InteractiveFly: GeneBrief

Hexamethylene bisacetamide inducible: Biological Overview | References

BIOLOGICAL OVERVIEW

Regulation of the positive transcription elongation factor, P-TEFb (see Cdk9), plays a major role in controlling mammalian transcription and this is accomplished in part by controlled release of P-TEFb from the 7SK snRNP that sequesters the kinase in an inactive state. This study demonstrates that a similar P-TEFb control system exists in Drosophila. An RNA previously suggested to be a 7SK homolog is, in fact, associated with P-TEFb, through the action of a homolog of the human HEXIM1/2 proteins (dHEXIM). In addition, a Drosophila La related protein (now called dLARP7) is shown to be the functional homolog of human LARP7. The Drosophila 7SK snRNP (d7SK snRNP) responded to treatment of cells with P-TEFb inhibitors and to nuclease treatment of cell lysates by releasing P-TEFb. Supporting a critical role for the d7SK snRNP in Drosophila development, dLARP7 and dHEXIM were found to be ubiquitously expressed throughout embryos and tissues at all stages. Importantly, knockdown of dHEXIM was embryonic lethal, and reduction of dHEXIM in specific tissues led to serious developmental defects. These results suggest that regulation of P-TEFb by the d7SK snRNP is essential for the growth and differentiation of tissues required during Drosophila development (Nguyen, 2012).

The highly orchestrated pattern of gene expression driving cellular differentiation and tissue development is to a large extent controlled at the level of transcription, and regulation of the elongation phase of transcription plays an important role. RNA polymerase II elongation control starts with the default action of negative factors including DRB sensitivity inducing factor (DSIF) and negative elongation factor (NELF) that block the movement of initiated polymerases into the body of genes. These promoter proximal paused polymerases are poised for a regulated release into productive elongation by the positive transcription elongation factor, P-TEFb (a dimer of Cdk9 and Cyclin T). The cyclin-dependent kinase activity of P-TEFb coordinates the modification and exchange of factors associated with the elongation complex. The large subunit of DSIF, Spt5, as well as the NELFe subunit is phosphorylated by P-TEFb triggering the release of NELF from the complex. DSIF remains in the transcription complex and is joined by factors that dramatically change the rate of elongation from essentially zero to an average rate of ~3.8 kb/min. The P-TEFb-mediated transition into productive elongation is a singular event occurring near every gene's 5'-end that commits the engaged polymerase to complete an mRNA (Nguyen, 2012).

A large body of evidence points to RNA polymerase II elongation control as a general process required for the biogenesis of essentially all mRNAs. Treatment of cells with P-TEFb inhibitors blocks mRNA production and most transcription by RNA polymerase II in nuclei isolated from the cells and the process is reproduced utilizing in vitro systems derived from Drosophila and mammalian nuclear extracts regardless of the identity of the promoter used. Strong support for the generality of the process was found in the results of ChIP-Seq analyses that pinpointed the position of RNA polymerase II across mammalian and Drosophila genomes. Promoter proximal paused polymerases were found on a large number of Drosophila genes and on most mammalian genes. These included not only genes expressed at moderate to high levels of expression, but also genes with very low expression. The implication of these studies is that P-TEFb mediated release of the poised polymerases into productive elongation could be the rate limiting step of transcription on a large fraction of genes. Together all evidence points to P-TEFb not only being required for mRNA production, but also suggest that directed P-TEFb action could be a principle regulated step. In fact, c-myc which is a major regulator of many genes has been demonstrated to function at the level of elongation (Nguyen, 2012).

Because of the critical role that P-TEFb plays in regulating gene expression metazoans have developed a complex regulatory system that involves controlled sequestration and release of P-TEFb from an inhibitory complex. This complex is built on a 7SK snRNA scaffold (Marz, 2009) that constitutively contains a La related protein, LARP7 (Krueger 2008; Markert, 2008; He, 2008). 7SK is one of a few snRNAs that are capped by the addition of a single methyl group on the gamma phosphate on the 5'-end of the RNA. The methyl phosphate capping enzyme MEPCE responsible for the modification is also an integral part of the 7SK snRNP. In HeLa cells, about half of the 7SK snRNP contains these two proteins along with a heterogeneous array of hnRNP proteins. In the other half of the 7SK snRNPs, the hnRNPs are replaced by a double-stranded RNA-binding protein, HEXIM1 or HEXIM2 and this protein interacts with and inhibits P-TEFb (Michels, 2004; Yik, 2003; Byers, 2005; Yik, 2005). Both of the 7SK snRNPs distinguish themselves from all other snRNPs by being readily extracted from mild detergent treated nuclei at low salt indicating that they are not tightly bound to chromatin. The P-TEFb not in the 7SK snRNP, on the other hand, is only extracted by higher salt, indicating that it is associated with chromatin and suggesting that it is actually engaged in functional interactions. These biochemical properties suggest that P-TEFb could be extracted from the 7SK snRNP at the precise time and location needed to activate expression of specific genes (Nguyen, 2012).

Two proteins have been demonstrated to directly cause release of P-TEFb from the 7SK snRNP. One is HIV-1 Tat, a virally encoded transactivator which was the first protein found to functionally interact with P-TEFb. A crystal structure of Tat bound to P-TEFb revealed an extensive, stable interface between the viral and host protein. Tat also has an RNA-binding domain with specificity toward the nascent HIV transcript, TAR, which ultimately results in recruitment of P-TEFb and activation of viral transcription. Expression of Tat alone in human cells leads to the release of P-TEFb from the 7SK snRNP. This release does not require any modifications of Tat or the proteins or RNA in the 7SK snRNP because recombinant Tat can extract P-TEFb from highly purified 7SK snRNPs immunoprecipitated from cell extracts. Although Tat interacts directly with the 7SK RNA and the RNA-binding domain of Tat has a modest effect on the release of P-TEFb from the 7SK snRNP, the P-TEFb-binding domain is sufficient for efficient release. The second protein, Brd4, is a bromodomain-containing protein that binds to acetylated histones normally associated with active regions of transcription. Brd4 can be found in complex with P-TEFb and this is mediated by a small domain found in the C-terminus of Brd4. Just like Tat, this domain leads to the release of P-TEFb from the 7SK snRNP when expressed in human cells. Furthermore, it can also extract P-TEFb directly from the immunoprecipitated 7SK snRNPs. Because Brd4 does not have an RNA-binding domain it is clear that P-TEFb binding is the key property required. A number of other transcription factors can interact with P-TEFb and some of them may also have the ability to extract P-TEFb from the 7SK snRNP. The fact that at least some factors have the ability to extract P-TEFb provides additional support for a model in which the P-TEFb used to activate a gene is taken directly from the 7SK snRNP. This would guard against accidental activation of poised polymerases on genes that should be silent while maintaining a relatively high level of P-TEFb in a potentially active state (Nguyen, 2012).

Drosophila is an excellent model system to examine the role that RNA polymerase II elongation control takes in regulating development. P-TEFb was first identified in Drosophila and fly HSP70 genes were among the first genes shown to have promoter proximal paused polymerase. Elegant studies from several labs have demonstrated that these poised polymerases are prevalent across the fly genome and are especially enriched on developmentally controlled genes. The rapid induction provided by poised polymerases is useful in synchronous activation of particular genes across a population of cells and they also can act as insulators when placed between enhancers and another promoter. Unfortunately, only a little is known about the 7SK snRNP in Drosophila. One study identified a potential candidate for 7SK RNA (Gruber, 2008) and another demonstrated that the MEPCE homolog, Bin3, was associated with and stabilized the RNA (Singh, 2011). Importantly, it was not determined if P-TEFb was associated with the RNA. This study provides conclusive evidence that the RNA identified is indeed 7SK and that a Drosophila 7SK snRNP containing homologs of human P-TEFb, HEXIM1/2 and LARP7 exists. It is also shown that dHEXIM is essential for development and that the snRNP is widely expressed. Taken altogether, this suggests an important role of snRNP in development (Nguyen, 2012).

This study has provided strong evidence that the 7SK snRNP which has been demonstrated to control P-TEFb in mammals is also expressed in Drosophila. Bioinformatic, biochemical and molecular genetic methods led to the identification of a P-TEFb complex containing homologs of HEXIM1/2, LARP7 and 7SK. The d7SK snRNP responds to inhibition of P-TEFb in an immortal cell line by release of P-TEFb and dHEXIM suggesting that it is regulated by similar mechanisms as found in humans. Importantly, having identified most of the components of the d7SK snRNP and developed molecular probes for the proteins and RNA, allows for the complex to be studied during the development of an organism. Toward that end, it was demonstrated that components are expressed in a variety of tissues and embryonic developmental stages. Significantly, the key P-TEFb regulator, dHEXIM, is essential for proper Drosophila development (Nguyen, 2012).

The fact that the various components of the 7SK snRNP complex are found in both Drosophila and human is unlikely the result of convergent evolution. Rather, this strongly suggests that the complex characterized in this work is a true homolog of the one found in mammals. This is further supported by the fact that the fly 7SK snRNP complex is sensitive to P-TEFb inhibitors in a manner similar to that of human's. Thus, one would expect the complex to have similar biological function in both Drosophila and human at the molecular, cellular and developmental level (Nguyen, 2012).

Although it was possible to draw many similarities between the human and Drosophila 7SK snRNP, there were some differences found. Most of the detailed work on the 7SK snRNP in humans has been carried out in HeLa cells, and this study describes the biochemical characterization of complexes obtained from the Kc cell line. Both cell lines are immortal and have similar doubling times. As was found in the human system, P-TEFb is inhibited by the 7SK snRNP. Both human HEXIM1 and dHEXIM have the ability to bind to small dsRNAs in addition to 7SK. In HeLa cells, ~80% of HEXIM1 is found in a low molecular weight form out of the 7SK snRNP, and in Drosophila, 90%-95% of dHEXIM is not in the d7SK snRNP. A difference is that most of the 'free' dHEXIM is found in a high molecular weight form that is slightly smaller than the 7SK snRNP. The nature of the rapidly sedimenting form of dHEXIM is not known, but based on a number of the current results (gradient analyses and co-immunoprecipitions) it is not part of the 7SK snRNP. In both organisms, all 7SK is bound by LARP7, but only in Drosophila is some free LARP7 found. What role LARP7 might play out of the 7SK complex is not clear. Treatment of human or Drosophila cells with P-TEFb inhibitors leads to release of P-TEFb and HEXIM. While it has been determined that human 7SK undergoes a conformational change upon loss of P-TEFb and HEXIM1, this has not been examined for d7SK. However, the main structural elements of human 7SK are found in d7SK including two highly conserved regions that predict that the 5'-end of the RNA would pair with a region just upstream of the final stem and loop that is required for P-TEFb association with the 7SK snRNP (Nguyen, 2012).

Bin3, the Drosophila homolog of the human methyl phosphate capping enzyme (MEPCE) that methylates the gamma phosphate at the 5'-end of 7SK was recently demonstrated to be required for the stability of d7SK (Singh, 2011). A similar analysis on flies mutant for Bin3 was performed, and it was found that mutation of Bin3 lead to destabilization of d7SK without affecting U6 snRNA that is capped in a similar manner to 7SK snRNA. Bin3 was discovered as a protein that interacted with Bicoid, a homeodomain protein that directs early pattern formation. Besides playing a role in stabilizing d7SK, Bin3 was found in a complex with bicoid on the 3'-UTR of the caudal mRNA. It will be interesting to determine if Bin3's role in repressing translation of caudal mRNA is related to its role in the d7SK snRNP (Nguyen, 2012).

It is of interest that dLARP7 showed differential intra-nuclear distribution in a tissue-specific manner. In tissues like larval salivary glands, fat bodies and the adult ovary, dLARP7 displayed pan-nuclear localization, excluding nucleoli as is found for LARP7 in HeLa cells. In contrast, in the larval gut and gastric caecae, it localizes predominantly to the nucleoli, with diffuse staining in the nucleoplasm. Similar intra-nuclear localization was reported for the mammalian 7SK RNA, though the exact reason for the observed discrepancy is not clearly understood. For example, while a number of groups have showed that 7SK is pan-nuclear, colocalizing with splicing factor compartments, another study has shown that a majority of nuclear 7SK is localized to the nucleolus. While this discrepancy could be due to the age of the culture or the culture conditions applied, it is implicit in these data that the localization of 7SK snRNP is dynamic and might change in response to developmental and physiological needs. Accordingly, dLARP7 also showing these localizations trends reminiscent of 7SK snRNP suggests a greater biological meaning to differential expression and localization (Nguyen, 2012).

What has been learned about the involvement of the 7SK snRNP in Drosophila development? Although some tissues seem to have more or less dLARP7 and dHEXIM, both are ubiquitously expressed. Variation in expression may relate to the amount of the 7SK snRNP that is needed for each tissue to supply P-TEFb for its transcriptional program. The most important finding was that dHEXIM is absolutely essential for survival of the embryo and is required for development of all tissues tested. When knocked down early embryos fail to progress past the larval stage. When the early requirement is bypassed by expressing siRNA later in specific tissues, every tissue targeted was dramatically affected yielding severe phenotypes. These results support the idea that regulation of P-TEFb plays a critical role in Drosophila development which fits with the recent findings indicating that genes controlling development are usually loaded with polymerases poised for the P-TEFb-mediated transition into productive elongation (Nguyen, 2012).

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).

Search PubMed for articles about Drosophila Hexim

Byers, S. A., Price, J. P., Cooper, J. J., Li, Q. and Price, D. H. (2005). HEXIM2, a HEXIM1-related protein, regulates positive transcription elongation factor b through association with 7SK. J Biol Chem 280: 16360-16367. PubMed ID: 15713662

Gruber, A. R., Kilgus, C., Mosig, A., Hofacker, I. L., Hennig, W. and Stadler, P. F. (2008). Arthropod 7SK RNA. Mol Biol Evol 25: 1923-1930. PubMed ID: 18566019

He, N., Jahchan, N. S., Hong, E., Li, Q., Bayfield, M. A., Maraia, R. J., Luo, K. and Zhou, Q. (2008). A La-related protein modulates 7SK snRNP integrity to suppress P-TEFb-dependent transcriptional elongation and tumorigenesis. Mol Cell 29: 588-599. PubMed ID: 18249148

Krueger, B. J., Jeronimo, C., Roy, B. B., Bouchard, A., Barrandon, C., Byers, S. A., Searcey, C. E., Cooper, J. J., Bensaude, O., Cohen, E. A., Coulombe, B. and Price, D. H. (2008). LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated. Nucleic Acids Res 36: 2219-2229. PubMed ID: 18281698

Markert, A., Grimm, M., Martinez, J., Wiesner, J., Meyerhans, A., Meyuhas, O., Sickmann, A. and Fischer, U. (2008). The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and affects transcription of cellular and viral polymerase II genes. EMBO Rep 9: 569-575. PubMed ID: 18483487

Marz, M., Donath, A., Verstraete, N., Nguyen, V. T., Stadler, P. F. and Bensaude, O. (2009). Evolution of 7SK RNA and its protein partners in metazoa. Mol Biol Evol 26: 2821-2830. PubMed ID: 19734296

Michels, A. A., Fraldi, A., Li, Q., Adamson, T. E., Bonnet, F., Nguyen, V. T., Sedore, S. C., Price, J. P., Price, D. H., Lania, L. and Bensaude, O. (2004). Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor. EMBO J 23: 2608-2619. PubMed ID: 15201869

Nguyen, D., Fayol, O., Buisine, N., Lecorre, P. and Uguen, P. (2016). Functional interaction between HEXIM and Hedgehog signaling during Drosophila wing development. PLoS One 11: e0155438. PubMed ID: 27176767

Nguyen, D., Krueger, B. J., Sedore, S. C., Brogie, J. E., Rogers, J. T., Rajendra, T. K., Saunders, A., Matera, A. G., Lis, J. T., Uguen, P. and Price, D. H. (2012). The Drosophila 7SK snRNP and the essential role of dHEXIM in development. Nucleic Acids Res 40: 5283-5297. PubMed ID: 22379134

Singh, N., Morlock, H. and Hanes, S. D. (2011). The Bin3 RNA methyltransferase is required for repression of caudal translation in the Drosophila embryo. Dev Biol 352: 104-115. PubMed ID: 21262214

Yik, J. H., Chen, R., Nishimura, R., Jennings, J. L., Link, A. J. and Zhou, Q. (2003). Inhibition of P-TEFb (CDK9/Cyclin T) kinase and RNA polymerase II transcription by the coordinated actions of HEXIM1 and 7SK snRNA. Mol Cell 12: 971-982. PubMed ID: 14580347

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date revised: 25 September 2016

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