than one-tenth the size of sog. In fact, many patterning genes active in the early fly embryo contain small transcription units only a few kilobases in length. Small transcription units offer dual advantages in rapid patterning processes: essentially no lag in activation or repression (Bothma, 2011).

All five Snail target genes examined in this study exhibit Pol II elongation after the onset of repression. The number of transcripts produced during repression lag depends on the Pol II density across the transcription unit at the onset of repression. Whole-genome Pol II binding assays suggest that there are at least several Pol II complexes per kilobase. This estimate is based on comparing the total amount of Pol II within these genes to that present at the promoter of the uninduced hsp70 gene, for which there are accurate measurements. As a point of reference, the Pol II density on induced heat-shock genes is one complex per 75-100 bp, which is comparable to the footprint size, ∼50 bp, of an elongating Pol II complex. Thus, somewhere in the vicinity of ∼50 (or more) sog transcripts may be produced in a diploid cell after the onset of Snail repression. This represents a significant fraction of the steady-state expression of a typical patterning gene (∼200 transcripts per cell (Bothma, 2011).

Repression lag could impinge on a number of patterning processes, such as Notch signaling. The specification of the ventral midline of the central nervous system depends on the activation of Notch signaling in the ventralmost regions of the neurogenic ectoderm. Sca products somehow facilitate the activation of the Notch receptor, and repression lag could potentially disrupt this process by producing high steady-state levels of Sca in the mesoderm where Notch is normally inactive. Similar arguments might apply to the unwanted accumulation of Delta products in the mesoderm. Perhaps microRNAs are required to inhibit these transcripts and thereby facilitate localized activation of Notch signaling. Indeed, miR-1 is expressed in the presumptive mesoderm, at the right time and place to regulate Sca and/or Delta, and is known to be able to target Delta transcripts. Repression lag is potentially quite severe for Hox genes, particularly Antp and Ubx, which contain large transcription units (75–100 kb) that could take over an hour to silence after the onset of repression. It is conceivable that miRNAs encoded by the miR-iab4 gene, which are known to target Antp and Ubx transcripts, might inhibit postrepression transcripts (Bothma, 2011).

The precellular Drosophila embryo possesses a number of inherently elegant features for the detailed visualization of differential gene activity in development. Indeed, such studies were among the first to highlight the importance of transcriptional repression in the delineation of precise on/off patterns of gene expression. This study extends this rich tradition of visualization by providing the first dynamic view of gene silencing. The key feature of this method is the use of sequential 5′ and 3′ intronic probes to distinguish nascent transcripts produced by Pol II complexes shortly after their release from the promoter versus mature Pol II elongation complexes that have already transcribed 5′ intronic sequences. Elongating Pol II complexes have been shown to complete transcription after the onset of Snail repression and, as a result, moderately sized genes are fully silenced only after a significant lag. It is suggested that this repression lag represents a previously unrecognized constraint on the regulatory dynamics of the precellular embryo (Bothma, 2011).

Pcf11 is a termination factor in Drosophila that dismantles the elongation complex by bridging the CTD of RNA polymerase II to the nascent transcript

The mechanism by which Pol II terminates transcription in metazoans is not understood. This study shows that Pcf11 is directly involved in termination in Drosophila. dPcf11 is concentrated at the 3' end of the hsp70 gene in cells, and depletion of dPcf11 with RNAi causes Pol II to readthrough the normal region of termination. dPcf11 also localizes to most transcribed loci on polytene chromosomes. Biochemical analysis reveals that dPcf11 dismantles elongation complexes by a Pol II C-terminal domain (CTD) dependent but nucleotide-independent mechanism and that dPcf11 forms a bridge between the CTD and RNA. This bridge appears to be crucial because an anti-CTD antibody, which also dismantles the elongation complex, is found to bridge the CTD to RNA. dPcf11 was observed to inhibit transcription at low, but not high, nucleotide levels, suggesting that dPcf11 dismantles paused elongation complexes. These results provide a biochemical basis for the dependency of termination on pausing and the CTD in metazoans (Zhang, 2006).

Termination of Pol II transcription is an essential step in gene expression, but the mechanism is poorly understood. Besides its requirement for recycling use of Pol II, the choice of termination site can influence the availability of splice sites and polyadenylation sites in pre-mRNA. Half of the mRNAs in humans utilize alternate polyadenylation sites, and this can affect the location, stability, and coding potential of the transcripts. Pol II molecules that fail to terminate can inhibit function of downstream promoters by displacing proteins from the DNA. This so-called transcription interference can serve to regulate expression of some genes (Zhang, 2006 and references therein).

Pol II termination is coupled to polyadenylation by the polyadenylation signal in the nascent transcript. Two models have been proposed to explain this coupling. According to the torpedo model, cleavage of the nascent transcript, which precedes polyadenylation, generates an uncapped end on the residual transcript engaged with Pol II. This uncapped end is an entry point for a 5' to 3' exonuclease that chases down the Pol II and induces termination. The torpedo model received recent support with the finding that mutation of a 5' to 3' exonuclease, called Rat1, causes Pol II to readthrough terminators in yeast. Depletion of the homologous protein Xrn2 from human cells also impairs termination on a transiently transfected β globin gene (Zhang, 2006 and references therein).

An alternative model, originally called the antiterminator model but now generalized as the allosteric model, posits that the polyadenylation signal in the nascent transcript causes an allosteric change in Pol II that decreases the processivity of the elongation complex (EC). This could be due to the dissociation of an antiterminator from the EC or association of a factor that depresses processivity. Until recently, the strongest support for the allosteric model was provided by circumstances in which termination occurs in the absence of the cleavage reaction. Under these circumstances, the torpedo model for termination cannot apply, as there is no entry point for the 5' to 3' exonuclease (Zhang, 2006 and references therein).

Recently, a yeast protein called Pcf11 dismantles a yeast Pol II EC. This reaction depends on the CTD of Pol II, thus providing a possible reason for why deletion of the CTD impairs termination in human cells. The CTD corresponds to the unusual C-terminal domain of the largest Pol II subunit and is composed of multiple copies of a heptapeptide with the consensus YSPTSPS. yPcf11 appears to dismantle the EC by bridging the CTD to the nascent transcript. In yeast, mutations in yPcf11 impair both termination and polyadenylation. yPcf11 is in a complex called CF1A, which is involved in processing the 3' end of mRNAs. CF1A recognizes part of the tripartite polyadenylation signal in the GAL7 gene, thus providing a possible basis for how the polyadenylation signal might recruit or regulate the activity of yPcf11. Human Pcf11 is in a complex with at least 15 other polypeptides, and the complex is required for 3' end processing in vitro. The hPcf11 complex interacts with CF1m and CPSF, two proteins that recognize the polyadenylation signal in the nascent transcript. Nothing is known about the role of hPcf11 in termination (Zhang, 2006 and references therein).

Given the results that termination can occur prior to nascent transcript cleavage, and the discovery that yPcf11 could be the engine that drives the termination reaction in yeast it was asked whether Pcf11 is involved in termination in Drosophila (Zhang, 2006).

This study provides evidence that dPcf11 is directly involved in Pol II termination. Immunofluorescence microscopy and ChIP indicate that dPcf11 is concentrated at the 3' end of the hsp70 gene, and depletion of dPcf11 from Drosophila cells increases the level of Pol II normally detected downstream from the polyadenylation signal of hsp70. In addition, the N-terminal region of dPcf11 completely dismantles an elongation complex. This last result sets dPcf11 apart from all other proteins that have been implicated in Pol II termination and is strong evidence that dPcf11 is directly involved in termination. The detection of dPcf11 at most highly transcribed loci in polytene chromosomes suggests that dPcf11 is involved in termination at many genes. dPcf11 provides a basis for connecting three key aspects of termination: the CTD, the polyadenylation signal, and pausing (Zhang, 2006).

A crucial step in the termination reaction mediated by dPcf11 appears to be the formation of a bridge between the CTD and the nascent transcript, since this is the only functional aspect common to the CTD antibody and dPcf11, both of which dismantled the EC. Additional support for the importance of the bridge comes from the analysis of yeast Pcf11: mutations impairing RNA binding or CTD binding each inhibit the dismantling reaction. In addition, the dismantling reaction can be inhibited by hybridizing a DNA oligonucleotide to the nascent transcript in the region just outside from where RNA exits Pol II. Presumably, the oligonucleotide blocks formation of the bridge by interfering with the Pcf11-RNA interaction (Zhang, 2006).

Because the CTD antibody and Pcf11 are structurally unrelated, it is unlikely that the dismantling reaction involves Pcf11 directly recognizing part of the body of Pol II. How the formation of the bridge disrupts the elongation complex is a mystery. One possibility is that constraining the CTD or the RNA causes either of these or Pcf11 itself to contact the RNA exit channel in a way that destabilizes the EC. RNA-protein interactions in the RNA exit channel of bacterial RNA polymerase contribute to pausing and termination. The molecular contacts at the RNA exit channel of the Pol II EC may be uniquely suited for allosteric control of the EC, because it was observed that Rho, which normally functions in termination in bacteria, disrupts Pol II ECs, but not Pol I or Pol III ECs. Rho moves along RNA in a 5' to 3' direction, so it probably collides with the region of Pol II at the RNA exit channel (Zhang, 2006).

dPcf11 seems to interact with a relatively small region of the Drosophila CTD. This is in contrast with the yeast and human CTDs where Pcf11 could in principal coat almost all of the yeast CTD and half of the human CTD. The results from Drosophila suggest that the bridge does not have to form close to the body of the Pol II molecule to dismantle the EC. The binding of dPcf11 to the Drosophila CTD may not be dictated by the heptad per se but by a slightly larger motif that appears four times in the region where dPcf11 bound the CTD. This motif, PSYSPTSP, corresponds to the region of a peptide composed of two consensus heptads that was contacted by yPcf11 in a crystallized complex (Zhang, 2006).

Phosphorylation of the CTD could influence the activity of Pcf11. Phosphorylation of serine 2 in the CTD appears to increase the affinity of yPcf11 for the CTD. Importantly though, yPcf11 binds the unphosphorylated CTD, and there is evidence in yeast indicating that the CTD of Pol II is dephosphorylated just prior to termination. ChIP data indicate that the level of serine 2 phosphorylation increases as Pol II moves from the 5′ to the 3′ end of the hsp70 gene, and the same occurs on several yeast genes (Ahn, 2004). This rising level of serine 2 phosphorylation could contribute to the recruitment of Pcf11 near the 3′ end of the gene. However, the phosphates on the CTD might also antagonize the ability of Pcf11 to form a bridge with the nascent transcript due to electrostatic repulsion. The CTD phosphatase Ssu72 has been implicated in termination. Ssu72 might participate in termination by removing phosphates from the CTD so the bridge can form between the CTD and RNA (Zhang, 2006).

These results show that dPcf11 is concentrated near the polyadenylation signal of hsp70, similar to what was observed for several genes in yeast. Though Pcf11 binds RNA, it seems unlikely that Pcf11 alone recognizes the polyadenylation signal in the nascent transcript for two reasons: (1) equivalent crosslinking is observed to two unrelated RNAs, neither of which contained a polyadenylation signal; (2) amino acids 1-149 of Pcf11 lack any known RNA recognition motifs. Nevertheless, Pcf11 appears to have a surface that interacts specifically with RNA, because mutating one amino acid in yeast Pcf11 impaired RNA binding without affecting CTD binding (Zhang, 2006).

Yeast Pcf11 is part of a complex called CF1A, which contains three other subunits. One subunit, Rna15, recognizes part of the polyadenylation signal, thus providing a way to recruit yPcf11 to the end of the gene after the polyadenylation signal has been transcribed. Human Pcf11 is part of a complex called CFIIA, which itself does not appear to recognize the polyadenylation signal. CFIIA, however, interacts with CPSF and CFI_m, two proteins that recognize different parts of the polyadenylation signal in humans and that are involved in pre-mRNA 3' end processing. If CPSF and CFI_m are involved in recruiting Pcf11 to the 3' end of genes in metazoans, regulation is needed to prevent Pcf11 from prematurely terminating transcription. ChIP detects both CPSF and CFI_m well upstream of the polyadenylation site in the human G6PD gene), and earlier studies indicated that CPSF could be recruited to the 5' end of genes through association with TFIID (Zhang, 2006 and references therein).

The location of pause sites will be a key parameter in dictating where Pcf11 dismantles the elongation complex. As long as the EC is moving, it resists the action of Pcf11. It is suspected that this resistance arises because the RNA reeling out of an actively moving EC interferes with physical interactions that might be required for the dismantling reaction. There are ample data to indicate that pause sites are involved in selection of termination sites. Diverse mechanisms could be used by the cell to cause the EC to pause. These include the presence of pause sites that are intrinsic to the DNA sequence. Intrinsic pauses are found scattered throughout almost any stretch of DNA, so this could account for the stochastic selection of termination sites downstream from a polyadenylation signal. Specific proteins bound to the DNA could cause pausing as appears to be the case for the MAZ protein. Finally, nucleosomes cause ECs to pause. This could explain why chromatin remodeling factors appear to act as terminators (Zhang, 2006 and references therein).

It is concluded that dependence of the Pcf11 dismantling reaction on pausing and the CTD provide possible explanations for why these two things are important for termination. The specificity of termination probably arises from the combinatorial actions of factors that control pausing, the association of Pcf11 with the CTD, and the association of Pcf11 with the nascent transcript. These results provide direct support for an allosteric model of termination but certainly do not preclude possible contributions from an RNA exonuclease after cleavage of the nascent transcript. One possibility that has been proposed is that the exonuclease shortens the residual nascent transcript, forcing Pcf11 to bind close to the RNA exit channel (Zhang, 2006).

Paf1 coordinates histone modifications and changes in nucleosome structure with transcription activation and Pol II elongation

The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. The Drosophila homologs of three subunits of the yeast Paf1 complex have been identified and characterized and striking differences were found between the yeast and Drosophila complexes. Although Drosophila Paf1, Rtf1, and Cdc73 (Hyrax) colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure. Paf1 therefore directs the histone methyltransferase activity and links active transcription and modifications of chromatin structure. The data support a model in which the Drosophila Paf1 complex plays a key role in coordinating histone modifications and changes in nucleosome structure with transcription activation and Pol II elongation, thereby serving as a critical link between gene expression and chromatin structure (Adelman, 2006; full text of article).

Proper control of gene expression is necessary for the development, differentiation, and survival of the cell, and transcription regulation is a cornerstone of this process. The formation of mRNA in eukaryotes involves a complex multistep pathway wherein each step provides an opportunity for regulation. Once RNA polymerase II (Pol II) has been recruited to a promoter and initiates transcription, it must efficiently escape from the promoter-proximal region and transcribe through a gene that is covered with nucleosomes. The nascent RNA must also be capped, spliced, polyadenylated, and exported to the cytoplasm before it can serve as a template for protein translation. Recent evidence from many laboratories indicates that there is a dynamic interplay between the protein complexes that carry out mRNA transcription, processing, and export, such that the efficiency of one step can have significant consequences for other steps in the pathway. For this reason, many factors that are required for the production of functional, mature RNA and were initially thought to directly stimulate Pol II transcription elongation have since been shown to elicit their primary effects on cotranscriptional processing or RNA export. Thus, a major goal towards understanding the mechanisms of transcription regulation requires the identification of both the direct and indirect activities of the numerous factors implicated in RNA production (Adelman, 2006).

The yeast Paf1 complex is one example of a factor that has been linked to a number of transcription-related activities. Yeast Paf1 is a complex of at least five polypeptides (Paf1, Rtf1, Cdc73, Leo1, and Ctr9) that has been implicated in processes as divergent as transcription initiation and elongation, modification of histone tails, phosphorylation of the Pol II C-terminal domain (CTD), RNA processing, and export. Although yeast Paf1 was originally thought to be an alternate mediator based upon its direct interactions with Pol II, it has since been found to be recruited throughout the body of active genes and to associate with the elongation-competent form of Pol II. Additional roles for the Paf1 complex have been suggested by the association of Paf1 with several RNA processing and export factors, such as Ccr4, the major yeast deadenylase, and Hpr1, a component of the THO complex that is involved in the export of mRNAs (Adelman, 2006).

Components of the Paf1 complex are nonessential in yeast, but mutations in Paf1 subunits confer sensitivity to 6-azauracil and generate Spt^– phenotypes, which are generally thought to signify defects in transcription elongation. In vitro transcription assays with naked DNA templates suggested that Paf1 and Cdc73 might directly stimulate transcription elongation; however, it is not clear what effects Paf1 has on elongation rates in vivo. In Saccharomyces cerevisiae, deletion of Paf1 or Cdc73 did not alter the distribution of Pol II on an active gene but dramatically decreased the chromatin immunoprecipitation (ChIP) signal observed for serine 2-phosphorylated (Ser2-P) Pol II. Consistent with a Ser2 phosphorylation defect, recruitment of 3' cleavage and processing factors was impaired in the paf1Delta strain and poly(A) tail length was modestly shortened (Adelman, 2006).

A link between the Paf1 complex and the chromatin architecture within transcribed regions has been suggested by genetic interactions between Paf1 components and Chd1, subunits of the yeast FACT complex (see Drosophila FACT complex), and histone assembly factors in the Hir/Hpc pathway. The packaging of template DNA into nucleosomes is known to represent a formidable obstacle to Pol II elongation in vitro, an obstacle that is overcome in vivo by a number of proteins that facilitate Pol II elongation by modifying chromatin structure and/or stability. Examples of factors that have been implicated in transcription through nucleosomes are chromatin remodeling enzymes, such as Chd1 and Swi/Snf, and histone-binding proteins like Spt6 and FACT. The ensemble of these complexes appear to help disassemble nucleosomes to promote efficient Pol II transcription through bound DNA and then to reassemble nucleosomes after the passage of Pol II. Both Spt6 and FACT have recently been shown to help maintain the proper balance between assembly and disassembly of nucleosomes during active transcription by Pol II, with the loss of these factors leading to a net failure to reassemble nucleosomes in the wake of transcription (Adelman, 2006).

The yeast Paf1 complex is required for ubiquitination of histone H2B at lysine 123 in the promoter-proximal region of activated genes. This ubiquitination event is a prerequisite for the methylation of histone H3 (at lysine residues 4 and 79) that accompanies active transcription in yeast; thus, the latter processes are defective in cells lacking functional Paf1. In addition, the Paf1 complex has been reported to be critical for the recruitment of the yeast SET2 histone methyltransferase complex to actively transcribed genes, leading to methylation of histone H3 at residue lysine 36 (Adelman, 2006 and references therein).

Although the yeast Paf1 complex has been studied extensively, a number of important questions remain unanswered. Key questions concern the nature of the interactions between the subunits of the Paf1 complex and their associations with Pol II, as well as the importance of Pol II binding in Paf1 function. A pivotal issue concerns the fact that deletion of Rtf1 or Cdc73 has been reported to reduce the association of all Paf1 components with the Pol II and chromatin yet lead to much weaker phenotypes than does deletion of the other Paf1 components. These results have led some to propose that the critical role of Paf1 occurs when the complex is not chromatin associated; however, the other potential activities of Paf1 have yet to be clearly identified. Furthermore, the subunit composition of the Paf1 complex in human cells appears to differ from that in yeast, since the human Rtf1 protein does not appear to stably associate with the other members of the Paf1 complex (Adelman, 2006).

To address these issues and to investigate the activity of Paf1-associated proteins in Drosophila, the Drosophila homologs of the yeast Paf1, Rtf1, and Cdc73 proteins were identified and characterized. In vivo analyses of the Drosophila Paf1 complex uncover both important similarities to and differences from the reported functions of Paf1 in yeast and provide insight into the connections among histone methylation, nucleosome stability, and transcription activation in a metazoan organism. Strikingly, the Drosophila Paf1 homolog is a previously annotated gene that encodes an essential protein, suggesting that the role of Paf1 has evolved and become more critical in metazoans. Rtf1 is not stably associated with the Drosophila Paf1 and Cdc73 proteins in vivo and shows only a weak interaction with Pol II. Moreover, when Paf1-depleted cells are assayed by tandem RNA interference (RNAi)-ChIP, no changes were observed in the level of Ser2-P Pol II on the Hsp70 gene, in contrast to results obtained with yeast. Interestingly, it appears that major effects of Paf1 depletion are the loss of H3-K4 trimethylation near the Hsp70 promoter and a significant decrease in the recruitment of Spt6 and FACT to the body of the Hsp70 gene, suggesting that Drosophila Paf1 may coordinate the activities of elongating Pol II with factors that maintain the proper chromatin architecture during transcription (Adelman, 2006).

This study shows that the most striking similarities between the yeast and Drosophila Paf1 complexes are their association with elongating RNA Pol II and their roles in gene activation, while the nature of the Pol II association and the composition of the Paf1 complex reflect marked differences between the species. The global view provided by Drosophila polytene chromosomes shows that the chromosome-associated Paf1 and Rtf1 proteins colocalize with active Pol II. This result supports the idea that these proteins participate in most, if not all, Pol II transcription. Remarkably, Paf1 and Rtf1 do appear to be separable from actively elongating Pol II under conditions of heat shock. Although Paf1 and Rtf1 are recruited actively to heat shock loci upon heat stress, these factors also remain associated with a number of additional sites on the chromosome, while Pol II is localized almost exclusively at heat shock loci under these conditions. These data suggest that Paf1 and Rtf1 may remain bound to the chromosome at activated genes through interactions with additional proteins (Adelman, 2006).

It has been suggested that, in yeast, while the Paf1 complex is entirely nuclear in its localization, it has cellular functions that are independent of elongating Pol II. The nucleolar association of Paf1 and Rtf1 observed on Drosophila polytene chromosomes could possibly represent such a function. At the nucleolar organizer, Paf1 shows broad labeling while the Rtf1 signal is restricted to the nucleolar periphery in a manner that is largely nonoverlapping. Interestingly, although the yeast Paf1 complex does not show strong nucleolar association normally, in an Rtf1 mutant, the Paf1 complex shows a strong association that is postulated to be a manifestation of its normal role in nuclear processing or export (Adelman, 2006).

By using ChIP experiments, this study obtained a higher-resolution view of the localization of Paf1, Rtf1, and Cdc73 at the Hsp70 gene. The lack of a ChIP signal at Hsp70 under uninduced conditions demonstrates that the presence of engaged Ser-5-P Pol II or the associated elongation factors such as Spt5 and TFIIS is not sufficient to recruit Paf1, Rtf1, or Cdc73. Upon heat induction, recruitment of all three proteins was observe primarily within the coding regions of active Drosophila genes, rather than regions upstream of the promoter, or downstream of the site for cleavage and polyadenylation. The reduction in the Paf1 signal downstream of the polyadenylation site, which accompanies a decrease in the Pol II signal, likely signifies that Paf1 dissociates from chromatin within this region, consistent with recent results obtained with yeast. However, it is noted that the absence of a significant Paf1 signal obtained with a given primer pair may simply indicate that the interactions of Paf1 with a particular region are transient (Adelman, 2006).

The Paf1 complex in S. cerevisiae has been reported to be required for full Ser-2 phosphorylation of the Pol II CTD. This role of Paf1 in CTD phosphorylation regulation also appears consistent with the fact that rtf1Delta mutants show synthetic lethality with CTD kinase and phosphatase mutants in CTK1 and FCP1. The lack of a Ser-2-P Pol II signal detected in yeast Paf1 mutants resulted in reduced recruitment of cleavage and polyadenylation factors, causing a defect in the polyadenylation of nascent transcripts. However, although depletion of Drosophila Paf1 or Rtf1 has a marked effect on induced Hsp70 RNA levels, no change was seen in the levels of Ser2-P Pol II on the Hsp70 gene in Paf1 or Rtf1 RNAi-treated cells, indicating a difference between the functions of Paf1 in yeast and a metazoan system (Adelman, 2006).

Another fundamental difference that observed between Drosophila and yeast Paf1 complexes is the relationship of the Paf1 and Rtf1 subunits in providing anchorage of the complex to Pol II. In yeast, it has been shown that the association of Paf1 with Pol II and active chromatin depends on the presence of Rtf1. In contrast, this study found that the recruitment of Paf1 to activated Drosophila Hsp70 is independent of Rtf1, while Rtf1 recruitment is dependent on Paf1. These results may reflect the evolution of a more important role for the Paf1 protein in metazoans in providing affinity of the complex for Pol II, while Rtf1 became a more loosely bound component of the complex (Adelman, 2006).

The role was investigated of Drosophila Paf1 in the modification of histones within actively transcribed regions. Whereas yeast Paf1 has been implicated in regulating the bulk levels of methylation of histone H3 at lysine residues 4 and 79, an effect was observed of Paf1 depletion on the trimethylation of H3-K4, but not on di- or trimethylation of H3-K79. Similarly, it was observed that trimethylation of H3-K4 occurred within the promoter-proximal region of Hsp70 and Hsp26 upon heat shock and could be seen to increase from 2.5 to 10 min after heat induction, but no significant levels of H3-K79 dimethylation were observed within the active Hsp70 gene. The latter result differs from results from other systems which link H3-K79 dimethylation with active transcription. However, it is consistent with recent data suggesting that both Grappa, the Drosophila H3-K79 methyltransferase, and the signal corresponding to H3-K79 dimethylation are localized to both active and intergenic regions of Drosophila polytene chromosomes. An alternative possibility is that the apparent differences between yeast and Drosophila result from the experimental systems used; RNAi treatments in Drosophila decrease, but do not completely abolish, their target, and thus the small amount of remaining protein may be sufficient to carry out certain functions. Conversely, the deletion mutants used to investigate yeast Paf1 entirely remove an important protein for many generations of cell growth, raising the possibility that some observed effects are indirect or secondary in nature (Adelman, 2006).

It is interesting that although H3-K4 trimethylation depends upon Paf1 and the recruitment of Paf1 is temporally similar to H3-K4 methylation, the distribution of Paf1 appears to be spatially distinct from the promoter region where the strongest trimethylated H3-K4 signals are observed. Thus, the results suggest that the effects of Paf1 mutants on the modification of promoter-proximal nucleosomes (including the ubiquitination of H2B-K123) may occur through indirect mechanisms. These data are consistent with reports on yeast that indicate that the distribution of Paf1 subunits does not strictly correlate with the patterns of ubiquitinated H2B or methylated histone H3. The localization of H3-K4 trimethylation reported in this study is in agreement with the recently described distribution of Trithorax, a Drosophila H3-K4 methyltransferase. Furthermore, recent studies employing a Drosophila Trithorax mutant fly line suggest that a multiprotein complex that contains Trithorax plays a role in Hsp70 gene activation. However, whether the role of Trithorax in Hsp70 activation is direct or indirect remains to be established. It is noted that no effect of Paf1 depletion is observed on the rates of Pol II recruitment, or distribution over the gene, suggesting that H3-K4 trimethylation may serve as a mark of transcription activation rather than a prerequisite for gene activation (Adelman, 2006).

These studies have provided new insights into the increased importance of the Paf1 complex in a metazoan system. It is significant that Paf1 is recruited in a manner that is spatially and temporally identical to that of chromatin-associated factors Spt6 and FACT. In agreement with the strong colocalization of Paf1 with these nucleosome-associated factors, it was shown that depletion of Paf1 significantly reduces the recruitment of both Spt6 and the FACT subunit SSRP1. A relationship among Paf1, Rtf1, and FACT is consistent with findings that an rtf1Delta mutation shows synthetic lethality with POB3, a subunit of the yeast FACT complex. Moreover, the FACT complex has been shown to interact with the Paf1 complex and the chromodomain-containing Chd1 protein at actively transcribed genes. In vitro, FACT has been shown to function optimally to facilitate transcription through nucleosomes when it is present at approximately one molecule of FACT per two nucleosomes; the effectiveness of FACT in promoting elongation is decreased dramatically below this threshold. If these results reflect the situation in vivo, the greater than 50% decrease in FACT levels at the active Hsp70 gene in Paf1-depleted cells would result in a rather pronounced effect on transcription through nucleosomes (Adelman, 2006).

Furthermore, recent evidence obtained with yeast has shown that mutations of Spt6 or the FACT subunit Spt16 lead to aberrant chromatin architecture in the wake of elongating Pol II, presumably due to defects in reassembly of nucleosome structure. The failure to efficiently repackage transcribed DNA results in transcription initiation from cryptic sites and a reduction in levels of properly initiated and processed RNA. If a primary role of Drosophila Paf1 is to help stably recruit factors like Spt6 and FACT, then loss of Paf1 activity could also lead to the accumulation of nonfunctional or improperly processed RNA species. In support of this idea, a paper that was published during the preparation of this report states that mutations in yeast Spt6 alter the recruitment of Paf1 subunit Ctr9 and lead to defects in 3'-end processing of nascent RNA. It is thus tempting to speculate that the vast array of transcription elongation and RNA processing and export defects reported in yeast Paf1 mutant strains could result from perturbation of the nucleosome structure along actively transcribed genes. Moreover, it may be these chromatin and processing defects that account for the decrease in the amount of Hsp70 mRNA that accumulates in response to heat shock in Paf1- or Rtf1-depleted cells (Adelman, 2006).

Finally, the Paf1 gene in yeast is nonessential while the Paf1 gene in Drosophila is essential. This may reflect the more varied and demanding requirements of the transcription machinery in higher eukaryotes, where chromatin frequently plays a greater and more stringent role in regulation. This, in turn, may place a greater demand on the Paf1 complex, which appears to function at the interface between transcription and chromatin, perhaps serving as a platform that stimulates the association of a number of nucleosome-modifying complexes with actively elongating Pol II (Adelman, 2006).

In summary, the gene for Paf1 is a required Drosophila gene that colocalizes with actively elongating Pol II when chromatin associated and plays a critical role in the activation of stress-induced genes. Furthermore, recent data reveal that mutations in parafibromin, the human homolog of the Paf1 complex subunit Cdc73, are associated with an elevated risk of parathyroid carcinomas; thus, the Paf1 complex may be a key regulator of cellular control in metazoans. The connection between Paf1 and trimethylation of histone H3 at lysine 4 near the promoters of active genes is particularly interesting because a human homolog of Trithorax, the histone methyltransferase implicated in this activity, is ALL-1/MLL-1, which is associated with a number of acute leukemias. Future work to define the way in which Paf1 directs the histone methyltransferase activity of this key enzyme should provide insight into the interaction between active transcription and modifications of chromatin structure. The data support a model in which the Drosophila Paf1 complex plays a key role in coordinating histone modifications and changes in nucleosome structure with transcription activation and Pol II elongation, thereby serving as a critical link between gene expression and chromatin structure (Adelman, 2006).

Phosphorylation of histone H3 at Ser10 by JIL-1 facilitates RNA polymerase II release from promoter-proximal pausing in Drosophila

The Drosophila JIL-1 kinase is known to phosphorylate histone H3 at Ser10 (H3S10) during interphase. This modification is associated with transcriptional activation, but its function is not well understood. Evidence is presented suggesting that JIl-1-mediated H3S10 phosphorylation is dependent on chromatin remodeling by the brahma complex and is required during early transcription elongation to release RNA polymerase II (Pol II) from promoter-proximal pausing. JIL-1 localizes to transcriptionally active regions and is required for activation of the E75A ecdysone-responsive and hsp70 heat-shock genes. The heat-shock transcription factor, the promoter-paused form of Pol II (Pol IIo^ser5), and the pausing factor DSIF (DRB sensitivity-inducing factor) are still present at the hsp70 loci in JIL-1-null mutants, whereas levels of the elongating form of Pol II (Pol IIo^ser2) and the P-TEFb kinase are dramatically reduced. These observations suggest that phosphorylation of H3S10 takes place after transcription initiation but prior to recruitment of P-TEFb and productive elongation. Western analyses of global levels of both forms of Pol II further suggest that JIL-1 plays a general role in early elongation of a broad range of genes. Taken together, the results introduce H3S10 phosphorylation by JIL-1 as a hallmark of early transcription elongation in Drosophila (Ivaldi, 2007).

The eukaryotic cell packages its DNA wrapped around histone proteins to form nucleosomes, the basic units of chromatin. These nucleosomes assemble into higher-order chromatin structures through which the transcription machinery must navigate each time it is signaled to transcribe. Mechanisms have consequently evolved to maintain a flexible chromatin state that can readily respond to intrinsic and extrinsic stimuli and accordingly modulate gene expression. Most prominently, histone-modifying enzymes can methylate, acetylate, and phosphorylate various amino acid residues of histone N termini, thereby changing their affinity for different transcriptional regulators. ATP-dependent chromatin remodeling complexes can also be recruited to alter the position and accessibility of the nucleosome. The binding of specific transcription factors triggers a cascade of events during which these diverse chromatin modulators work in concert to allow the RNA polymerase II (Pol II) machinery to bind target genes, initiate transcription, and elongate the messenger RNA (mRNA). These regulators maintain tight control of transcription throughout the elongation process by continuously communicating with the C-terminal domain (CTD) of the largest subunit of Pol II (Ivaldi, 2007 and references therein).

The CTD of Pol II consists of a heptad repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) that is conserved from yeast to humans. It integrates transcriptional events by interacting with distinct regulatory proteins that recognize different patterns of CTD phosphorylation. When Pol II is first recruited to the promoter as part of the preinitiation complex, its CTD is hypophosphorylated. After Pol II disengages from the promoter, the CTD becomes phosphorylated at Ser5 (Pol IIo^ser5) by TFIIH, a general transcription factor that is part of the Pol II machinery. As part of an early elongation complex, Pol II progresses 20-40 base pairs (bp) downstream from the promoter. It then pauses in a process referred to as promoter-proximal pausing to allow for capping of the nascent mRNA. DRB sensitivity-inducing factor (DSIF, Spt5) and negative elongation factor (NELF) cooperate to repress transcription elongation and maintain this pause. Pol II is released once the P-TEFb kinase is recruited to relieve the negative effects of DSIF and NELF and phosphorylate the CTD at Ser2 (Pol IIo^ser2), marking the onset of productive elongation. The various transcriptional steps are associated with distinct histone modifications and chromatin remodeling complexes. Set1, the enzyme responsible for methylating Lys4 of histone H3 (H3K4) in Saccharomyces cerevisiae, is known to physically associate with the CTD of Pol II when it is phosphorylated at Ser5. At the same time, trimethylation of H3K4 has been found concentrated at the 5' end of transcribed genes. Methylation of Lys36 of H3 (H3K36), on the other hand, is associated with a later step in elongation; this mark accumulates further downstream from the promoter and associates with the CTD when phosphorylated at Ser2. Other modifications, such as lysine acetylation, arginine methylation, and serine phosphorylation, have also been associated with activation of gene expression. Of interest, phosphorylation of histone H3 at the Ser10 residue (H3S10) has been shown to be important for activation of transcription in yeast, Drosophila, and mammalian cells, but its precise role in this process is not well understood (Ivaldi, 2007 and references therein).

Several studies have suggested an important role for H3S10 phosphorylation in specific transcriptional responses to signaling stimuli. The yeast Snf1 kinase phosphorylates H3S10 upon activation of the INO1 gene. In mammalian fibroblasts, rapid phosphorylation of histone H3 concomitant with activation of immediate-early (IE) response genes takes place when cells are treated with growth factors and various stress-inducing agents. Further, Coffin-Lowry syndrome is characterized by impaired transcriptional activation of the c-fos gene and a loss of EGF-induced phosphorylation of histone H3S10. Treatment of immature rat ovarian granulosa cells with follicle-stimulating hormone produces rapid H3S10 phosphorylation in a PKA-dependent manner, suggesting a role for histone phosphorylation in cellular differentiation. Additionally, H3S10 phosphorylation follows the stimulation of the suprachiasmatic nucleus of rats with light and activation of hippocampal neurons. It further appears to play a central role during cytokine-induced gene expression mediated by IkappaB kinase α (IKK-α). What remains unclear from these studies is whether H3S10 phosphorylation is limited to mediating signal transduction events or whether it plays a more general role in the activation of gene expression in vertebrates (Ivaldi, 2007).

Studies in Drosophila suggest that this modification may be required for the transcription of most genes in this organism. Using the heat-shock response as a model system, it has been established that H3S10 phosphorylation patterns parallel those of active genes. Drosophila responds to a rise in temperature by rapidly increasing the transcription of heat-shock genes while repressing genes expressed previously. Before heat shock, phosphorylated H3S10 localizes to euchromatic regions of polytene chromosomes and colocalizes with Pol II. After heat shock, this modification redistributes to the active heat-shock loci and disappears from the rest of the chromosome, where genes are now repressed (Nowak, 2000; Ivaldi, 2007).

Despite these observations, the precise role of H3 phosphorylation in gene activation remains elusive. The mammalian MSK1 and MSK2 kinases, among others, have been shown to be responsible for H3S10 phosphorylation associated with transcription. The Drosophila homolog of MSK1/2, the JIL-1 threonine/serine kinase, has been shown to phosphorylate H3S10 in vitro. H3S10 phosphorylation levels in vivo are dramatically reduced in JIL-1^z2-null mutants. The JIL-1 protein localizes to interband regions of polytene chromosomes and is found up-regulated on the male X chromosome. Furthermore, the JIL-1^z2 allele enhances the phenotype of trx-G mutations. These data indirectly suggest that JIL-1-mediated H3S10 phosphorylation plays an important role in transcriptional activation (Ivaldi, 2007).

This study further characterizes the role of JIL-1-mediated H3S10 phosphorylation in transcription. JIL-1 is required for the transcription of the majority of, if not all, Drosophila genes. Mechanistic analyses place the phosphorylation event subsequent to transcription initiation but prior to productive elongation; JIL-1 plays an integral role in the release of Pol II from promoter-proximal pausing. The data therefore highlight H3S10 phosphorylation as a novel hallmark of early productive elongation in Drosophila (Ivaldi, 2007).

These results establish H3S10 phosphorylation by JIL-1 as a key event during early elongation of transcription in Drosophila. JIL-1 appears to interact with the transcription machinery at most or all actively transcribed regions on Drosophila polytene chromosomes, including active ecdysone and heat-shock genes. At the same time, expression levels of the hsp70 and E75A genes are decreased in JIL-1-null mutants. Importantly, when JIL-1 is mutated, a global decrease in the phosphorylation levels of elongating RNA polymerase II is observed, suggesting that JIL-1 is required for transcription of the majority of genes (Ivaldi, 2007).

The results further elucidate the timing of H3S10 phosphorylation within the framework of the cascade of events that lead to activation of transcription in eukaryotes. Phosphorylation of H3S10 is not required for transcription factor recruitment, since loss of JIL-1 does not affect binding of HSF at the hsp70 genes after heat shock. Also, H3S10 phosphorylation is dependent on BRM chromatin remodeling, which is required genome-wide prior to the recruitment of Pol II. Transcription initiation can take place independently of JIL-1, as shown by the normal levels of Pol IIo^ser5 and H3K4 methylation in JIL-1^z2 mutants, indicating that the chromatin environment in the absence of JIL-1 is still suitable for transcription initiation. However, productive elongation is impaired in these mutants, as is evident by the decrease in Pol IIo^ser2 levels. These findings introduce H3S10 phosphorylation as a new component of an increasingly complex chromatin environment that is required at the onset of transcription elongation in Drosophila, suggesting a role for JIL-1 in the release of Pol II from promoter-proximal pausing and facilitation of early elongation. Specifically, in JIL-1 mutants, P-TEFb is not detected at the induced hsp70 genes while levels of DSIF are maintained. In the absence of P-TEFb, neither DSIF nor Pol II can be phosphorylated, which is sufficient to block productive elongation. It is likely that Pol II arrests in a paused state and cannot elongate in these mutants. It is also possible that Pol II continues to elongate but is unable to communicate with the proper mRNA processing machinery, which is normally contingent on Ser2 phosphorylation of its CTD (Orphanides, 2002). In this case, the mRNA would be produced but quickly degraded, leading to the transcription defects observed in the Northern analyses. Further work is needed to distinguish between these two possibilities (Ivaldi, 2007).

Although JIL-1 is required for transcription, its presence is not sufficient to ensure gene activation, since JIL-1 is present at all previously transcribed genes that are silenced after heat shock, whereas phosphorylated H3S10 is found exclusively at the transcriptionally active heat-shock genes (Nowak, 2000). Nevertheless, recruitment of JIL-1 to the hsp70 gene is transcription dependent. One possibility is that JIL-1 can exist in both active and inactive states. Once recruited to activate a gene, it may eventually be repressed by inactivation rather than disassociation. Alternatively, the net levels of phosphorylated H3S10 could result from a delicate balance between kinase and phosphatase activities. It has been proposed previously that phosphatase 2A (PP2A) plays a major role in transcription-dependent H3S10 phosphorylation (Nowak, 2003). Therefore, even if JIL-1 is actively maintained at silent genes, its action may be counterbalanced by PP2A. Further studies are required to shed light on how JIL-1 activity can be regulated to affect transcription (Ivaldi, 2007).

In vertebrates, phosphorylation of H3S10 seems to be limited to transcription activation of specific genes in the context of particular signal transduction pathways. In fact, activation of the hsp70 genes by different stressors in mammalian cells is associated with distinct signaling pathways that are not always linked to H3S10 phosphorylation. Contrary to the Drosophila response, heat shock elicits histone H4 acetylation instead of H3S10 phosphorylation at the hsp70 loci in mouse fibroblasts. In contrast, both H3S10 phosphorylation and H4 acetylation are detected at the hsp70 genes upon arsenite treatment of the same cells (Thomson. 2004). Therefore, mammals appear to have more diverse mechanisms of transcription activation and may partially rely on H3S10 phosphorylation in a context-dependent manner. In yeast, substituting the H3 Ser10 for an Ala prevents the recruitment of the TATA-binding protein to the INO1 and GAL1 gene promoters, suggesting that H3S10 phosphorylation is required for the assembly of the preinitiation complex. It would be interesting to explore the significance of this apparent diversity across species (Ivaldi, 2007 and references therein).

The results presented in this study shed light on the mechanism of transcription regulation by H3S10 phosphorylation. It has been recently shown that H3S10 phosphorylation antagonizes the binding of the heterochromatin protein HP1 to histone H3 methylated in Lys9 (H3K9) during mitosis in mammalian cells. It was consequently proposed that JIL-1 maintains chromosome structure in Drosophila by counteracting heterochromatin formation and preventing its spreading into euchromatin. This model for JIL-1 activity could explain a lack of transcription in JIL-1^z2 mutants, since any ectopic heterochromatin would make the DNA inaccessible to the Pol II machinery. However, contrary to such a prediction, the current results show that heat-shock puffs are still formed in JIL-1^z2mutants, and transcription factors and the Pol II machinery retain the ability to bind despite the disruption of chromatin structure. Furthermore, transcription can be initiated, as is evident by the phosphorylation of Pol II at Ser5. This requires several components of the core transcription machinery and the procession of Pol II a few bases downstream from the promoter. These results suggest that, rather than contribute to global chromosome structure, JIL-1-mediated H3S10 phosphorylation may be required to maintain a local chromatin environment that serves as a platform for the recruitment of P-TEFb and the consequent release of Pol II from promoter-proximal pausing (Ivaldi, 2007).

It has become increasingly evident that transcription elongation is a rate-limiting step of gene expression that requires tight regulation. It was reported recently that the majority of gene promoters in human embryonic stem cells are occupied by a promoter-proximally paused Pol II, poised for productive elongation (Guenther, 2007). This suggests that the expression of these genes is predominantly regulated at the level of Pol II release rather than during preinitiation. The exact mechanism of P-TEFb recruitment, a key step in this process, remains to be determined. Several transcription regulators have been shown to recruit P-TEFb, but this is the first evidence of a histone modification required precisely at the timing of recruitment (Ivaldi, 2007).

The exact contribution of H3S10 phosphorylation to P-TEFb recruitment remains open to further investigation. Recent reports have shown that the ubiquitous protein 14-3-3 binds to H3 only when phosphorylated at Ser10, and this interaction could provide a mechanistic link between H3S10 phosphorylation and P-TEFb (Macdonald, 2005). It is possible that 14-3-3 interacts with P-TEFb directly or indirectly through other transcription regulators that are known to recruit it. Alternatively, 14-3-3 is known to interact with many chromatin-related proteins, thus providing another avenue to manipulate the local chromatin environment to support P-TEFb recruitment and early elongation. Further analyses will be necessary to test these hypotheses and clarify the role and mechanism of regulation of JIL-1 and H3S10 phosphorylation in gene expression (Ivaldi, 2007).

Set2 associates with hyperphosphorylated RNAPII

Recent reports have shown that Set2 from various organisms binds to the hyperphosphorylated CTD of RNAPII, implying that K36 methylation plays an important role in the transcription elongation process. The presence of both the WW and SRI domains suggested that Set2 may associate with RNAPII also in Drosophila. To address this issue, extracts prepared from control or Set2 RNAi embryos were immunoprecipitated with antibodies directed against Ser5-phosphorylated CTD, followed by immunoblotting with antibodies directed against Set2 and Ser5-phosphorylated CTD form of RNAPII. Immunoprecipitation of Ser5-phosphorylated CTD resulted in strong immunoreactivity of both phosphorylated CTD and Set2 in control embryos whereas no Set2 is detected in extracts from RNAi embryos. This result was corroborated by showing co-localization of Set2 and elongating RNAPII on salivary gland chromosomes. While these results demonstrate that Set2 is associated with the elongating form of RNAPII in Drosophila, the precise role of this association is currently unclear. However, the fact that a loss of Set2/K36 methylation results in mutant phenotypes associated with defects in the ecdysone response indicates that Set2/K36 methylation plays an important role in the ecdysone regulatory hierarchy (Stabell, 2007).

Histone H3 K36 methylation is mediated by a trans-histone methylation pathway involving an interaction between Set2 and histone H4

Set2-mediated H3 K36 methylation is an important histone modification on chromatin during transcription elongation. Although Set2 associates with the phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), the mechanism of Set2 binding to chromatin and subsequent exertion of its methyltransferase activity is relatively uncharacterized. This study identified a critical lysine residue in histone H4 that is needed for interaction with Set2 and proper H3 K36 di- and trimethylation. It was also determined that the N terminus of Set2 contains a histone H4 interaction motif that allows Set2 to bind histone H4 and nucleosomes. A Set2 mutant lacking the histone H4 interaction motif is able to bind to the phosphorylated CTD of RNAPII and associate with gene-specific loci but is defective for H3 K36 di- and trimethylation. In addition, this Set2 mutant shows increased H4 acetylation and resistance to 6-Azauracil. Overall, this study defines a new interaction between Set2 and histone H4 that mediates trans-histone regulation of H3 K36 methylation, which is needed for the preventative maintenance and integrity of the genome (Du, 2008).

Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7

RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr¹-Ser²-Pro³-Thr⁴-Ser⁵-Pro⁶-Ser⁷. Differential phosphorylation of serine-2 and serine-5 at the 5' and 3' regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, serine-7 phosphorylation has been revealed on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. These results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression (Chapman, 2007).

Differential phosphorylation of CTD residues of the large subunit of eukaryotic RNA polymerase II (Pol II) occurs during the transcription cycle and appears to orchestrate the recruitment, activation, and displacement of various factors involved in transcription and mRNA processing. A variety of kinases have been identified, with phosphorylation activity directed toward the amino acids tyrosine-1 (Abl1/2), serine-2 (CTDK1, CDK9, and DNA-PK), serine-5 (ERK1/2 and CDK7-9), and serine-7 (DNA-PK). The mammalian CTD is >99% conserved across species and possesses almost double the length of its yeast counterparts. A minimum length of CTD is required to support the growth of yeast or mammalian cells. However, this is dependent on the number and position of consensus and nonconsensus repeats, which suggests that CTD function is composed of both sequence and length. Of the 52 mammalian CTD repeats, 21 obey the consensus sequence and lie largely proximal to the Linker region. The distal C-terminal region deviates from this consensus, predominantly at position 7. These nonconsensus repeats may affect the binding of specific factors or may serve to prevent phosphorylation at the position of deviation. Indeed, studies in vivo suggest that they are equivalent to consensus repeats for functions such as splicing of the fibronectin extra domain I exon but not for maintenance of long-term cell viability (Chapman, 2007).

To investigate the role of the CTD repeat structure on its phosphorylation, a system was established that allows the comparison of CTDs of different lengths and repeat compositions in vivo. Recombinant polymerases are engineered with a point mutation conferring resistance to α-amanitin, allowing the endogenous polymerase to be inhibited (and degraded) after addition of α-amanitin but without affecting recombinant polymerase activity. Monoclonal antibodies (mAbs) were produced against the CTD phosphoserine epitopes Ser²-P, Ser⁵-P, and Ser⁷-P. In preparing these antibodies, earlier findings were considered that showed that the functional unit of the CTD is not the heptad repeat itself but is in a sequence lying within heptapeptide pairs. Thus, in the production and testing of these antibodies, a panel of di-heptapeptides with various modifications was used. Analysis of these antibodies and commercially available antibodies revealed that some recognition profiles were limited by modifications on neighboring repeats. For example, the α-Ser⁷-P antibody (4E12) is affected by upstream, but not downstream, Ser⁵-P (Chapman, 2007).

Combining these tools, the phosphorylation of wild-type (WT) CTD was compared with that of different lengths of consensus repeats. If all repeats are equally accessible to CTD kinases, intensities of phosphorylation signals should be expected for WT and mutants 1 to 8 proportional to CTD length. Dual labeling of membranes with α-Rpb1 antibody (mAb Pol3/3 recognizes an epitope outside the CTD) and with α-phospho-CTD antibody reveals forms of different mobility -- the rapidly migrating, unmodified IIa form and the slower, modified IIo form. For mutants containing 16 to 24 consensus repeats, the majority of Pol II is not efficiently phosphorylated and accumulates in the IIa form. Within the IIo form, Ser²-P appears in a sharp, slow migrating band, whereas in longer CTDs, Ser⁵-P appears largely in a band migrating between the Ser²-P band and IIa, which suggests that at least two populations of phosphorylated CTD exist in vivo at any time: Ser²-P alone and Ser⁵-P alone. These data are supported by both the recognition profiles of the antibodies and previous work showing a shift in IIo to a faster migrating form upon treatment with a Ser²-kinase inhibitor. Antibody raised against Ser⁷-P revealed the existence of this epitope in vivo, which is distributed among the major Ser²-P and Ser⁵-P reactive bands. The epitope is lacking from the Ser⁵-P band that appears just above the IIa form. Strong reactivity of α-Ser⁷-P is detectable for a band between IIa and IIo. Furthermore, although Ser²-P and Ser⁵-P appear in all mutants, Ser⁷-P appears only in mutants with more than 24 repeats (Chapman, 2007).

To investigate the effect of nonconsensus repeats on the distribution of phosphorylation, a panel of CTD mutants was analyzed for their reactivity against phospho-CTD antibodies. α-Ser⁷-P does not recognize a mutant lacking Ser⁷ but strongly recognizes mutants containing Ser⁷ substituted with glutamic acid (S7E), indicating either that this antibody recognizes a CTD conformation or that S7E can structurally mimic Ser⁷-P for antibody recognition. Furthermore, replacement of Ser⁷ with alanine prevents recognition of the intermediate band between IIa and IIo by α-Ser⁵-P, suggesting that this form may be Ser⁷-P-dependent (Chapman, 2007).

Because deviations from serine at position 7 in the WT CTD are concentrated in its distal region, chimeras were produced to assess the effect of proximal and distal positioning of nonconsensus repeats. The two chimeras of consensus repeats, and repeats containing S7E substitutions, produce a form that migrates between IIa and IIo. The proximal positioning of nonconsensus repeats (S7A and S7T/K) affects the appearance of a form similar in mobility to the intermediate IIo Ser^5/7-P-reactive band seen in mutants of >35 pure consensus repeats (Chapman, 2007).

To determine whether Ser⁷ phosphorylation is a physiological event during the transcription cycle, chromatin immunoprecipitation (ChIP) experiments were conducted. A detailed example is shown for the T cell receptor beta (TCRβ) gene locus. Ser⁷ was phosphorylated on transcribing Pol II, appearing strongly at the promoter and increasing toward the 3' region of TCRβ. The differences in Ser² phosphorylation that was observe, compared with earlier data, may result from the antibodies used, because the H5 antibody preferentially recognizes repeats with phosphorylated Ser² and Ser⁵ (Chapman, 2007).

Given that Ser⁷ is phosphorylated across TCRβ and all other genes tested (GAPDH, RPLPO, and RPS27), the ability of synthetic polymerases to transcribe and produce mature mRNA from the c-myc and pes1 genes was analyzed. The effect on c-myc and pes1 mRNA levels of Ser⁷ substitution to E or K/T appears dependent on its position, either proximal or distal to the Linker, suggesting again that functional differences exist between these regions. Substitution of Ser⁷ to the non-phosphoacceptor, alanine, did not obviously affect mRNA levels, nor did it affect the long-term growth of cell lines, although viability was compromised. This may be due to the effect of this mutation on small nuclear RNA genes (Chapman, 2007).

ChIP experiments revealed that S7E-containing mutants do not stably associate with any of the genes tested, providing an explanation for the deficit in mRNA observed for mutants containing S7E in the Linker-proximal region. Mutants containing either 48 consensus or S7A repeats appear to be recruited to protein coding genes at similar levels (Chapman, 2007).

It is concluded that the nature of the amino acid at position 7 of the Linker-proximal CTD region is important in steps involved in the stable association of Pol II with class II genes. Accumulation of Ser⁷-P in the 3' region of the TCRβ gene may suggest a role in transcription and/or 3' RNA processing of some protein-coding genes. Previous models can now be expanded for the cycle of CTD modification across genes that are transcribed by RNA polymerase II, not only to show how potential phosphorylation patterns change from 5' to 3' regions across a gene but also to speculate as to the region of the CTD in which they occur. Phosphorylation of Ser⁷ in the proximal part of CTD and replacement of Ser⁷ by other amino acids in the distal part of CTD may constitute an added layer of gene regulation by mammalian RNA polymerases (Chapman, 2007).

Drosophila MCRS2 associates with RNA polymerase II complexes to regulate transcription

Drosophila MCRS2 (dMCRS2; MCRS2/MSP58 and its splice variant MCRS1/p78 in humans) belongs to a family of forkhead-associated (FHA) domain proteins. Whereas human MCRS2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 has been largely uncharacterized. Recent data show that MCRS2 is purified as part of a complex containing the histone acetyltransferase MOF (males absent on first) in both humans and flies. MOF mediates H4K16 acetylation and regulates the expression of a large number of genes, suggesting that MCRS2 could also have a function in transcription regulation. This study shows that dMCRS2 copurifies with RNA polymerase II (RNAP II) complexes and localizes to the 5' ends of genes. Moreover, dMCRS2 is required for optimal recruitment of RNAP II to the promoter regions of cyclin genes. In agreement with this, dMCRS2 is required for normal levels of cyclin gene expression. A model is proposed whereby dMCRS2 promotes gene transcription by facilitating the recruitment of RNAP II preinitiation complexes (PICs) to the promoter regions of target genes (Anderson, 2010).

The initiation of mRNA transcription involves the assembly of a transcription preinitiation complex (PIC), which as a minimum includes RNA polymerase II (RNAP II), Mediator, and six general transcription factors (TFIIA, -B, -D, -E, -F, and -H) at the core promoter DNA region. PIC assembly is initiated by the binding of the TATA box binding protein (TBP) subunit of TFIID to the promoter, which is stabilized in the presence of TFIIA and Mediator. Subsequently, TFIIB binds to and stabilizes the TFIIA-TFIIB-Mediator-DNA complex and functions as an adaptor that recruits the preformed RNAP II-TFIIF complex to the promoter. TFIIE and TFIIH then join to form the complete PIC (Anderson, 2010).

Once the PIC has been assembled on the promoter, transcription initiation occurs in several steps, which involve extensive phosphorylation of the C-terminal domain (CTD) of RNAP II. Early on in the transition from preinitiation to elongation, phosphorylation of Ser5s in the CTD heptapeptide repeats takes place, and this depends on the activity of the TFIIH-associated kinase cyclin-dependent kinase 7 (Cdk7; mammals)/Kin28 (yeast). Subsequently, Ser2s are phosphorylated by the elongation phase kinase Cdk9 (mammals)/CTDK-1 (yeast) to generate elongation-proficient RNAP II complexes. Another Cdk, Cdk8, can negatively regulate RNAP II transcription, partially via its inhibitory effect on Cdk7 activity. More recently, it has been suggested that Cdk11^p110 regulates RNAP II transcription in humans. Thus, Cdk11^p110 binds to hypo- and hyper-phosphorylated RNAP II, and antibody-mediated repression of Cdk11^p110 activity results in inhibition of RNAP II transcription (Anderson, 2010 and references therein).

In addition to the phosphorylation events that control RNAP II activity, modification of the chromatin structure represents an important mechanism for regulating gene expression. When the chromatin is in its repressed state, the DNA is wrapped tightly around the histones, creating a barrier to the assembly of the RNAP II PIC at the promoter region. Activation of gene expression is associated with a number of histone modifications that loosen the chromatin structure, including acetylation, methylation, ubiquitylation, and phosphorylation. Histone H3 and H4 acetylations are particularly frequent toward the 5' ends of actively transcribed genes and presumably facilitate the initial assembly of the PICs at the promoter region. MOF (males absent on first) is a histone H4 lysine 16 (H4K16)-specific histone acetyltransferase (HAT) in both mammals and Drosophila. MOF is part of several complexes, including the Drosophila male-specific lethal (MSL) complex, which is required for X chromosome dosage compensation, the mammalian counterpart of the MSL complex, and the MOF-MSL1v1 complex, which mediates p53 acetylation at K120. In addition, MOF copurifies with a number of other proteins, such as the forkhead-associated (FHA) domain-containing protein MCRS2, NSL1-3 (for nonspecific lethal 1 to 3), and MBD-R2, as part of the NSL complex (Anderson, 2010).

This study focuses on the function of Drosophila MCRS2 (dMCRS2), the Drosophila ortholog of human MCRS2 (also known as MSP58). Whereas human MCRS1 and -2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 is largely uncharacterized. In addition to the recent observation that human and Drosophila MCRS2s form complexes with MOF (Cai, 2010; Prestel, 2010; Mendjan, 2006; Raja, 2010), several other reports suggest that MCRS1 and -2 proteins could function in transcription regulation via interactions with the transcriptional repressor Daxx or the basic region leucine zipper factor Nrf1 (Anderson, 2010).

Drosophila MCRS2 and its human homologue, MCRS2, are 59% identical, with the highest level of homology being in the FHA domain. Whereas dMCRS2 is largely uncharacterized, MCRS1 and -2 have been linked with a variety of cellular processes, RNAP I-dependent transcription, transcriptional repression, and cell cycle control, though these functions remain poorly understood (Anderson, 2010).

This study shows that dMCRS2 is an essential nuclear protein required for cell cycle progression and growth during development. The data show that dMCRS2 physically associates with Cdk11 and RNAP II and colocalizes with RNAP II PICs on polytene chromosomes in vivo. Consistent with this, dMCRS2 is required for optimal binding of RNAP II components to the cyclin promoter regions and for normal levels of cyclin gene expression (Anderson, 2010).

The demonstration of colocalization of dMCRS2 with RNAP II on numerous sites on polytene chromosomes is in agreement with a recent ChIP-seq analysis, which revealed that dMCRS2 is present on the promoters of over 4,000 genes, correlating with 55% of active genes (Raja, 2010). Furthermore, gene expression profiling studies show that dMCRS2 depletion elicits the downregulation of over 5,000 genes. This essential function as a broad-specificity transcriptional regulator is reflected by the extreme growth defect of dMCRS2-depleted cells both in vivo and in cell culture and in the fact that dMCRS2 has been recovered as a hit in RNAi screens for diverse cellular functions such as centrosome maturation and Hedgehog signaling (Anderson, 2010).

In accordance with its pleiotropic function, dMCRS2 can be purified with a number of proteins, from NSL components to members of the RNAP II machinery. Moreover, dMCRS2 colocalizes with RNAP II PICs on polytene chromosomes in vivo, suggesting that it may regulate an early step in the recruitment and/or assembly of RNAP II PICs. This is consistent with the majority of dMCRS2 binding to the promoter regions of autosomal and X-linked genes and the fact that dMCRS2 is required for the loading of RNAP II components to cyclin gene promoters. Thus, dMCRS2 appears to be an important transcriptional regulator, and the data represent the first evidence for a physical connection between dMCRS2 and the core transcriptional machinery. While the results suggest that dMCRS2 associates with RNAP II complexes via protein-protein interactions, future studies will need to establish the exact molecular nature of this connection (Anderson, 2010).

Interestingly, MCRS2 and dMCRS2 copurify with the MOF HAT independently of the dosage compensation MSL complex. Furthermore, it was observed that dMCRS2 coimmunoprecipitates and colocalizes extensively with MOF on polytene chromosomes. MOF, as well as binding to the 3' ends of MSL targets along the male X chromosome, is also found on numerous promoter regions, both on the X chromosome and on autosomes in both sexes. Since MCRS2 also binds to promoters, it is possible that dMCRS2 and MOF could function in concert in transcriptional regulation. However, despite the evidence that MOF regulates a broad range of both X-linked and autosomal genes, no physical connection between the putative dMCRS2-MOF NSL complex and RNAP II complexes has been established so far. This study shows that both dMCRS2 and MOF associate with core RNAP II complexes in cultured cells (Anderson, 2010).

dMCRS2 may promote transcription by different mechanisms. Through its HAT activity, dMCRS2-associated MOF may create a relaxed chromatin state favorable to PIC assembly, either by inducing the physical weakening of DNA/histone or histone/histone interactions or by promoting the recruitment of bromodomain-containing factors. dMCRS2 may also induce PIC formation by recruiting the preformed RNAP II/TFIIF complex and/or promoting transcription elongation through the recruitment of CK2 and the FACT complex, which facilitates transcription elongation by remodeling chromatin. However, whether these different dMCRS2-containing complexes regulate common target genes or whether they represent distinct transcriptional regulators remains to be investigated. In summary, a model is proposed where dMCRS2 binds to multiple sites along the chromosomes and promotes the recruitment of RNAP II PICs to target genes (Anderson, 2010).

Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? This study investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. It was found that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC; Drosophila RB, E2F and Myb), a tissue-specific version of the mammalian MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription (Chen, 2011).

The results suggest a stepwise series of developmentally programmed events as terminal differentiation genes convert from a transcriptionally silent state in precursor cells to full expression in differentiating spermatocytes. In precursor cells, differentiation genes are repressed and associated with background levels of hypophosphorylated Pol II and H3K4me3. These genes also display elevated levels of H3K27me3 and Polycomb at the promoter region, suggesting that they are acted upon by the PcG transcriptional silencing machinery. Notably, the differentiation genes studied in precursor cells in this study did not show the hallmark bivalent chromatin domains enriched for both the repressive H3K27me3 mark and the active H3K4me3 mark that have been characterized for a cohort of differentiation genes in mammalian ESCs (Chen, 2011).

The cell fate switch from proliferating spermatogonia to the spermatocyte differentiation program initiates both global and local changes in the transcriptional regulatory landscape, starting a cell type-specific gene expression cascade that eventually leads to robust transcription of the terminal differentiation genes. Globally, soon after the switch from spermatogonia to spermatocytes, core subunits of the PRC2 complex are downregulated, including E(z), the enzyme that generates the H3K27me3 mark. Locally, after male germ cells become spermatocytes, Pol II accumulates at the terminal differentiation gene promoters, although these genes still remain transcriptionally silent, with low H3K4me3 and high Polycomb protein levels near their promoters (Chen, 2011).

The next step awaits the expression of spermatocyte-specific forms of core transcription machinery and chromatin-associated regulators, including homologs of subunits of both the general transcription factor TFIID (tTAFs) and the MIP/dREAM complex (Aly and other testis-specific components of tMAC). The tMAC complex acts either locally or globally, perhaps at the level of chromatin or directly through interaction with tTAFs, to allow recruitment of tTAFs to promoters of target terminal differentiation genes. The action of tTAFs then allows full and robust transcription of the terminal differentiation genes, partly by displacing Polycomb from their promoters (Chen, 2011).

Strikingly, the two major PcG protein complexes appear to be regulated differently by the germ cell developmental program: whereas the PRC2 components E(z) and Su(z)12 are downregulated, the PRC1 components Polycomb, Polyhomeotic and dRing continue to be expressed in spermatocytes. The global downregulation of the epigenetic 'writer' E(z) in spermatocytes might facilitate displacement of the epigenetic 'reader', the PRC1 complex, from the differentiation genes, with the local action of tTAFs at promoters serving to select which genes are relieved of PRC1. In addition, the tTAFs act at a second level to regulate Polycomb by recruiting and accompanying Polycomb and several other PRC1 components to a particular subnucleolar domain in spermatocytes. It is not yet known whether sequestering of PRC1 to the nucleolus by tTAFs plays a role in the activation of terminal differentiation genes, perhaps by lowering the level of PRC1 that is available to exchange back on to differentiation gene promoters. Conversely, recruitment of PRC1 to the nucleolar region might have a separate function, such as in chromatin silencing in the XY body as observed in mammalian spermatocytes (Chen, 2011).

The findings indicate that, upon the switch from spermatogonia to spermatocytes, the terminal differentiation genes go through a poised state, marked by presence of both active Pol II and repressive Polycomb, before the genes are actively transcribed. Stalled Pol II and abortive transcript initiation are emerging as a common feature in stem/progenitor cells. This mechanism may prime genes to rapidly respond to developmental cues or environmental stimuli. Stalled Pol II could represent transcription events that have initiated elongation but then pause and await further signals, as in the regulation of gene expression by the androgen receptor or by heat shock. Alternatively, Pol II might be trapped at a nascent preinitiation complex, without melting open the DNA, as found in some instances of transcriptional repression by Polycomb. Although ChIP analyses did not have the resolution to distinguish whether Pol II was stalled at the promoter or had already initiated a short transcript, the results with antibodies specific for unphosphorylated Pol II suggest that Pol II is trapped in a nascent preinitiation complex. The PRC1 component dRing has been shown to monoubiquitylate histone H2A on Lys119 near or just downstream of the transcription start site. It is proposed that in early spermatocytes, before expression of the tTAFs and tMAC, the local action of PRC1 in causing H2AK119ub at the terminal differentiation gene promoters might block efficient clearing of Pol II from the preinitiation complex and prevent transcription elongation (Chen, 2011).

Removal of PRC1 from the promoter and full expression of the terminal differentiation genes in spermatocytes require the expression and action of tMAC and tTAFs. Cell type-specific homologs of TFIID subunits have been shown to act gene-selectively to control developmentally programmed gene expression. For example, incorporation of one subunit of the mammalian TAF4b variant into TFIID strongly influences transcriptional activation at selected promoters, directing a generally expressed transcriptional activator to turn on tissue-specific gene expression (Chen, 2011).

The local action of the tTAFs to relieve repression by Polycomb at target gene promoters provides a mechanism that is both cell type specific and gene selective, allowing expression of some Polycomb-repressed genes while keeping others silent. Similar developmentally programmed mechanisms may also reverse PcG-mediated epigenetic silencing in other stem cell systems. Indeed, striking parallels between the current findings and recent results from mammalian epidermis suggest that molecular strategies are conserved from flies to mammals. In mouse epidermis, the mammalian E(z) homolog Ezh2 is expressed in stem/precursor cells at the basal layer of the skin. Strikingly, as was observed for E(z) and Su(z)12 in the Drosophila male GSC lineage, the Ezh2 level declines sharply as cells cease DNA replication and the epidermal differentiation program is turned on. Overexpression of Ezh2 in epidermal precursor cells delays the onset of terminal differentiation gene expression, and removal of the Ezh2-generated H3K27me3 mark by the Jmjd3 (Kdm6b) demethylase is required for epidermal differentiation (Chen, 2011 and references therein).

In particular, the results suggest a possible explanation for the conundrum that, although PcG components are bound at many transcriptionally silent differentiation genes in mammalian ESCs, loss of function of PcG components does not cause loss of pluripotency but instead causes defects during early embryonic differentiation. In Drosophila male germ cells, events during the switch from precursor cell proliferation to differentiation are required to recruit Pol II to the promoters of differentiation genes. Without this differentiation-dependent recruitment of Pol II, loss of Polycomb is not sufficient to precociously turn on terminal differentiation genes in precursor cells. Rather, Polycomb that is pre-bound at the differentiation gene promoters might serve to delay the onset of their transcription after the mitosis-to-differentiation switch. Robust transcription must await the expression of cell type- and stage-specific components of the transcription machinery. These might in turn guide gene-selective reversal of Polycomb repression to facilitate appropriate differentiation gene expression in specific cell types (Chen, 2011).

Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling

The kinetics and magnitude of cytokine gene expression are tightly regulated to elicit a balanced response to pathogens and result from integrated changes in transcription and mRNA stability. Yet, how a single microbial stimulus induces peak transcription of some genes (TNFalpha) within minutes whereas others (IP-10) require hours remains unclear. This study dissected activation of several lipopolysaccharide (LPS)-inducible genes in macrophages, an essential cell type mediating inflammatory response in mammals. A key difference between the genes is the step of the transcription cycle at which they are regulated. Specifically, at TNFalpha, RNA Polymerase II initiates transcription in resting macrophages, but stalls near the promoter until LPS triggers rapid and transient release of the negative elongation factor (NELF) complex and productive elongation. In contrast, no NELF or polymerase is detectible near the IP-10 promoter before induction, and LPS-dependent polymerase recruitment is rate limiting for transcription. It was further demonstrated that this strategy is shared by other immune mediators and is independent of the inducer and signaling pathway responsible for gene activation. Finally, as a striking example of evolutionary conservation, the Drosophila homolog of the TNFalpha gene, eiger, displayed all of the hallmarks of NELF-dependent polymerase stalling. It is proposed that polymerase stalling ensures the coordinated, timely activation the inflammatory gene expression program from Drosophila to mammals (Adelman, 2010).

The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65-Prp19 complex

Pre-mRNA splicing is frequently coupled to transcription by RNA polymerase II (RNAPII). This coupling requires the C-terminal domain of the RNAPII largest subunit (CTD), although the underlying mechanism is poorly understood. Using a biochemical complementation assay, an activity was identified that stimulates CTD-dependent splicing in vitro. This activity was purified from HeLa cells and found to consist of a complex of two well-known splicing factors: U2AF65 and the Prp19 complex (PRP19C). Evidence is provided that both U2AF65 and PRP19C are required for CTD-dependent splicing activation, that U2AF65 and PRP19C interact both in vitro and in vivo, and that this interaction is required for activation of splicing. Providing the link to the CTD, it was shown that U2AF65 binds directly to the phosphorylated CTD, and that this interaction results in increased recruitment of U2AF65 and PRP19C to the pre-mRNA. These results not only provide a mechanism by which the CTD enhances splicing, but also describe unexpected interactions important for splicing and its coupling to transcription (David, 2011).

A number of interactions linking pre-mRNA processing and the CTD have been documented. For capping, the functional connection with the CTD is straightforward; the guanylytransferase and methyltransferase enzymes necessary for capping both bind to the S5- phosphorylated CTD, which allosterically activates guanylytransferase activity (McCracken, 1997; Yue, 1997; Ho, 1999). Connections between the polyadenylation machinery and the CTD have also been demonstrated. Human CstF50 was shown to interact physically with both the phosphorylated and unphosphorylated CTD, an interaction that appears important for efficient cleavage/polyadenylation in vivo (Fong, 2001). The yeast CFI subunit Pcf11 also interacts with the S2-phosphorylated CTD, the functional importance of which was suggested by a genetic interaction between a Pcf11 allele and an RBP1 CTD truncation allele (Licatalosi, 2002). Also, a CTD phosphatase, Ssu72, was shown recently to be important for transcription-coupled 3' processing in vitro (Xiang, 2010; David, 2011 and references therein).

The machinery that carries out pre-mRNA splicing is considerably more complex than those responsible for capping and polyadenlyation. The spliceosome, the protein-RNA assembly that catalyzes intron removal, contains at least 150 proteins and undergoes dynamic changes in conformation and protein composition during the series of events that begin with splice site recognition and end after the execution of the two catalytic steps. In vitro, spliceosome assembly proceeds through the formation of a series of stable intermediate complexes, which are biochemically separable and amenable to proteomic analysis (Wahl, 2009). Among the earliest steps in spliceosome assembly is recognition of the 5' and 3' splice sites by the U1 snRNP and U2AF, respectively. U2AF is a dimer comprised of U2AF65 and U2AF35. U2AF65 binds to polypyrimidine-rich sequences found near the 3' end of most introns and promotes stable U2 snRNP association with the pre-mRNA, an activity that requires its N-terminal arginine-serine-rich (RS) domain. U2AF35 contacts a well-conserved AG dinucleotide at the 3' end of the intron and can interact with exon-bound SR proteins; both interactions can stabilize U2AF binding to suboptimal polypyrimidine tracts. Later steps in spliceosome assembly involve the activity of numerous additional factors, including the U4/U6.U5 tri-snRNP and the Prp19 complex, or PRP19C (Wahl, 2009). PRP19C was first discovered in yeast, where it was shown to be an essential splicing factor that does not tightly associate with snRNPs (Hogg, 2010). PRP19C, which consists of four polypeptides that form a salt-stable core (CDC5L, PRLG1, Prp19, and SPF27) and three more loosely associated polypeptides (HSP73, CTNNBL1, and AD002) (Grote, 2010), is found at the core of catalytically activated spliceosomes and plays a critical but poorly understood role in activation of the spliceosome. Because PRP19C does not contain any proteins known to bind RNA, it is likely that PRP19C recruitment to the spliceosome occurs through protein-protein interactions with RNA-bound factors, although no such interaction has yet been described (David, 2011 and references therein).

Most of what is known about the process of spliceosome assembly has come from the use of in vitro systems that are uncoupled from transcription, leaving the role of the transcriptional machinery in the process relatively poorly understood. However, a few physical interactions between splicing factors and the CTD have been documented. The yeast U1 snRNP component Prp40 was shown to bind to the phosphorylated CTD through multiple WW domains. In humans, splicing factors that have been shown to bind directly to the CTD include CA150, PSF, and p54/NRB. Of these, support for a functional significance to the CTD interaction has been provided only for PSF, which can be recruited to promoters by strong transcriptional activators to promote splicing in a CTD-dependent manner in vivo (David, 2011 and references therein).

In order to study the functional connections between the CTD and pre-mRNA splicing, a fusion between the CTD and the SR protein SRSF1 (formerly ASF/SF2) was constructed. This allowed recruitment of the CTD to splicing substrates harboring SRSF1-binding sites independent of transcription. Using this fusion protein, which is called SRSF1-CTD, in in vitro splicing assays, an increase was observed in splicing kinetics in its presence when compared with SRSF1 alone. In addition, a HeLa nuclear fraction (NF20-40) was found to be capable of activating splicing of one substrate, IgMA3, in HeLa S-100 in the presence of SRSF1-CTD but not SRSF1, suggesting that NF20-40 contains a factor capable of functionally interacting with the CTD. The factor responsible for this activity was purified and characterized, and it was found to consists of a complex containing both U2AF65 and PRP19C. U2AF65 and PRP19C interact directly in vitro and in an RNA-independent manner in vivo. Additionally, U2AF65 binds directly to the phosphorylated CTD, increasing U2AF association with the pre-mRNA and recruitment of PRP19C. U2AF65 thus bridges the transcriptional machinery and later stages of spliceosomal assembly through novel interactions with the RNAPII CTD and PRP19C (David, 2011).

A model is presented for activation of CTD-dependent splicing by a U2AF-PRP19C complex. At promoters, RNAPII is present in preinitiation complexes, but the CTD is unphosphorylated and unable to recruit splicing factors. Transcription initiation results in CTD phosphorylation by multiple kinases, resulting in the association of splicing factors (including SR proteins), U1 snRNP through unknown interactions, and the U2AF-PRP19C complex via a direct interaction with U2AF65. RNAPII-associated U1 and SR proteins recognize a transcribed exon, resulting in its tethering to the RNAPII elongation complex through multiple interactions. Transcription of the 3' splice site results in a transition from protein-protein interactions between U2AF65 and the p-CTD to protein-RNA interactions, resulting in efficient recognition of the 3' splice site. This facilitates rapid transition to a mature spliceosomal complex promoted by U2AF65-associated PRP19C (David, 2011).

RNA polymerase II kinetics in polo polyadenylation signal selection

Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, it was shown that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. These results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation (Pinto, 2011).

Since many genes in flies and other eukaryotes possess alternative pA sites in terminal exons, the possibility that general transcription kinetics might also have a role in alternative polyadenylation was investigated. To do this, the well-characterized C4 fly mutant, which has a 50% decrease in Pol II elongation rate, was used. Interestingly, it was shown that this results in increased utilization of the polo pA1 site. In particular, in C4 flies, polo pA1 is used 3.5-fold more efficiently than in the wild type. It was further demonstrated that the effect of Pol II kinetics in alternative pA site selection is likely to be general as analysis of five additional Drosophila genes possessing alternative terminal exon pA sites showed a similar switch to upstream pA site usage in the C4 mutant. This clearly indicates that Pol II kinetics has an important role in alternative polyadenylation site selection. Significantly, human Pol II carrying the equivalent mutation to the Drosophila C4, when transfected into human cells, affects alternative splicing of a FN minigene. Inclusion of the alternative EDI exon in this system increases ~4-fold, a similar level to that obtained for alternative polo pA signal selection. C4 flies also show a difference in Pol II occupancy across the polo gene. Thus, in wild-type flies Pol II levels are reduced downstream of pA1, while in C4 flies this pattern is disrupted. Presumably, the slow Pol II enables the pA1 signal on the nascent transcript to be exposed to the polyadenylation machinery for a longer time before Pol II transcribes pA2. Therefore, pA1 will be processed before pA2 is transcribed pointing to a mechanism that relies on the rule of 'first come, first served'. When the Pol II elongation rate is higher, as in wild-type flies, it transcribes through pA1 and pA2 more efficiently, so that the polyadenylation machinery processes both pA signals on the nascent transcript. This resembles the mechanism described above for EDI alternative splicing where slow Pol II preferentially includes the alternative EDI exon which is normally excluded, because it allows the machinery time to assemble on the spliceosome. The results now indicate that both alternative polyadenylation and alternative splicing depend on Pol II kinetics. In view of recent findings that highlight the importance of alternative pA signal selection, the results now suggest a general molecular mechanism for this process (Pinto, 2011).

The physiological consequences of correct polo pA signal choice in vivo as shown in this study are profound: pA2 is essential for abdominal histoblast proliferation, development of the adult epidermis and viability of the transgenic flies. The lethality and strong abdominal phenotype observed in gfp-poloΔpA2;polo⁹ flies are due to the fact that these flies lack polo pA2 mRNA and consequently cannot express sufficient levels of Polo protein (from polo pA1 mRNA) for flies to survive the pupa stage. This phenotype is in agreement with earlier studies showing that polo¹/polo² individuals express low levels of Polo protein and present an abnormal development of the abdomen. It is anticipated that the C4 mutant would display a similar phenotype to gfp-polo ΔpA2 flies since it downregulates pA2 usage. However, a developmental defect was observed similar to the so-called 'Ubx effect'. It is noted that slow Pol II elongation will impact on many genes so that any phenotype observed is likely to derive from complex genetic effects. It is also noted that polo pA2 mRNA is still produced at significant levels in the C4 mutant, which may produce sufficient protein for development of the abdomen, as opposed to the ΔpA2 transgenic flies that lack polo pA2 mRNA and show a decrease in Polo protein production in the histoblasts (Pinto, 2011).

Two genes in humans (hap and Bzw1) where alternative distal pA site usage results in increased levels of protein production have been previously described. In T cells and cancer cell lines, proximal pA site selection in the 3′UTR results in a relief from microRNA repression with the same final effect of an increase in protein production. However, a considerable proportion of genes do not follow this pattern, pointing to the existence of other regulatory elements in the different 3′UTRs. Presumably, polo is one such gene since it is shown that its expression is not regulated by dme-mir-8 and dme-mir-1016 overexpression. Instead regulated polo expression relies on the fact that the choice of pA1 by the transcriptional/processing machinery leads to a decrease in the translation of Polo. This indicates that Polo protein expression is modulated by pA signal selection and by translational control through the 3′UTR, suggesting the presence of regulatory elements in the different 3′UTRs. As Polo is a master regulator of the cell cycle, it is predicted that the consequences of this type of control will be critical to the cell (Pinto, 2011).

Histoblasts have a very high proliferation rate and go through very rapid cell cycles at the onset of metamorphosis, therefore would be predicted to be especially sensitive to Polo levels. The lack of polo pA2 mRNA in ΔpA2 flies leads to a reduction in Polo protein levels with the consequent block of histoblast proliferation. This will in turn result in a subsequent failure to correctly develop the adult epidermis. Consistent with these studies, loss of Polo has been shown to cause G₂ arrest (Pinto, 2011).

Interestingly, it was also shown that, in flies where Polo is overexpressed, the shorter mRNA produced by pA1 usage is more abundant than the longer mRNA produced by pA2 selection. This suggests that Polo controls its own expression levels by an auto-regulatory loop, where higher levels of Polo lead to preferential recognition of pA1. As this mRNA is not efficiently translated, this will lead to a decrease in the protein levels produced. It has been shown that upregulation of su(f) leads to an auto-regulatory feedback loop through the recognition of a weak intronic pA site. This results in the production of a truncated polypeptide leading to a shut down in gene expression. In the case of polo, a more subtle process is evident, as pA1 selection generates a functional transcript, even though it is not efficiently translated into protein (Pinto, 2011).

Taken together, these results suggest that Pol II kinetics have an important role in pA site selection. Moreover, the results reinforce the view that tight regulation at the level of pA signal selection is necessary for the cell. The importance of precisely choosing the correct pA signal to control cell viability during development is underlined by these studies (Pinto, 2011).

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ~40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, 'bivalent' ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation (Min, 2011).

Determining the presence and status of Pol II at bivalent genes is important for understanding the mechanisms that govern their transcription. Bivalent genes that are targets of the PRC2 complex, but not PRC1, retain promoter-proximal engaged Pol II that is more tightly confined to a region near the TSS and also display dramatically reduced levels of elongating Pol II. These results suggest that transcription at bivalent genes bound by PRC2 is regulated after the initiation step but very early in elongation. Although PRC2 activity has been shown to function in recruiting PRC1 and in DNA looping, no connections to distinct steps in transcription have been made previously (Min, 2011).

The bivalent genes that are occupied by both the PRC2 and the PRC1 complexes display much less promoter-proximal Pol II, suggesting a preinitiation block to earlier steps in transcription at these genes. This is consistent with the ability of PRC1 to compact nucleosomes, interfere with specific functions of the transcriptional apparatus, or both. The stronger repression of bivalent genes through the additional activity of PRC1 is also evident in that these genes show more robust retention of repressive chromatin through differentiation than PRC1^-,2⁺ bivalent genes. Nonetheless, these genes are not completely silent. Both bivalent gene classes were found to exhibit significant levels of productive elongation that are higher than at PRC-bound genes with H3K27me3 but not H3K4me3 (Min, 2011).

The transcriptional activities of most genes are proportional to the levels of H3K4me3 on their promoter regions. In marked contrast, the transcriptional activity and divergent transcription of both classes of bivalent genes can vary widely, but the amount of H3K4me3 on the promoter remains at a level that is almost equal to the genome-wide average. This constant presence of H3K4me3 modifications across the promoter region may suggest that bivalent genes are poised for further activation. Recently, H3K4me3 modifications have been shown to evoke a dynamic cycle of histone acetylation and deacetylation at the promoters of inactive genes, which may facilitate the cross-talk between different histone modifications to prepare for activation. In addition, the high affinity of the TAF3 subunit of TFIID for the H3K4me3-modified histone tail may assist activation for bivalent genes that are highly enriched for CpG islands but lacking a TATA box (Min, 2011).

Many developmental regulatory genes are targets of PRC2 and PRC1 in ESCs, and most are transcribed at a modest level but do not feature a significant peak of paused Pol II. Interestingly, another study has shown that these genes retain a high level of Ser5-phosphorylated Pol II at the promoter relative to the gene body. Although paused Pol II is Ser5-phosphorylated, these promoters could possibly contain a form of Pol II that either has not fully entered elongation or is backtracked and unable to elongate in a run-on assay (Min, 2011).

Most developmental regulatory genes are not highly expressed in ESCs; however, it is noted that regulators of multiple lineages show some 'promiscuous transcription,' and this shares some similarities with what has been observed in the hematopoietic system. Interestingly, the regulators of neural and neuroectodermal lineages are among the highly expressed regulators in ESCs, supporting the hypothesis that ESCs in culture have an 'aptitude' for neural differentiation (Min, 2011).

OCT4, SOX2, and NANOG play important roles in preventing activation of specific lineage differentiation pathways, as well as forming the positive feedback transcription network for maintaining and establishing the pluripotent and self-renewal potentials in ESCs. Oct4 and Nanog are actively transcribed in ESCs but still exhibit a rate-limiting step at pausing. Rapid, synchronous, and high levels of activation correlate better with genes that possess paused Pol II over genes that do not. Taken together, it is speculated that pausing provides a responsive transcriptional regulatory step for controlling the level of critical core pluripotency transcription factors in ESCs (Min, 2011).

The profile of engaged RNA polymerases provides both a measure of transcription and a means of identifying those steps that are slow and regulated. This comparison of ESCs and MEFs establishes that transcription elongation is often controlled by dynamically tuning the release of the paused Pol II. However, an important class of regulated genes in ESCs that show bivalent histone modifications is modulated at both elongation and stages prior to elongation. Identification of the molecular targets of upstream activators or repressors and the role of these targets in modulating the rate-limiting steps of transcription will be essential to fully elucidate the mechanisms governing the regulation of the ESC state (Min, 2011).

Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development

Chromatin modifications are associated with many aspects of gene expression, yet their role in cellular transitions during development remains elusive. This study used a new approach to obtain cell type-specific information on chromatin state and RNA polymerase II (Pol II) occupancy within the multicellular Drosophila melanogaster embryo. The relationship between chromatin modifications and the spatio-temporal activity of enhancers was directly assessed. Rather than having a unique chromatin state, active developmental enhancers show heterogeneous histone modifications and Pol II occupancy. Despite this complexity, combined chromatin signatures and Pol II presence are sufficient to predict enhancer activity de novo. Pol II recruitment is highly predictive of the timing of enhancer activity and seems dependent on the timing and location of transcription factor binding. Chromatin modifications typically demarcate large regulatory regions encompassing multiple enhancers, whereas local changes in nucleosome positioning and Pol II occupancy delineate single active enhancers. This cell type-specific view identifies dynamic enhancer usage, an essential step in deciphering developmental networks (Bonn, 2012).

A method was developed to batch isolate tissue-specific chromatin for immunoprecipitation (BiTS-ChIP), which uses a transgene to express a tagged nuclear protein specifically in the cell type of interest. Entire embryos are covalently cross-linked, and intact, fixed nuclei are isolated and sorted by FACS to obtain pure populations of nuclei from specific cell types; the average purity of all samples used in this study was 97.4%. To generate a widely applicable protocol, the ChIP procedure was optimized to use less chromatin, thereby allowing multiple ChIP experiments to be performed from a single FACS sort (Bonn, 2012).

The results support a multistep model for enhancer activation: H3K4me1 may indicate enhancers in an intermediate state, in which they are susceptible to subsequent repression via H3K27 trimethylation or activation via H3K79 trimethylation, H3K27 acetylation and Pol II recruitment. Chromatin modifications typically cover relatively large regions of >2 kb that encompass multiple enhancer elements. The deposition of H3K79me3 or H3K27ac may therefore place an entire regulatory region in a permissive state, and the activity of individual enhancer elements contained within these regions appear to be determined by the timing of transcription factor occupancy, nucleosome remodeling and Pol II association (Bonn, 2012). It has been previously reported that transcription factor occupancy alone is sufficient to predict spatio-temporal enhancer activity; this study shows that histone modifications and Pol II occupancy information alone can accurately predict the activity status of regulatory regions. Integrating chromatin modification and transcription factor occupancy data within the same cell type and at the same stage of development will provide a very accurate way to distinguish functional transcription factor binding events from nonfunctional occupancy and should facilitate better modeling of tissue-specific gene expression and the underlying cis-regulatory networks during development. Considering the high resolution, precision and sensitivity of the data afforded by BiTS-ChIP, this method provides a powerful approach to decipher transcriptional networks and should be widely applicable to other species and complex tissues (Bonn, 2012).

Argonaute-bound small RNAs from promoter-proximal RNA polymerase II

Argonaute (Ago) proteins mediate posttranscriptional gene repression by binding guide miRNAs to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, a mouse embryonic stem cell system was adapted to express a single epitope-tagged Ago protein family member in an inducible manner. This paper reports the small RNA profile of Ago-deficient cells and shows that Ago-dependent stability is a common feature of mammalian miRNAs. Using this criteria and immunopurification, an Ago-dependent class of noncanonical miRNAs was identified derived from protein-coding gene promoters, which were named transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II (RNAPII) complexes produces hairpin RNAs that are processed in a DiGeorge syndrome critical region gene 8 (Dgcr8)/Drosha-independent but Dicer-dependent manner. TSS-miRNA activity is detectable from endogenous levels and following overexpression of mRNA constructs. Finally, evidence is presented of differential expression and conservation in humans, suggesting important roles in gene regulation (Zamudio, 2014).

Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. This study investigated whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. The hypophosphorylated C-terminal domain of Pol II was found to be incorporated into mediator condensates; phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. It was also found that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference (Guo, 2019).

Previous studies have shown that the hypophosphorylated C-terminal domain (CTD) of Pol II can interact with mediator and that Pol II and mediator occur in condensates at super-enhancers (Cho, 2018; Sabari, 2018), but have not established whether the CTD contributes to Pol II interactions with mediator condensates. To investigate whether the Pol II CTD is incorporated into mediator condensates, the human mediator complex and recombinant Pol II CTD fused to GFP (full-length GFP-CTD52 and the truncated forms GFP-CTD26 and GFP-CTD10) were purified, and condensate formation was measured in an in vitro droplet assay. Mediator droplets incorporated and concentrated GFP-CTD52 to a much greater extent than they did the truncated forms or control GFP. The interaction of the CTD with mediator was further investigated using MED1, which is the largest subunit of the mediator complex. MED1 has proven to be a useful surrogate for mediator condensates in previous studies, has an exceptionally large intrinsically disordered region (IDR) that contributes to condensate formation and has been shown to associate with Pol II in human cells. Droplet assays revealed that the IDR of MED1 fused to mCherry (mCherry-MED1-IDR) formed condensates that incorporated and concentrated GFP-CTD52 to a greater extent than did the truncated forms or GFP alone. The GFP-CTD52 and MED1-IDR condensates exhibited liquid-like fusion behaviour, and showed evidence of dynamic internal rearrangement and internal-external exchange of molecules by fluorescence recovery after photobleaching (FRAP), which is consistent with liquid-liquid phase-separated condensates. These results are consistent with the idea that the CTD of Pol II contributes to the incorporation of Pol II into mediator condensates (Guo, 2019).

Attempts were made to determine whether splicing-factor condensates occur at genes associated with super-enhancers because these genes are transcribed at especially high rates. RNA splicing can occur co-transcriptionally, and some nuclear speckles have previously been reported to occur in the vicinity of highly transcribed genes. Eight components of the splicing apparatus were selected, and immunofluorescence microscopy with concurrent nascent RNA fluorescence in situ hybridization (FISH) was used for Nanog and Trim28 to determine whether the splicing apparatus occurs in puncta in the vicinity of these genes associated with super-enhancers. The results showed that all eight splicing factors occur in puncta at these two genes. To gain additional insights into splicing-factor puncta that colocalize with Pol II, mouse embryonic stem cells (mouse ES cells) that were engineered to express endogenously tagged proteins were studied using lattice light-sheet imaging in live cells. It has previously been shown that large numbers of mediator and Pol II molecules can occur in puncta and that these puncta sometimes colocalize. Using a similar approach, this study found that large numbers of SRSF2 molecules occur in puncta and some of these (approximately 15%) overlap with Pol II puncta. Treatment of cells with pladienolide B (an inhibitor of splicing) -- that reduced splicing as determined by a splicing reporter -- reduced the levels of splicing factors but not of Pol II in puncta at Trim28 DNA FISH foci. This treatment also led to the incorporation of splicing factors into 'mega-speckles' at some distance from the gene, a phenomenon that has previously been observed when splicing is inhibited. These results suggest that functional RNA-splicing apparatus is present in condensates at active genes associated with super-enhancers (Guo, 2019).

Actively transcribed genes may become associated with nuclear speckles or obtain splicing apparatus stored in speckles that are thought to be phase-separated. In live-cell imaging, this study found that the SRSF2 puncta exhibited features of liquid-like condensates: all of these puncta showed evidence of dynamic internal rearrangement. Internal-external exchange of molecules by FRAP were sensitive to treatment with 1,6-hexanediol, and some would occasionally fuse. These results are consistent with previous reports on speckle behaviour and suggest that the SRSF2-containing puncta that come into contact with active genes associated with super-enhancers are liquid-like condensates. At highly transcribed genes (such as those driven by super-enhancers), large numbers of Pol II molecules may be engaged in transcription elongation and these might serve to recruit into condensates some portion of the apparatus that is otherwise located in speckles (Guo, 2019).

Next, whether hypophosphorylated Pol II tends to be associated with MED1 condensates was investigated, and whether, by contrast, hyperphosphorylated Pol II tends to be associated with SRSF2 condensates. Using immunofluorescence and antibodies against the hypophosphorylated CTD of Pol II or the CTD of Pol II phosphorylated at serine 2, this prediction was confirmed: MED1 puncta more frequently overlapped with signals for the hypophosphorylated CTD, whereas SRSF2 puncta more frequently overlapped with signals for the serine-2-phosphorylated CTD. A control experiment showed that there was essentially no overlap between SRSF2 puncta and the heterochromatin protein HP1a, and strong overlap between SRSF2 puncta visualized using independent methods. An independent experimental approach that used chromatin immunoprecipitation followed by sequencing (ChIP-seq) with antibodies against MED1, SRSF2 and the two phosphoforms of Pol II also confirmed that MED1 tends to occupy super-enhancers and promoters together with Pol II that contains a hypophosphorylated CTD, whereas SRSF2 is observed across the transcription unit and is prominent at the ends of genes together with Pol II that contains a serine-2-phosphorylated CTD (Guo, 2019).

If the formation or maintenance of splicing-factor condensates is dependent on the phosphorylation of the Pol II CTD, it would be expected that inhibition of CTD phosphorylation in cells would prevent the formation of splicing-factor condensates at genes driven by super-enhancers. Indeed, inhibition of CTD phosphorylation by the cyclin-dependent kinase (CDK) inhibitor D-ribofuranosylbenzimidazole (DRB) caused a marked reduction in the occupancy of multiple components of splicing-factor condensates (SRSF2, SRSF1, SF3B1, U2AF2 and PRPF8) at Nanog or Trim28 DNA FISH foci, and a washout of the drug led to a partial reestablishment of most of these splicing factors within two hours. By contrast, treatment with DRB had minor effects on mediator and Pol II condensates. These results suggest that phosphorylation of the Pol II CTD is necessary for the formation of splicing-factor condensates at these genes in vivo, although it is possible that altered phosphorylation of other substrates of CDKs may contribute to these observations (Guo, 2019).

The transition of Pol II from initiation to elongation is accompanied by phosphorylation of the CTD heptapeptide repeat by CDK7 and CDK9. Phosphorylation of the CTD has previously been shown to affect its interaction with hydrogels formed by the low-complexity domains of FUS, EWS and TAF15 (FET) proteins, which suggests that phosphorylation may affect the condensate-interacting properties of the CTD. Whether phosphorylation of the CTD by CDK7 or CDK9 would affect its incorporation into mediator condensates was investigated. CTD phosphorylation by either CDK7 or CDK9 caused a reduction in CTD incorporation into mediator droplets. Similarly, CTD phosphorylation caused a reduction in the incorporation of the CTD into MED1-IDR droplets. These results are consistent with a model in which phosphorylation of the Pol II CTD causes eviction from mediator condensates (Guo, 2019).

The observation that CTD phosphorylation is necessary for the formation of splicing-factor condensates at highly transcribed genes suggests that CTD phosphorylation might enhance the partitioning of the Pol II CTD into condensates formed by splicing components. To investigate this idea, four human splicing factors (SRSF2, SRSF1, U2AF2 and HNRNPA1) were selected as surrogates for the more complex splicing-factor condensates, and their condensate-forming properties were explored. Each of the four purified human proteins fused to mCherry formed phase-separated droplets. SRSF2 is one of multiple proteins involved in pre-mRNA splicing that contain serine-arginine (SR) dipeptide repeats, and it has an especially large SR-rich domain; therefore used SRSF2 was used as a core component to study whether it could concentrate the other three factors into heterotypic droplets. Indeed, all of these factors could form binary heterotypic droplets with SRSF2. It was then asked whether phosphorylation of the Pol II CTD influences the incorporation of the CTD into splicing-factor condensates in vitro using recombinant SRSF1 and SRSF2. The results showed that the unphosphorylated CTD was not efficiently incorporated into SRSF1 or SRSF2 droplets, whereas the CDK7- or CDK9-phosphorylated CTD was incorporated and concentrated in both SRSF1 and SRSF2 droplets. The ability of SRSF2 to incorporate the phosphorylated CTD was dependent on the length of the CTD—as expected for a high-valency condensate interaction, and consistent with models in which the truncation of the CTD leads to splicing defects. It is concluded that phosphorylation of the Pol II CTD leads to a switch in the preference of the CTD for interactions between mediator condensates and condensates that contain proteins with SR-rich domains (Guo, 2019).

The results indicate that phosphorylation of the Pol II CTD alters the condensate-partitioning behaviour of Pol II and may thus drive an exchange of Pol II from condensates that are involved in transcription initiation to condensates that are involved in RNA splicing at genes associated with super-enhancers. This model is consistent with evidence from previous studies that suggests that large clusters of Pol II can fuse with mediator condensates in cells; that phosphorylation dissolves CTD-mediated Pol II clusters; that CDK9 and cyclin T can interact with the Pol II CTD through a phase-separation mechanism; that Pol II is no longer associated with mediator during transcription elongation, and that nuclear speckles containing splicing factors can be observed at loci with high transcriptional activity. Previous studies have shown that the Pol II CTD can interact with components of the transcription-initiation apparatus and RNA-processing machinery in a phosphoform-specific manner, but did not explore the possibility that these components occur in condensates or that phosphorylation of the Pol II CTD alters the partitioning behaviour of Pol II between these condensates. The results reveal that mediator condensates and splicing-factor condensates occur at the same genes driven by super-enhancers, and suggest that the transition of Pol II from interactions with components involved in initiation to those involved in splicing can be mediated, in part, through a change in condensate partitioning that is regulated by phosphorylation of the CTD. These results also suggest that phosphorylation may be one of the mechanisms that regulate the condensate-partitioning of proteins in processes in which protein function involves eviction from one condensate and migration to another (Guo, 2019).

Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II

Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing (see Drosophila Pol II). The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)-which consists of CDK9 (see Drosophila Cdk9) and cyclin T1-is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation. The sequence determinant on P-TEFb that facilitates this action is currently unknown. This study identified a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase -- DYRK1A (see Drosophila Minibrain) -- also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD-which in isolation does not phase separate, despite being a low-complexity domain-is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH. By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II (Lu, 2018).

A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting

Live-cell imaging has revealed unexpected features of gene expression. Using improved single-molecule RNA microscopy, this study shows that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. It was additionally observed that the HIV-1 promoter displays stochastic fluctuations on two time scales, which are referred to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. It is proposed that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells (Tantale, 2016).

Search PubMed for articles about Drosophila Pol II

Adelman, K., et al. (2005). Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol. Cell 17(1): 103-12. PubMed ID: 15629721

Adelman, K., Wei, W., Ardehali, M. B., Werner, J., Zhu, B. Reinberg, D. and Lis, J. T. (2006). Drosophila Paf1 modulates chromatin structure at actively transcribed genes. Mol. Cell. Biol. 26(1): 250-60. PubMed ID: 16354696

Abakir, A., Giles, T. C., Cristini, A., Foster, J. M., Dai, N., Starczak, M., Rubio-Roldan, A., Li, M., Eleftheriou, M., Crutchley, J., Flatt, L., Young, L., Gaffney, D. J., Denning, C., Dalhus, B., Emes, R. D., Gackowski, D., Correa, I. R., Jr., Garcia-Perez, J. L., Klungland, A., Gromak, N. and Ruzov, A. (2020). N(6)-methyladenosine regulates the stability of RNA:DNA hybrids in human cells. Nat Genet 52(1): 48-55. PubMed ID: 31844323

Adelman, K., et al. (2010). Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling. Proc. Natl. Acad. Sci. 106(43): 18207-12. PubMed ID: 19820169

Akhtar, J., Renaud, Y., Albrecht, S., Ghavi-Helm, Y., Roignant, J. Y., Silies, M. and Junion, G. (2021). m⁶A RNA methylation regulates promoter- proximal pausing of RNA polymerase II. Mol Cell 81(16): 3356-3367. PubMed ID: 34297910

Andersen, D. S., et al. (2010). Drosophila MCRS2 associates with RNA polymerase II complexes to regulate transcription. Mol. Cell. Biol. 30(19): 4744-55. PubMed ID: 20679484

Ardehali, M. B. and Lis, J. T. (2009). Tracking rates of transcription and splicing in vivo. Nat. Struct. Mol. Biol. 16: 1123-1124. PubMed ID: 19888309

Arzate-Mejia, R. G., Josue Cerecedo-Castillo, A., Guerrero, G., Furlan-Magaril, M. and Recillas-Targa, F. (2020). In situ dissection of domain boundaries affect genome topology and gene transcription in Drosophila. Nat Commun 11(1): 894. PubMed ID: 32060283

Andrulis, E. D., Werner, J., Nazarian, A., Erdjument-Bromage, H., Tempst, P. and Lis, J. T. (2002). The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila. Nature 420: 837-841. PubMed ID: 12490954

Baillat, D., Hakimi, M. A., Naar, A. M., Shilatifard, A., Cooch, N. and Shiekhattar, R. (2005). Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell 123(2): 265-276. PubMed ID: 16239144

Barbieri, E., Trizzino, M., Welsh, S. A., Owens, T. A., Calabretta, B., Carroll, M., Sarma, K. and Gardini, A. (2018). Targeted enhancer activation by a subunit of the integrator complex. Mol Cell 71(1): 103-116 e107. PubMed ID: 30008316

Bieniasz, P.D., et al. (1999). Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription. Proc. Natl. Acad. Sci. 96: 7791-7796. PubMed ID: 10393900

Boehm, A. K., Saunders, A., Werner, J. and Lis, J. T. (2003). Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol Cell Biol. 23(21): 7628-37. PubMed ID: 14560008

Boehning, M., Dugast-Darzacq, C., Rankovic, M., Hansen, A. S., Yu, T., Marie-Nelly, H., McSwiggen, D. T., Kokic, G., Dailey, G. M., Cramer, P., Darzacq, X. and Zweckstetter, M. (2018). RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat Struct Mol Biol 25(9): 833-840. PubMed ID: 30127355

Bollen, M., Peti, W., Ragusa, M. J. and Beullens, M. (2010). The extended PP1 toolkit: designed to create specificity. Trends Biochem Sci 35: 450-458. PubMed ID: 20399103

Bonn, S., et al. (2012). Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development. Nat. Genet. 44(2): 148-56. PubMed ID: 22231485

Bothma, J. P., Magliocco, J. and Levine, M. (2011). The Snail repressor inhibits release, not elongation, of paused Pol II in the Drosophila embryo. Curr. Biol. 21: 1571-1577. PubMed ID: 21920753

Buckley, M. S., Kwak, H., Zipfel, W. R. and Lis, J. T. (2014). Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation. Genes Dev 28: 14-19. PubMed ID: 24395245

Cai, Y., et al. (2010). Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex. J. Biol. Chem. 285: 4268-4272. PubMed ID: 20018852

Chapman, R. D., et al. (2007). Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7. Science 318(5857): 1780-2. PubMed ID: 18079404

Cazalla, D., Xie, M. and Steitz, J. A. (2011). A primate herpesvirus uses the integrator complex to generate viral microRNAs. Mol Cell 43(6): 982-992. PubMed ID: 21925386

Chen, K., Johnston, J., Shao, W., Meier, S., Staber, C. and Zeitlinger, J. (2013). A global change in RNA polymerase II pausing during the Drosophila midblastula transition. Elife 2: e00861. PubMed ID: 23951546

Chen, X., Lu, C., Prado, J. R., Eun, S. H. and Fuller, M. T. (2011). Sequential changes at differentiation gene promoters as they become active in a stem cell lineage. Development 138(12): 2441-50. PubMed ID: 21610025

Cho, W. K., Spille, J. H., Hecht, M., Lee, C., Li, C., Grube, V. and Cisse, II (2018). Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361(6400): 412-415. PubMed ID: 29930094

Chopra, V. S., Hong, J. W. and Levine, M. (2009). Regulation of Hox gene activity by transcriptional elongation in Drosophila. Curr. Biol. 19(8): 688-93. PubMed ID: 19345103

Ciurciu, A., Duncalf, L., Jonchere, V., Lansdale, N., Vasieva, O., Glenday, P., Rudenko, A., Vissi, E., Cobbe, N., Alphey, L. and Bennett, D. (2013). PNUTS/PP1 regulates RNAPII-mediated gene expression and is necessary for developmental growth. PLoS Genet 9: e1003885. PubMed ID: 24204300

Conrad, T., Cavalli, F. M., Vaquerizas, J. M., Luscombe, N. M. and Akhtar, A. (2012). Drosophila dosage compensation involves enhanced Pol II recruitment to male X-linked promoters. Science 337(6095): 742-6. PubMed ID: 22821985

Cubenas-Potts, C., Rowley, M. J., Lyu, X., Li, G., Lei, E. P. and Corces, V. G. (2017). Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture. Nucleic Acids Res 45(4): 1714-1730. PubMed ID: 27899590

Dahlberg, O., Shilkova, O., Tang, M., Holmqvist, P. H. and Mannervik, M. (2015) P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development. PLoS Genet 11: e1004971. PubMed ID: 25679530

David, C. J., Boyne, A. R., Millhouse, S. R. and Manley, J. L. (2011). The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65-Prp19 complex. Genes Dev. 25(9): 972-83. PubMed ID: 21536736

Devaiah, B. N. and Singer, D. S. (2012). Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation. J Biol Chem 287: 38755-38766. PubMed ID: 23027873

Doamekpor, S. K., Sanchez, A. M., Schwer, B., Shuman, S. and Lima, C. D. (2014). How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes. Genes Dev 28: 1323-1336. PubMed ID: 24939935

Du, H. N., Fingerman, I. M. and Briggs, S. D. (2008). Histone H3 K36 methylation is mediated by a trans-histone methylation pathway involving an interaction between Set2 and histone H4. Genes Dev. 22(20): 2786-98. PubMed ID: 18923077

Eberle, A.B., Jordán-Pla, A., Gañez-Zapater, A., Hessle, V., Silberberg, G., von Euler, A., Silverstein, R.A. and Visa, N. (2015). An interaction between RRP6 and SU(VAR)3-9 targets RRP6 to heterochromatin and contributes to heterochromatin maintenance in Drosophila melanogaster. PLoS Genet 11: e1005523. PubMed ID: 26389589

Eissenberg, J. C., Shilatifard, A., Dorokhov, N., Michener, D. E.. (2007). Cdk9 is an essential kinase in Drosophila that is required for heat shock gene expression, histone methylation and elongation factor recruitment. Mol. Genet. Genomics. 277(2): 101-14. PubMed ID: 17001490

Elrod, N. D., Henriques, T., Huang, K. L., Tatomer, D. C., Wilusz, J. E., Wagner, E. J. and Adelman, K. (2019). The integrator complex attenuates promoter-proximal transcription at protein-coding genes. Mol Cell 76(5):738-752. PubMed ID: 31809743

Fant, C. B., Levandowski, C. B., Gupta, K., Maas, Z. L., Moir, J., Rubin, J. D., Sawyer, A., Esbin, M. N., Rimel, J. K., Luyties, O., Marr, M. T., Berger, I., Dowell, R. D. and Taatjes, D. J. (2020). TFIID enables RNA polymerase II promoter-proximal pausing. Mol Cell. PubMed ID: 32229306

Ferrari, F., Plachetka, A., Alekseyenko, A. A., Jung, Y. L., Ozsolak, F., Kharchenko, P. V., Park, P. J. and Kuroda, M. I. (2013). 'Jump Start and Gain' model for dosage compensation in Drosophila based on direct sequencing of nascent transcripts. Cell Rep 5: 629-636. PubMed ID: 24183666

Flanagan, J. F., Mi, L.-Z., Chruszcz, M., Cymborowski, M., Clines, K. L., Kim, Y., Minor, W., Rastinejad, F., Khorasanizadeh, S. (2005). Double chromodomains cooperate to recognize the methylated histone H3 tail. Nature 438: 1181-1185. PubMed ID: 16372014

Fong, N. and Bentley, D. L. (2001). Capping, splicing, and 3' processing are independently stimulated by RNA polymerase II: different functions for different segments of the CTD. Genes Dev. 15: 1783-1795. PubMed ID: 11459828

Gardini, A., Baillat, D., Cesaroni, M., Hu, D., Marinis, J. M., Wagner, E. J., Lazar, M. A., Shilatifard, A. and Shiekhattar, R. (2014). Integrator regulates transcriptional initiation and pause release following activation. Mol Cell 56(1): 128-139. PubMed ID: 25201415

Gawlinski, P., Nikolay, R., Goursot, C., Lawo, S., Chaurasia, B., Herz, H. M., Kussler-Schneider, Y., Ruppert, T., Mayer, M., and Grosshans, J. (2007). The Drosophila mitotic inhibitor Fruhstart specifically binds to the hydrophobic patch of cyclins. EMBO Rep. 8: 490-496. PubMed ID: 17431409

Gaykalova, D. A., Kulaeva, O. I., Volokh, O., Shaytan, A. K., Hsieh, F. K., Kirpichnikov, M. P., Sokolova, O. S. and Studitsky, V. M. (2015). Structural analysis of nucleosomal barrier to transcription. Proc Natl Acad Sci U S A 112: E5787-5795. PubMed ID: 26460019

Ghosh, S. K., Missra, A., Gilmour, D. S. (2011). Negative elongation factor accelerates the rate at which heat shock genes are shut off by facilitating dissociation of heat shock factor. Mol. Cell Biol. 31(20): 4232-43. PubMed ID: 21859888

Gibbs, E. B., Lu, F., Portz, B., Fisher, M. J., Medellin, B. P., Laremore, T. N., Zhang, Y. J., Gilmour, D. S. and Showalter, S. A. (2017). Phosphorylation induces sequence-specific conformational switches in the RNA polymerase II C-terminal domain. Nat Commun 8: 15233. PubMed ID: 28497798

Gilchrist, D. A., et al. (2011). Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation. Cell 143(4): 540-51. PubMed ID: 21074046

Gomez-Orte, E., Saenz-Narciso, B., Zheleva, A., Ezcurra, B., de Toro, M., Lopez, R., Gastaca, I., Nilsen, H., Sacristan, M. P., Schnabel, R. and Cabello, J. (2019). Disruption of the Caenorhabditis elegans Integrator complex triggers a non-conventional transcriptional mechanism beyond snRNA genes. PLoS Genet 15(2): e1007981. PubMed ID: 30807579

Gonzalez, A., Jafari, S., Zenere, A., Alenius, M. and Altafini, C. (2019). Thermodynamic model of gene regulation for the Or59b olfactory receptor in Drosophila. PLoS Comput Biol 15(1): e1006709. PubMed ID: 30653495

Grosshans, J., Muller, H.A., and Wieschaus, E. (2003). Control of cleavage cycles in Drosophila embryos by fruhstart. Dev. Cell 5: 285-294. PubMed ID: 12919679

Grote, M., et al. (2010). Molecular architecture of the human Prp19/CDC5L complex. Mol. Cell Biol. 30: 2105-2119. PubMed ID: 20176811

Guenther, M. G., Levine, S. S., Boyer, L. A., Jaenisch, R., and Young, R. A. (2007). A chromatin landmark and transcription initiation at most promoters in human cells. Cell 130: 77-88. PubMed ID: 17632057

Guo, Y. E., Manteiga, J. C., Henninger, J. E., Sabari, B. R., Dall'Agnese, A., Hannett, N. M., Spille, J. H., Afeyan, L. K., Zamudio, A. V., Shrinivas, K., Abraham, B. J., Boija, A., Decker, T. M., Rimel, J. K., Fant, C. B., Lee, T. I., Cisse, II, Sharp, P. A., Taatjes, D. J. and Young, R. A. (2019). Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572(7770):543-548. PubMed ID: 31391587

Ho, C. K. and Shuman, S. (1999). Distinct roles for CTD Ser-2 and Ser-5 phosphorylation in the recruitment and allosteric activation of mammalian mRNA capping enzyme. Mol. Cell 3: 405-411. PubMed ID: 10198643

Hogg, R., McGrail, J. C. and O'Keefe, R. T. (2010). The function of the NineTeen Complex (NTC) in regulating spliceosome conformations and fidelity during pre-mRNA splicing. Biochem. Soc. Trans. 38: 1110-1115. PubMed ID: 20659013

Ivaldi, M. S., Karam, C. S. and Corces, V. G. (2007). Phosphorylation of histone H3 at Ser10 facilitates RNA polymerase II release from promoter-proximal pausing in Drosophila. Genes Dev. 21(21): 2818-31. PubMed ID: 17942706

Jerebtsova, M., Klotchenko, S. A., Artamonova, T. O., Ammosova, T., Washington, K., Egorov, V. V., Shaldzhyan, A. A., Sergeeva, M. V., Zatulovskiy, E. A., Temkina, O. A., Petukhov, M. G., Vasin, A. V., Khodorkovskii, M. A., Orlov, Y. N. and Nekhai, S. (2011). Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1. Mol Cell Biochem 347: 79-87. PubMed ID: 20941529

Kamieniarz-Gdula, K., Gdula, M. R., Panser, K., Nojima, T., Monks, J., Wisniewski, J. R., Riepsaame, J., Brockdorff, N., Pauli, A. and Proudfoot, N. J. (2019). Selective and roles of vertebrate PCF11 in premature and full-length transcript termination. Mol Cell 74(1): 158-172. PubMed ID: 30819644

Kessler, R., Tisserand, J., Font-Burgada, J., Reina, O., Coch, L., Attolini, C. S., Garcia-Bassets, I. and Azorin, F. (2015). dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes. Nat Commun 6: 7049. PubMed ID: 25916810

Kirchner, J., Gross, S., Bennett, D. and Alphey, L. (2007). Essential, overlapping and redundant roles of the Drosophila protein phosphatase 1 alpha and 1 beta genes. Genetics 176: 273-281. PubMed ID: 17513890

Kwak, H. and Lis, J. T. (2013). Control of transcriptional elongation. Annu Rev Genet 47: 483-508. PubMed ID: 24050178

Lai, F., Gardini, A., Zhang, A. and Shiekhattar, R. (2015). Integrator mediates the biogenesis of enhancer RNAs. Nature 525(7569): 399-403. PubMed ID: 26308897

Laitem, C., Zaborowska, J., Tellier, M., Yamaguchi, Y., Qingfu, C., Egloff, S., Handa, H. and Murphy, S. (2015). CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription. Transcription: 0. PubMed ID: 26399478

Larschan, E., Bishop, E. P., Kharchenko, P. V., Core, L. J., Lis, J. T., Park, P. J. and Kuroda, M. I. (2011). X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila. Nature 471: 115-118. PubMed ID: 21368835

Louder, R. K., He, Y., Lopez-Blanco, J. R., Fang, J., Chacon, P. and Nogales, E. (2016). Structure of promoter-bound TFIID and model of human pre-initiation complex assembly. Nature 531(7596): 604-609. PubMed ID: 27007846

Lee, C., et al. (2008). NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila. Mol. Cell Biol. 28(10): 3290-300. PubMed ID: 18332113

Li, L. M. and Arnosti, D. N. (2011). Long- and short-range transcriptional repressors induce distinct chromatin states on repressed genes. Curr. Biol. 21: 406-412. PubMed ID: 21353562

Licatalosi, D. D., et al. (2002). Functional interaction of yeast pre-mRNA 3? end processing factors with RNA polymerase II. Mol. Cell 9: 1101-1111. PubMed ID: 12049745

Lindstrom, D. L. and Hartzog, G. A. (2001). Genetic interactions of Spt4-Spt5 and TFIIS with the RNA polymerase II CTD and CTD modifying enzymes in Saccharomyces cerevisiae. Genetics 159: 487-497. PubMed ID: 11606527

Lindstrom, D. L., et al. (2003). Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. Mol. Cell Biol. 23: 1368-1378. PubMed ID: 12556496

Lin, H., Chen, M., Kundaje, A., Valouev, A., Yin, H., Liu, N., Neuenkirchen, N., Zhong, M. and Snyder, M. (2015). Reassessment of Piwi binding to the genome and Piwi impact on RNA Polymerase II distribution. Dev Cell 32: 772-774. PubMed ID: 25805139

Lis, J. T., et al. (2000). P-TEFb kinase recruitment and function at heat shock loci. Genes Dev. 14: 792-803. PubMed ID: 10766736

Liu, B., Xu, Q., Wang, Q., Feng, S., Lai, F., Wang, P., Zheng, F., Xiang, Y., Wu, J., Nie, J., Qiu, C., Xia, W., Li, L., Yu, G., Lin, Z., Xu, K., Xiong, Z., Kong, F., Liu, L., Huang, C., Yu, Y., Na, J. and Xie, W. (2020). The landscape of RNA Pol II binding reveals a stepwise transition during ZGA. Nature 587(7832): 139-144. PubMed ID: 33116310

Lu, F., Portz, B. and Gilmour, D. S. (2019). The C-terminal domain of RNA polymerase II is a multivalent targeting sequence that supports Drosophila development with only consensus heptads. Mol Cell 73(6):1232-1242. PubMed ID: 30765194

Lu, H., Yu, D., Hansen, A. S., Ganguly, S., Liu, R., Heckert, A., Darzacq, X. and Zhou, Q. (2018). Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. Nature 558(7709): 318-323. PubMed ID: 29849146

Macdonald, N., et al. (2005). Molecular basis for the recognition of phosphorylated and phosphoacetylated histone h3 by 14-3-3. Mol. Cell 20: 199-211. PubMed ID: 16246723

Mancebo. H. S., et al. (1997). P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes Dev. 11(20): 2633-44. PubMed ID: 9334326

Marshall, N. F. and D. H. Price. (1995). Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J. Biol. Chem. 270: 12335-12338. PubMed ID: 7759473

McCracken, S., et al. (1997). 5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 11: 3306-3318. PubMed ID: 9407024

Mendjan, S., et al. (2006). Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila. Mol. Cell 21: 811-823. PubMed ID: 16543150

Min, I. M., et al. (2011). Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev. 25(7): 742-54. PubMed ID: 21460038

Murakami, K., Elmlund, H., Kalisman, N., Bushnell, D. A., Adams, C. M., Azubel, M., Elmlund, D., Levi-Kalisman, Y., Liu, X., Gibbons, B. J., Levitt, M. and Kornberg, R. D. (2013). Architecture of an RNA polymerase II transcription pre-initiation complex. Science 342: 1238724. Abstract

Missra, A. and Gilmour, D. S. (2010). Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex. Proc. Natl. Acad. Sci. 107(25): 11301-6. PubMed ID: 20534440

Muse, G. W., et al. (2007). RNA polymerase is poised for activation across the genome. Nat. Genet. 39(12): 1507-11. PubMed ID: 17994021

Ni, Z., Schwartz, B. E., Werner, J., Suarez, R.-R. and Lis, J. T. (2004). Coordination of transcription, RNA processing, and surveillance by P-TEFb kinase on heat shock genes. Mol. Cell 13: 55-65. PubMed ID: 14731394

Ni, Z., Saunders, A., Fuda, N. J., Yao, J., Suarez, J. R., Webb, W. W. and Lis, J. T. (2008). P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo. Mol. Cell Biol. 28(3): 1161-70. PubMed ID: 18070927

Nowak, S. J. and Corces, V. G. (2000). Phosphorylation of histone H3 correlates with transcriptionally active loci. Genes Dev. 14: 3003-3013. PubMed ID: 11114889

Nowak, S. J., Pai, C. Y., and Corces, V. G. (2003). Protein phosphatase 2A activity affects histone H3 phosphorylation and transcription in Drosophila melanogaster. Mol. Cell. Biol. 23: 6129-6138. PubMed ID: 12917335

Oktaba, K., Zhang, W., Lotz, T. S., Jun, D. J., Lemke, S. B., Ng, S. P., Esposito, E., Levine, M. and Hilgers, V. (2014). ELAV links paused Pol II to alternative polyadenylation in the Drosophila nervous system. Mol Cell 57(2):341-8. PubMed ID: 25544561

Orphanides, G. and Reinberg, D. (2002). A unified theory of gene expression. Cell 108: 439-451. PubMed ID: 11909516

Patel, A. B., Louder, R. K., Greber, B. J., Grunberg, S., Luo, J., Fang, J., Liu, Y., Ranish, J., Hahn, S. and Nogales, E. (2018). Structure of human TFIID and mechanism of TBP loading onto promoter DNA. Science 362(6421). PubMed ID: 30442764

Pathak, R. U., Srinivasan, A. and Mishra, R. K. (2014). Genome-wide mapping of matrix attachment regions in Drosophila melanogaster. BMC Genomics 15: 1022. PubMed ID: 25424749

Peng, J., Marshall, N. F. and Price, D. H. (1998). Identification of a Cyclin subunit required for the function of Drosophila P-TEFb. J. Biol. Chem. 273: 13855-13860. PubMed ID: 9593731

Petesch, S. J. and Lis, J. T. (2008). Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci. Cell 134: 74-84. PubMed ID: 18614012

Pinto, P. A., et al. (2011). RNA polymerase II kinetics in polo polyadenylation signal selection. EMBO J. 30(12): 2431-44. PubMed ID: 21602789

Prestel, M., et al. (2010). The activation potential of MOF is constrained for dosage compensation. Mol. Cell 38: 815-826. PubMed ID: 20620953

Raj, A., Chimata, A. V. and Singh, A. (2020). Motif 1 Binding Protein suppresses wingless to promote eye fate in Drosophila. Sci Rep 10(1): 17221. PubMed ID: 33057115

Raja, S. J., et al. (2010). The nonspecific lethal complex is a transcriptional regulator in Drosophila. Mol. Cell 38: 827-841. PubMed ID: 20620954

Rosin, L. F., Nguyen, S. C. and Joyce, E. F. (2018). Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in Drosophila nuclei. PLoS Genet 14(7): e1007393. PubMed ID: 30001329

Rowley, M. J., Nichols, M. H., Lyu, X., Ando-Kuri, M., Rivera, I. S. M., Hermetz, K., Wang, P., Ruan, Y. and Corces, V. G. (2017). Evolutionarily conserved principles predict 3D chromatin organization. Mol Cell 67(5): 837-852. PubMed ID: 28826674

Rowley, M. J., Lyu, X., Rana, V., Ando-Kuri, M., Karns, R., Bosco, G. and Corces, V. G. (2019). Condensin II counteracts Cohesin and RNA Polymerase II in the establishment of 3D chromatin organization. Cell Rep 26(11): 2890-2903. PubMed ID: 30865881

Rubtsova, M. P., Vasilkova, D. P., Moshareva, M. A., Malyavko, A. N., Meerson, M. B., Zatsepin, T. S., Naraykina, Y. V., Beletsky, A. V., Ravin, N. V. and Dontsova, O. A. (2019). Integrator is a key component of human telomerase RNA biogenesis. Sci Rep 9(1): 1701. PubMed ID: 30737432

Rudenko, A., Bennett, D. and Alphey, L. (2003). Trithorax interacts with type 1 serine/threonine protein phosphatase in Drosophila. EMBO Rep 4: 59-63. PubMed ID: 12524522

Rudenko, A., Bennett, D. and Alphey, L. (2004). PP1beta9C interacts with Trithorax in Drosophila wing development. Dev Dyn 231: 336-341. PubMed ID: 15366010

Sabari, B. R., Dall'Agnese, A., Boija, A., Klein, I. A., Coffey, E. L., Shrinivas, K., Abraham, B. J., Hannett, N. M., Zamudio, A. V., Manteiga, J. C., Li, C. H., Guo, Y. E., Day, D. S., Schuijers, J., Vasile, E., Malik, S., Hnisz, D., Lee, T. I., Cisse, II, Roeder, R. G., Sharp, P. A., Chakraborty, A. K. and Young, R. A. (2018). Coactivator condensation at super-enhancers links phase separation and gene control. Science 361(6400). PubMed ID: 29930091

Sawarkar, R., Sievers, C. and Paro, R. (2012). Hsp90 globally targets paused RNA polymerase to regulate gene expression in response to environmental stimuli. Cell 149: 807-818. PubMed ID: 22579285

Shah, N., Maqbool, M. A., Yahia, Y., El Aabidine, A. Z., Esnault, C., Forne, I., Decker, T. M., Martin, D., Schuller, R., Krebs, S., Blum, H., Imhof, A., Eick, D. and Andrau, J. C. (2018). Tyrosine-1 of RNA polymerase II CTD controls global termination of gene transcription in mammals. Mol Cell 69(1): 48-61 e46. PubMed ID: 29304333

Skaar, J. R., Ferris, A. L., Wu, X., Saraf, A., Khanna, K. K., Florens, L., Washburn, M. P., Hughes, S. H. and Pagano, M. (2015). The Integrator complex controls the termination of transcription at diverse classes of gene targets. Cell Res 25(3): 288-305. PubMed ID: 25675981

Smith, E. R., Winter, B., Eissenberg, J. C. and Shilatifard, A. (2008). Regulation of the transcriptional activity of poised RNA polymerase II by the elongation factor ELL. Proc. Natl. Acad. Sci. 105(25): 8575-9. PubMed ID: 18562276

Srinivasan, S., Dorighi, K. M. and Tamkun, J. W. (2008). Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II. PLoS Genet 4: e1000217. PubMed ID: 18846226

Stabell, M., Larsson, J., Aalen, R. B. and Lambertsson, A. (2007). Drosophila dSet2 functions in H3-K36 methylation and is required for development. Biochem. Biophys. Res. Commun. 359(3): 784-9. PubMed ID: 17560546

Stadelmayer, B., Micas, G., Gamot, A., Martin, P., Malirat, N., Koval, S., Raffel, R., Sobhian, B., Severac, D., Rialle, S., Parrinello, H., Cuvier, O. and Benkirane, M. (2014). Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes. Nat Commun 5: 5531. PubMed ID: 25410209

Sung, H.-w. et al. (2012). Number of nuclear divisions in the Drosophila blastoderm controlled by onset of zygotic transcription. Curr. Biol. 23(2): 133-8. PubMed ID: 23290555

Tantale, K., Mueller, F., Kozulic-Pirher, A., Lesne, A., Victor, J. M., Robert, M. C., Capozi, S., Chouaib, R., Backer, V., Mateos-Langerak, J., Darzacq, X., Zimmer, C., Basyuk, E. and Bertrand, E. (2016). A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting. Nat Commun 7: 12248. PubMed ID: 27461529

Tatomer, D. C., Elrod, N. D., Liang, D., Xiao, M. S., Jiang, J. Z., Jonathan, M., Huang, K. L., Wagner, E. J., Cherry, S. and Wilusz, J. E. (2019). The Integrator complex cleaves nascent mRNAs to attenuate transcription. Genes Dev 33(21-22): 1525-1538. PubMed ID: 31530651

Tee, W. W., Shen, S. S., Oksuz, O., Narendra, V. and Reinberg, D. (2014). Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs. Cell 156: 678-690. PubMed ID: 24529373

Teves, S. S. and Henikoff, S. (2013). Transcription-generated torsional stress destabilizes nucleosomes. Nat Struct Mol Biol 21(1): 88-94. PubMed ID: 24317489

Thomson, S., Hollis, A., Hazzalin, C. A., and Mahadevan, L. C. (2004). Distinct stimulus-specific histone modifications at hsp70 chromatin targeted by the transcription factor heat shock factor-1. Mol. Cell 15: 585-594. PubMed ID: 15327774

Vorobyeva, N. E., Nikolenko, J. V., Nabirochkina, E. N., Krasnov, A. N., Shidlovskii, Y. V. and Georgieva, S. G. (2012). SAYP and Brahma are important for 'repressive' and 'transient' Pol II pausing. Nucleic Acids Res 40: 7319-7331. PubMed ID: 22638575

Wahl, M. C., Will, C. L. and Luhrmann, R. (2009). The spliceosome: design principles of a dynamic RNP machine. Cell 136: 701-718. PubMed ID: 19239890

Wang, X., Lee, C., Gilmour, D. S. and Gergen, J. P. (2007). Transcription elongation controls cell fate specification in the Drosophila embryo. Genes Dev. 21(9): 1031-6. PubMed ID: 17473169

Wang, X., Hang, S., Prazak, L. and Gergen, J. P. (2010). NELF potentiates gene transcription in the Drosophila embryo. PLoS One 5(7): e11498. PubMed ID: 20634899

Washington, K., Ammosova, T., Beullens, M., Jerebtsova, M., Kumar, A., Bollen, M. and Nekhai, S. (2002). Protein phosphatase-1 dephosphorylates the C-terminal domain of RNA polymerase-II. J Biol Chem 277: 40442-40448. PubMed ID: 12185079

Wen, Y. and Shatkin, A. J. (1999). Transcription elongation factor hSPT5 stimulates mRNA capping. Genes Dev 13: 1774-1779. PubMed ID: 10421630

Wu, C.-H., et al. (2003). NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes Dev. 17: 1402-1414. PubMed ID: 12782658

Xiang, K., et al. (2010). Crystal structure of the human symplekin-Ssu72-CTD phosphopeptide complex. Nature 467(7316): 729-33. PubMed ID: 20861839

Xie, M., Zhang, W., Shu, M. D., Xu, A., Lenis, D. A., DiMaio, D. and Steitz, J. A. (2015). The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3' ends. Genes Dev 29(14): 1552-1564. PubMed ID: 26220997

Xu, J., Grant, G., Sabin, L. R., Gordesky-Gold, B., Yasunaga, A., Tudor, M. and Cherry, S. (2012). Transcriptional pausing controls a rapid antiviral innate immune response in Drosophila. Cell Host Microbe 12: 531-543. PubMed ID: 23084920

Yao, J., et al. (2006). Dynamics of heat shock factor association with native gene loci in living cells. Nature 442(7106): 1050-3. PubMed ID: 16929308

Yeo, M., Lee, S. K., Lee, B., Ruiz, E. C., Pfaff, S. L. and Gill, G. N. (2005). Small CTD phosphatases function in silencing neuronal gene expression. Science 307: 596-600. PubMed ID: 15681389

Visconti, R., Palazzo, L., Della Monica, R. and Grieco, D. (2012). Fcp1-dependent dephosphorylation is required for M-phase-promoting factor inactivation at mitosis exit. Nat Commun 3: 894. PubMed ID: 22692537

Yoh, S, M,, Cho, H., Pickle, L., Evans, R. M. and Jones, K. A. (2007). The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. Genes Dev. 21(2): 160-74. PubMed ID: 17234882

Yue, Z., et al. (1997). Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II. Proc. Natl. Acad. Sci. 94: 12898-12903. PubMed ID: 9371772

Zabidi, M. A., Arnold, C. D., Schernhuber, K., Pagani, M., Rath, M., Frank, O. and Stark, A. (2015). Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation. Nature 518(7540): 556-559. PubMed ID: 25517091

Zamudio, J. R., Kelly, T. J. and Sharp, P. A. (2014). Argonaute-bound small RNAs from promoter-proximal RNA polymerase II. Cell 156: 920-934. PubMed ID: 24581493

Zeitlinger, J., et al. (2007). RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo. Nat. Genet. 39(12): 1512-6 . PubMed ID: 17994019

Zhang, Z. and Gilmour, D. S. (2006). Pcf11 is a termination factor in Drosophila that dismantles the elongation complex by bridging the CTD of RNA polymerase II to the nascent transcript. Mol. Cell 21(1): 65-74. PubMed ID: 16387654

Zouaz, A., Auradkar, A., Delfini, M. C., Macchi, M., Barthez, M., Ela Akoa, S., Bastianelli, L., Xie, G., Deng, W. M., Levine, S. S., Graba, Y. and Saurin, A. J. (2017). The Hox proteins Ubx and AbdA collaborate with the transcription pausing factor M1BP to regulate gene transcription. EMBO J. PubMed ID: 28871058

date revised: 15 August 2023

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