InteractiveFly: GeneBrief

RNA polymerase II 215kD subunit: Biological Overview | References

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).

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).

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).

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).

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).

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, 2006).

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, 2006).

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, 2006).

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, 2006).

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, 2006).

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, 2006).

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, 2006).

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, 2006 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, 2006).

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, 2006).

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, 2006).

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).

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).

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