Cyclin dependent kinase 9


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

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

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

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

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

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

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

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

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

Pol II elongation factors Elongin-A and Cdk9 are essential for optimal Ubx and Abd-B expression

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

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

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

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

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

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

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

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

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

ChIP-chip and conventional ChIP assays suggest that the Abd-B promoter region might also contain a stalled form of Pol II. As seen for Ubx, reduced levels of Cdk9 and Elo-A cause significant enhancements in the Abd-BM1/+ mutant phenotype. In particular, Abd-BM1/+ heterozygotes display a weak transformation of posterior abdominal segments into anterior segments, particularly the seventh abdominal segment (A7) into A6 (ectopic partial pigmentation) and A6 to A5 (ectopic bristles in A6 sternite). These phenotypes are augmented by reductions in either Cdk9 or Elo-A activity. Double heterozygotes display a more complete A7-to-A6 transformation, as well as an increase in the number of bristles in A6, suggesting a more severe A6-to-A5 transformation. These segmental transformations are weakly enhanced (not suppressed) by lower levels of Spt4 and Spt5. In contrast, mutations in the negative elongation factor Nelf-E strongly suppress the Abd-BM1 phenotype, which is consistent with enhanced transcription of Abd-B. Triple heterozygotes, Abd-BM1/+; Cdk9/+; Elo-A/+, display an even more dramatic transformation of A7 to A6 and A6 to A5. Thus, as seen for Ubx, reduced levels of Cdk9 and Elo-A cause a significant diminishment in Abd-B+ gene activity (Chopra, 2009).

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

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

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

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

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

Targets of Activity

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, 1998a). Moreover, immunodepletion experiments show that the vast majority of Cdk9 is associated with a cyclin T subunit (Peng, 1998a), 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, 1998a), 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 ┬Ám. 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).

Positive transcription elongation factor b (P-TEFb) is a kinase that phosphorylates the carboxyl-terminal domain (CTD) of RNA Polymerase II (Pol II). Flavopiridol, a highly specific P-TEFb kinase inhibitor, dramatically reduces the global levels of Ser2--but not Ser5--phosphorylated CTD at actively transcribed loci on Drosophila polytene chromosomes under both normal and heat shocked conditions. Brief treatment of Drosophila cells with flavopiridol leads to a reduction in the accumulation of induced hsp70 and hsp26 RNAs. Surprisingly, the density of transcribing Pol II and Pol II progression through hsp70 in vivo are nearly normal in flavopiridol-treated cells. The major defect in expression is at the level of 3' end processing. A similar but more modest 3' processing defect was also observed for hsp26. It is proposed that P-TEFb phosphorylation of Pol II CTD coordinates transcription elongation with 3' end processing, and failure to do so leads to rapid RNA degradation (Ni, 2004).

P-TEFb contributes to Pol II phosphorylation at the promoter and during most or all of the elongation phase of transcription

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).

Protein interactions: Characterization of Cyclin T and Cdk9

Production of full-length runoff transcripts in vitro and functional mRNA in vivo is sensitive to the drug 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). P-TEF functions in a DRB-sensitive manner to allow RNA polymerase II elongation complexes to efficiently synthesize long transcripts. Nuclear extracts of Drosophila melanogaster Kc cells have been fractionated and three activities, P-TEFa, factor 2, and P-TEFb, have been identified that are directly involved in reconstructing DRB-sensitive transcription. P-TEFb is essential for the production of DRB-sensitive long transcripts in vitro, while P-TEFa and factor 2 are stimulatory. P-TEFb activity is associated with a protein comprising two polypeptide subunits with apparent molecular masses of 124 and 43 kDa. Using a P-TEFb-dependent transcription system, it has been shown that P-TEFb acts after initiation and is the limiting factor in the production of long run-off transcripts (Marshall, 1995).

The entry of RNA polymerase II into a productive mode of elongation is controlled, in part, by the postinitiation activity of positive transcription elongation factor b (P-TEFb). Removal of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II abolishes productive elongation. Correspondingly, P-TEFb can phosphorylate the CTD of pure RNA polymerase II. Furthermore, P-TEFb can phosphorylate the CTD of RNA polymerase II when the polymerase is in an early elongation complex. Both the function and kinase activity of P-TEFb are blocked by the drugs 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and H-8. P-TEFb is distinct from transcription factor IIH (TFIIH) because the two factors have no subunits in common, P-TEFb is more sensitive to DRB than is TFIIH, and most importantly, TFIIH cannot substitute functionally for P-TEFb. It is proposed that phosphorylation of the CTD by P-TEFb controls the transition from abortive into productive elongation mode (Marshall, 1996).

A full-length cDNA encoding the large subunit of Drosophila P-TEFb (Cyclin T) was cloned on the basis of sequence information obtained from the pure protein. The large subunit from purified P-TEFb was excised from an SDS gel and subjected to a peptide sequencing protocol. Based on the peptide sequence information, a 4.3-kb cDNA was cloned from Drosophila embryonic mRNA. The open reading frame was determined by knowing the size of P-TEFb large subunit and by finding a consensus sequence for the translational start site. The cDNA encodes a 118-kDa protein with an isoelectric point of 9.5 and a putative nuclear localization signal from amino acids 872-876. The 5'-untranslated region is relatively long, and the 3'-untranslated region is terminated by a poly(A) tail. Northern analysis indicates embryonic and adult flies contain mRNAs 5, 5.5, and 7 kb in length (Peng, 1998a).

Comparison of the new Drosophila sequence to those in the data bases using a Blast search has indicated that the amino-terminal region has highest similarity to Drosophila cyclin C and cyclin H2 proteins. Although the amino-terminal region of the large subunit of Drosophila P-TEFb shows only 14% identity to that of cyclin C and 12% identity to that of cyclin H, it is predicted to have two 5-helix folds that are conserved in cyclin proteins. The cyclin-CDK interaction is partly dictated by the first cyclin fold in which helix 3 forms the hydrophobic core as illustrated by the crystal structure of cyclin A and CDK2. The first five helices, especially helix 3, are the most conserved sequences in the cyclin box of the large subunit of Drosophila P-TEFb. The carboxyl terminus of the large subunit of Drosophila P-TEFb did not show high similarity to any other protein in the data bases. All these data suggest that the large subunit is a novel cyclin. Because the kinase subunit of P-TEFb associates with a cyclin subunit and shows similarity to other cyclin-dependent kinases it has been termed Cdk9. A strong interaction is found between the two proteins (Peng, 1998a).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Functional interaction between HEXIM and Hedgehog signaling during Drosophila wing development

Studying the dynamic of gene regulatory networks is essential in order to understand the specific signals and factors that govern cell proliferation and differentiation during development. This also has direct implication in human health and cancer biology. The general transcriptional elongation regulator P-TEFb regulates the transcriptional status of many developmental genes. Its biological activity is controlled by an inhibitory complex composed of HEXIM and the 7SK snRNA. This study examines the function of HEXIM during Drosophila development. It was found that HEXIM affects the Hedgehog signaling pathway. HEXIM knockdown flies display strong phenotypes and organ failures. In the wing imaginal disc, HEXIM knockdown initially induces ectopic expression of Hedgehog (Hh) and its transcriptional effector Cubitus interuptus (Ci). In turn, deregulated Hedgehog signaling provokes apoptosis, which is continuously compensated by apoptosis-induced cell proliferation. Thus, the HEXIM knockdown mutant phenotype does not result from the apoptotic ablation of imaginal disc but rather from the failure of dividing cells to commit to a proper developmental program due to Hedgehog signaling defects. Furthermore, ci was shown to be a genetic suppressor of hexim. Thus, HEXIM ensures the integrity of Hedgehog signaling in wing imaginal disc, by a yet unknown mechanism (Nguyen, 2016).

Transcription of protein-coding genes is mediated by RNA polymerase II (RNA Pol II) whose processivity is tightly controlled by the positive transcription elongation factor b (P-TEFb) after transcriptional initiation. This kinase promotes productive transcription elongation by catalyzing the phosphorylation of a number of regulatory factors, namely the Negative elongation factor (NELF), the DRB-sensitivity inducing factor (DSIF), as well as the C-terminal domain (CTD) of RNA Pol II. In human cells, P-TEFb forms two alternative complexes, which differ in size, components, and enzymatic activity. A 'small complex' (SC), composed of CyclinT and CDK9, corresponds to the catalytically active P-TEFb. In contrast, P-TEFb is kept in a catalytically inactive state and forms a 'large complex' (LC) when bound by a macromolecular complex containing the 7SK snRNA, Bicoid-interacting protein 3 (BCDIN3), La-related protein 7 (LARP7), and Hexamethylene bis-acetamide inducible protein 1 (HEXIM1). The formation of the LC is reversible and P-TEFb can switch back and forth between LC and SC in a very dynamic manner. Thus, HEXIM, together with other factors, acts as a sink of active P-TEFb which regulates its biological availability at target genes in response to the transcriptional demand of the cell. Although HEXIM target genes are not known, many lines of evidence strongly support a connection between developmental pathways or diseases and the control of transcription by HEXIM (Nguyen, 2016).

Transcriptional pause was initially described in the late 80s for the Drosophila HSP90 gene, where transcription stalls shortly after the elongation start and RNA Pol II accumulates at the 5' end of the gene, which is thus poised for transcription. It has been proposed that this phenomenon may be more general, as virtually all developmental genes in Drosophila and approximately 20 to 30 percent of genes in human and mouse show similar properties. The release from pause and the transition to productive elongation is under the control of the NELF factor, and so to P-TEFb, which is in turn controlled by HEXIM. Given that these genes already completed transcriptional initiation and that mRNA synthesis started, release from pause allows for a very fast and synchronized transcriptional response with low transcriptional noise (Nguyen, 2016).

It has been proposed that sustained pause may be a potent mechanism to actually repress gene transcription. This leads to the apparent paradox where transcriptional repression requires transcriptional initiation. Therefore, knockdown of the transcriptional pausing factor HEXIM would release transcription and reveal the regulation of poised genes (Nguyen, 2016).

HEXIM1 has been initially identified as a 359 aa protein whose expression is induced in human vascular smooth muscle cells (VSMCs) following treatment with hexamethylene bis-acetamide (HMBA) which is a differentiating agent. It is also called estrogen down-regulated gene 1 (EDG1) due to its decreased expression by estrogen in breast cancer cells. Ortholog of HEXIM1 in mice and chickens is activated in heart tissue during early embryogenesis, and was so named cardiac lineage protein 1 (CLP-1). HEXIM1 is involved in many kinds of cancer, viral transcription of HIV-1, cardiac hypertrophy, and inflammation. Overall, HEXIM defects are strongly associated with imbalance in the control of proliferation and differentiation. The CLP-1/HEXIM1 null mutation is embryonic lethal in mice, and results in early cardiac hypertrophy. Heterozygous littermates are still affected but with a less severe phenotype and survived up to adulthood. Moreover, Mutation in the carboxy-terminal domain of HEXIM1 causes severe defects during heart and vascular development by reducing the expression of vascular endothelial growth factor (VEGF), which is essential for myocardial proliferation and survival. Overexpression of HEXIM1 in breast epithelial cells and mammary gland decreases estrogen-driven VEGF expression, whereas it is strongly increased in loss of function mutant. As reported recently, HEXIM1 expression is required for enhancing the response to tamoxifen treatment in breast cancer patients. In addition, increased HEXIM1 expression correlates with a better prognosis and decreases probability of breast cancer recurrence. Additionally, terminal differentiation of murine erythroleukemia cells induced by HMBA or DMSO correlates with elevated levels of both HEXIM1 mRNA and protein. Furthermore, in neuroblastoma cells, HEXIM1 overexpression inhibits cell proliferation and promotes differentiation. Moreover, HEXIM1 modulates the transcription rate of NF-κB, an important regulator of apoptosis, cell proliferation, differentiation, and inflammation. However, despite theses advances, the dissection of HEXIM functions was mostly approached on a biochemical basis, and to date, very little is known about its physiological and developmental relevance in an integrated model. In order to address this important point, an in vivo model was developed (Nguyen, 2012) and it shown that a similar P-TEFb regulation pathway also exists in Drosophila, and that HEXIM is essential for proper development (Nguyen, 2016 and references therein).

In Drosophila, the Hedgehog (Hh) signaling pathway controls cell proliferation, differentiation and embryo patterning. The Hh activity is transduced to a single transcription factor, Cubitus interruptus (Ci), the Drosophila homolog of Gli. Wing imaginal discs can be subdivided into two compartments based on the presence of Hh protein. The posterior compartment (P) expresses Engrailed (En), which activates Hh and represses ci expression. The anterior compartment (A) expresses ci. The full length Ci protein (called Ci155) is constitutively cleaved into a truncated protein acting as a transcriptional repressor (Ci75) of hh and Decapentaplegic (dpp) genes. Hh inhibits the proteolytic cleavage of Ci, which then acts as a transcriptional activator of a number of target genes [Patched (ptc) and dpp, to name a few]. Thus, Ci155 is accumulated at the boundary between the A and P compartments where there are high levels of Hh, and it is absent in P compartment. Ci regulates the expression of Hh target genes in a manner dependent on Hh levels. In addition to proteolytic cleavage, the biological activity of Ci is also modulated by phosphorylation and nucleo-cytoplasmic partitioning. The mis-regulation of any components of the Hh pathway usually modifies the Ci155 levels, and results in developmental defects (Nguyen, 2016).

This paper examines the function of HEXIM during Drosophila development. HEXIM knockdown is shown to disrupt organ formation. In the wing disc, this latter effect is mediated by a strong ectopic induction of Hh signaling followed by apoptosis. The death of proliferative cells is subsequently compensated by proliferation of the neighboring cells: this is the mechanism of apoptosis-induced cell proliferation. Ci, the transcriptional effector of Hh pathway, is highly accumulated at both mRNA and protein levels in cells where HEXIM is knocked-down. Thus, the severe phenotype of HEXIM mutants resulted from Hh-related wing patterning defects. Furthermore, it was also shown that ci acts as a genetic suppressor of hexim, suggesting that HEXIM is an interacting factor of the Hh signaling pathway. This is the first time that the physiological function of HEXIM has been addressed in a whole organism (Nguyen, 2016).

Given that HEXIM is a general regulator of transcription elongation, the transcription machinery of mutant cells is eventually expected to be strongly affected that leads to cell death. One would argue that the rn>RNAi Hex mutant phenotype (undeveloped wing) is likely to be a simple consequence of a severe demolition of the wing pouch. However, this study clearly shows that the whole tissue is not ablated, although HEXIM mutant displays significant levels of apoptosis. Indeed, dying cells are efficiently replaced by new ones through apoptosis-induced proliferation (AIP) to such extent that the wing disc, including the wing pouch, increases strongly in size but still fails to promote the proper development of the wing. Thus, the phenotype is not a consequence of reduced size of the wing pouch, but rather cells fail to commit to a proper developmental program. The ectopic induction of Hh is one (among other) clear signature of abnormal development (Nguyen, 2016).

Two lines of evidence support a functional connection between HEXIM and Hedgehog signaling: (1) Ci expression is induced early, and (2) ci is a genetic suppressor of hexim. Although Hh is supposed to be mainly anti-apoptotic, there are a few reports indicating that it can promote apoptosis during development. For example when Ptc is deleted, there is increasing apoptosis in hematopoietic cells or Shh increases cell death in posterior limb cells. In this study, the induction of Hedgehog signaling is a primary event that precedes the wave of apoptosis, in HEXIM knockdown mutants. Given that cells subject to patterning defects often undergo apoptosis, the ectopic expression of Hh is probably the molecular event that triggers apoptosis in the wing disc. Then, the subsequent AIP will produce new cells and fuel a self-reinforcing loop of Hh activation and apoptosis (since HEXIM expression is continuously repressed). Accordingly, in rn>RNAi Hex mutant, cells undergoing AIP survive but fail to differentiate. This is supported by previous reports where deregulation of Hedgehog signaling, through modifications of Ci expression levels, leads to developmental defects. The phenotype of double knockdown mutants of Ci and HEXIM can be simply explained as following: cells lack the ability to respond to Hedgehog signaling and become blind to Hh patterning defect, thus leading to a Ci-like phenotype (Nguyen, 2016).

Although it cannot be excluded that Ci155 expression is directly affected by HEXIM, the extended expression domain of 155 in rn>RNAi Hex mutants may also indirectly result from increased levels of Hh. Indeed, the breadth of the AP stripe is defined in part by a morphogenetic gradient of Hh, with a decreasing concentration towards the anterior part of the wing disc. Thus, the augmented levels of Hh induced in rn>RNAi mutants could in principle explain the broader Ci expression at the AP stripe. To summarize, HEXIM knockdown increases Hh expression, potentially through regulation of P-TEFb complex, leading to patterning defects and a wave of apoptosis followed by compensatory proliferation (Nguyen, 2016).

A genetic screen in Drosophila showed that the two components of the small P-TEFb complex, Cdk9 and Cyclin T, are strong activators of the Hh pathway, but so far, no evidence directly connects HEXIM to Hh pathway. To this regard, the current work clearly establishes this connection. It is then tempting to speculate that by knocking-down HEXIM, the levels of active P-TEFb will be eventually increased that leads to an ectopic activation of the Hh pathway. More work is needed to specifically address this mechanistic point (Nguyen, 2016).

Interestingly, when carried out in the eye discs, GMR>RNAi Hex mutants display an extreme but rare phenotype with protuberances of proliferating cells piercing through the eyes. Although these few events could not be characterized any further, the parallel with the proliferating cells, which fail to differentiate in the wing disc, is striking. Of note, the role of HEXIM in the balance between proliferation and differentiation is not quite novel. Indeed, HEXIM was previously reported to be up-regulated upon treatment of HMBA, a well known inducer of differentiation. This paper shows that the regulatory role of HEXIM during development is mediated via controlling the Hedgehog signaling pathway. This is the first study that has addressed this phenomenon in vivo and in a non-pathological context (Nguyen, 2016).

Among other functions, HEXIM acts as a regulator of the P-TEFb activity which is in turn a general regulator of elongation (Nguyen, 2012). The availability of the P-TEFb activity mediates transcriptional pausing, a mechanism by which RNA pol II pauses shortly after transcription initiation and accumulates at the 5' end of genes. Transcription may then or may not resume, depending on a number of inputs. In these cases, RNA pol II appears 'stalled' at the 5' end of genes. Release from transcriptional pausing is fast and allows a more homogeneous and synchronized transcription at the scale of an imaginal disc or organ. In the other hand, a lack of release from transcriptional pausing is also a potent way to silence transcription. Interestingly, genome wide profiling of RNA pol II revealed a strong accumulation at the 5' end of 20% to 30% of the genes, most of which involved in development, cell proliferation and differentiation. In this context, HEXIM knockdown would be expected to have strong developmental defects. Such effects have been seen in all tissues tested so far (Nguyen, 2016).

The patterning of WT wing disc is set by a morphogenetic gradient of Hh, with high levels in the P compartments and no expression in the A compartment. It is therefore tempting to speculate that the Hh coding gene would be in a transcriptionally paused state in the anterior part of the wing pouch, that would be released upon HEXIM knockdown. This simple molecular mechanism, although speculative, would account for the induction of the ectopic expression of Hh in the anterior part of the wing pouch and the subsequent loops of apoptosis and AIP, ultimately leading to the wing developmental defects. Attempts were made to see whether the distribution of RNA Pol II along hh and ci is compatible with a transcriptional pause by using a number of RNA Pol II ChIP-Seq datasets that have been generated, together with RNA-Seq data, over the past few years. This study has processed these datasets and computed the stalling index (SI) for all genes. The SI is computed after mapping ChIP-Seq reads on the reference genome and corresponds to the log ratio of the reads density at the 5' end of the gene over the reads density along the gene body. Although these datasets clearly reveal a number of 'stalled' genes (>>100), no evidence of paused RNA Pol II was found for hh and ci (SI value of order 0), which were instead being transcribed. It is noted, however, that these datasets have been generated from whole embryos and S2 cell line. Given that Hh and Ci define morphogenetic gradients, their expression (and their transcriptional status) is likely highly variable between cells located in the different sub-regions of a disc, which may therefore not be reflected in these datasets (Nguyen, 2016).

Apart from the developmental function of HEXIM that is addressed in this work and the connection between HEXIM and Hedgehog signaling, the current results may also be of interest for human health studies. First, Hedgehog is a major signaling pathway that mediates liver organogenesis and adult liver regeneration after injury. In a murine model of liver regeneration, the Hedgehog pathway promotes replication of fully differentiated (mature) hepatocytes. Thus, addressing whether a connection between HEXIM and Hh exists would provide a mechanistic link between the control of gene expression and adult liver regeneration. Second, deregulated Hedgehog signaling is a common feature of many human tumors, and is found in at least 25% of cancers. In addition, recent data showed that aberrant Hedgehog signaling activates proliferation and increases resistance to apoptosis of neighboring cells and thus helps create a micro-environment favorable for tumorigenesis. Since its discovery, deregulated HEXIM expression is often associated to cancers and other diseases. Adding a new connection between HEXIM and Hedgehog signaling will shed more light into the role of HEXIM in abnormal development and cancer (Nguyen, 2016).

Surprisingly, although the biochemical interactions between HEXIM and its partners have been thoroughly described, very little is known about its biological function. Thus, this is the first time that the functional impact of HEXIM has been addressed in an integrated system (Nguyen, 2016).

Cyclin dependent kinase 9: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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