cubitus interruptus

cis-Regulatory Sequences and Functions

Regulatory sequences responsible for the normal pattern of ci expression have been located. Separate elements regulate ci expression in embryos and imaginal discs. Mutants that delete a portion of an upstream regulatory region express ci ectopically in the posterior compartments of their wing imaginal discs and have wings with malformed posterior compartments. A proximal promoter element drives ci expression in the ventral midline (Schwartz, 1995).

Deformed, ci, and engrailed itself are targets of Engrailed. Engrailed is involved in an auto-regulatory loop in anterior compartments of parasegments, and both deformed and ci are limited to posterior compartments by engrailed function in adjacent anterior compartments. Engrailed binding sites have been found in the promoters of both engrailed and ci (Saenz-Robles, 1995).

Transcriptional Regulation

Evidence that the Engrailed protein normally represses ci in anterior parasegmental compartments (Eaton, 1990) includes the expansion of ci expression into anterior compartment cells that lack Engrailed function, diminution of ci expression upon overexpression of Engrailed protein in anterior compartment cells, and the ability of Engrailed protein to bind to the ci regulatory region in vivo and in vitro (Schwartz, 1995).

A dominant interaction between combgap and engrailed/invected mutations that gives rise to a gap in vein L4 strongly suggests that Cg and En/Inv act together to repress posterior cubitus interruptus transcription. Posterior expression of En represses the transcription of ci resulting in anterior specific expression. En has been shown to interact directly with the ci regulatory elements. In cg mutant wing imaginal discs, weak ectopic expression of ci-lacZ reporter constructs are found in posterior cells, thus Cg may act in concert with En to repress posterior ci. Hypomorphic mutants in either cg or en/inv can give rise to the reduction in vein L4 that is characteristic of ectopic ci expression (Svendsen, 2000).

Many proteins with multiple C2H2 zinc finger motifs like those found in cg have been shown to be transcription factors, DNA-binding proteins or chromatin proteins. The widespread localization of Cg on salivary gland chromosomes is consistent with all of these activities. While the data have not yet established direct action of Cg on the ci regulatory elements, binding of Cg to the ci region of polytene chromosomes suggests that Cg could be a direct regulator of ci transcription. Direct binding of Cg (produced in E. coli) to DNA from the ci regulatory region has not been detected. However, given that the transcriptional regulation of ci is likely to be complex, Cg may not act at the level of direct DNA binding. The involvement of the Pc-group genes in the repression of ci suggests that intricate regulatory modes are necessary to maintain the correct levels and spatial patterns of ci transcription during imaginal disc development. Furthermore, the ci-regulatory regions have been shown to be subject to transvection effects, indicating that interchromosomal interactions also govern ci regulation. Thus Cg may act at any level, from generally influencing the chromosome pairing through to direct binding of ci enhancer elements. Finally, the positive and negative effects of cg mutants on ci transcription and the genetic interaction with en/inv suggest that Cg may be required in conjunction with other transcription factors for the function of ci enhancers and that Cg may not specify activation or repression itself (Svendsen, 2000).

In legs and antennae, overall Ci levels are decreased in the anterior compartment, resulting in circumferential overgrowth of the anterior compartment and ectopic anterior expression of the morphogens wg and dpp. Similar effects on leg morphology have been previously reported when wg and dpp were ectopically activated in anterior cells. The rescue of cg mutant leg defects by additional expression of ci in the anterior compartment using the Gal4/UAS system indicates that the phenotypes result from a reduction of Ci-75 leading to the derepression of wg and dpp. Thus, it is concluded that the Cg protein is critical for the proper levels and spatial patterns of Ci and that the A/P limb patterning defects in cg mutants are due largely, if not completely, to mis-regulation of ci (Svendsen, 2000).

The effects of cg mutants on ci expression are seen only in the anterior compartment of legs but in both anterior and posterior compartments in wings. What is the basis for this difference? While anterior Ci is reduced in both limbs, ectopic posterior Ci is only seen in wings. One possibility is that the alleles that have been studied may have different effects on cg expression in anterior versus posterior compartments and/or legs versus wings. However, little Cg imaginal disc staining was seen in cg²/cg², suggesting little or no Cg protein is produced, and so the phenotype may be near null (there are no deficiencies uncovering the cg locus, so this could not be tested genetically). Cg may not be required for repression of ci in the posterior compartment of leg discs, or alternatively, there may be a much lower threshold for Cg function in legs. The different effects on ci expression in cg mutant leg and wing imaginal discs suggest that while the broad framework is similar, there may be unique aspects to A/P patterning in dorsal versus ventral limbs. The predominance of wing phenotypes in ci^W and similar ci regulatory mutations also suggests a difference in the way ci is regulated in wings versus legs. Another difference was seen in the effects of reduced Ci levels on the expression of dpp. A greater reduction of Ci staining is seen in the anterior compartments of wings compared with leg imaginal discs; paradoxically, ectopic expression of dpp is seen in all cg mutant leg imaginal discs but none is seen in cg²/cg² wing imaginal discs. Although there are limits to how accurately real levels of Ci may be inferred from histochemical staining in different tissues, the simple conclusion is that dpp responds to different thresholds of Ci in legs and wings, and that the effect of cg is indirect (Svendsen, 2000).

The posterior wing venation defect in cg hypomorphs is very similar to that found in ci mutants and this phenotype is enhanced in cg/+;ci/+ transheterozygotes. These ci mutants, however, are gain-of-function mutants; they show ectopic expression of ci in the posterior. In fact, direct misexpression of ci in the posterior using the UAS/Gal4 system can also produce the same vein defects as seen in these mutants and in cg mutants. Analysis of cg mutant discs reveals ectopic ci expression in the posterior, indicating that the cg posterior phenotype is almost certainly the direct result of deregulation of ci expression in this compartment (Campbell, 2000).

Ci expression is also abnormal in the anterior of cg mutant discs, being found at much lower levels than in wild-type discs. Loss of ci expression in the wing results in hedgehog gain-of-function phenotypes, including overgrowth and misexpression of dpp. Reduced Ci levels in the leg also result in the characteristic overgrowth phenotype, with ectopic expression of wg and dpp, found following ubiquitous expression of Hh -- i.e., the same phenotype as that found in cg mutant leg discs. Support for the proposal that the anterior combgap phenotype in the leg is also the direct result of deregulation of ci expression, in this case lowered levels of expression, comes from the observation that raising ci levels in cg mutant leg discs using the UAS/Gal4 system can suppress the overgrowth and ectopic dpp expression (Campbell, 2000).

One difference between ci and cg mutants is that wing discs from the former have a hedgehog gain-of-function phenotype with overgrowth and ectopic dpp in the anterior, while the latter do not show overgrowth and only very weak ectopic dpp. It is possible that the leg and wing are differentially sensitive to Ci levels and the Ci levels are still high enough in the wing in cg mutants to repress most dpp expression. Protein levels detected with antibody staining in ci hypomorphs and cg mutants are too low to detect significant differences with confidence, so the reason for the difference between ci and cg wings remains to be determined. Ci is also required during embryogenesis, but the putative null cg mutant survives to the early pupal stage. This suggests either that lower levels of Ci are sufficient for embryonic but not larval development or that cg RNA is maternally supplied. The first possibility is supported by the observation that hypomorphic ci mutants are not embryonic lethal and survive to the early pupal stage. However, in situ analysis reveals that CG RNA is maternally supplied so that the question of whether cg is required during embryogenesis will require the generation of germline clones (Campbell, 2000).

Full-length Ci acts as a transcriptional activator and there is evidence that the lowered levels of Ci in cg mutants also compromises Ci function as an activator. Although, dpp is misexpressed in cg discs, the level of expression, even at the compartment border, is lower than that found in wild-type discs. A similar phenomenon has been demonstrated for loss of ci in the wing and it appears that the high levels of dpp in wild-type discs require activation by Ci-155, as well as the absence of Ci-75. Thus, the lower levels of dpp in cg discs are presumably due to lower levels of Ci-155. Another gene directly activated by Ci is en in late third instar wing discs. Ci-dependent en activation in the anterior compartment does not occur in cg mutant cells, again presumably because the level of the Ci-155 activator form is too low (Campbell, 2000).

Thus Cg is required to activate ci expression to its normal levels in the anterior compartment and to repress ci expression in the posterior. The Cg protein contains multiple zinc fingers and is most probably a DNA-binding protein that would be expected to bind to elements at the ci locus. However, understanding the mechanism by which it regulates ci expression requires further studies. It is possible that Cg functions as a standard transcription factor and activates ci transcription in the anterior and represses it in the posterior. If this is the case, its activity must be modified in either the anterior or posterior compartments. Analysis of the Cg protein outside of the zinc fingers does not reveal any classical activator or repressor domains, but as these are often not well defined it is impossible to determine whether the protein has these activities without more-detailed studies (Campbell, 2000).

An argument against such a direct involvement of Cg in transcription is the well-documented role of En in regulating ci expression. En is a transcription factor that represses expression of several genes including ci, dpp and wg, and has been shown to bind to elements at the ci locus. It would appear likely that En is the primary factor that represses transcription of ci in the posterior. If this is the case, the function of Cg in regulating transcription may be indirect and may be to assist the binding of other transcription factors to the ci gene. If so, the misexpression of Ci in the posterior of cg mutant discs would be due to a lowered ability of En to bind in the absence of Cg protein, while the lowered Ci levels in the anterior would be due to a lowered ability to bind a currently unidentified transcriptional activator of ci. There are several possible mechanisms by which Cg might affect the binding of other factors. For example, there may be direct physical interactions between Cg and these other factors. Alternatively, Cg action could be more indirect, for example, it could modify chromatin structure at the ci locus producing a more open conformation. Further studies are required to test these possibilities (Campbell, 2000).

The differentiation of cells in the Drosophila eye is precisely coordinated in time and space. Each ommatidium is founded by a photoreceptor (R8) cell. These R8 founder cells are added in consecutive rows. Within a row, the nascent R8 cells appear in precise locations that lie out of register with the R8 cells in the previous row. The bHLH protein Atonal determines the development of the R8 cells. The expression of atonal is induced shortly before the selection of a new row of R8 cells and is initially detected in a stripe. Subsequently, atonal expression resolves into regularly spaced clusters (proneural clusters) that prefigure the positions of the future R8 cells. The serial induction of atonal expression, and hence the increase in the number of rows of R8 cells, requires Hedgehog function. In addition to this role, Hedgehog signaling is also required to repress atonal expression between the nascent proneural clusters. This repression has not been previously described and appears to be critical for the positioning of Atonal proneural clusters and, therefore, the position of R8 cells. The two temporal responses to Hedgehog are due to direct stimulation of the responding cells by Hedgehog itself (Dominguez, 1999).

The initial expression of ato in the eye discs occurs in a strip of cells anterior to the morphogenetic furrow. The levels of Ato within this stripe vary, with enhanced Ato expression corresponding to the approximate position of proneural clusters. Behind the furrow, the only cells that express ato are the future R8 cells. In mature R8 cells, the expression of ato is repressed. When ato and hh expressions are compared, it appears that the refinement of ato expression occurs in cells close to the hh-expressing cells, whereas the continuous stripe of ato, which is believed to be induced by Hh, is 5-7 ommatidial rows in front of the first row of hh-expressing cells. This observation suggests that Hh acts at a distance to induce ato. Such a long-range action of Hh could either be direct or indirect (relay by a secondary signal) (Dominguez, 1999).

In the eye disc, the Ci protein is expressed dynamically, with the highest levels of Ci protein overlapping with Ato expression. Accordingly, misexpression of high levels of Ci in clones of cells showed that Ci is able to induce Ato. The Ci accumulation in cells ahead of the furrow depends on Hh, because cells lacking smo activity have low uniform levels of Ci. Loss of Hh reception in more posterior regions results in the failure to downregulate Ci levels and consequently mutant cells have inappropriately high Ci protein levels when compared to wild-type neighbors. This indicates that Hh stimulates (at long-range) and inhibits (at short-range) Ci accumulation (Dominguez, 1999).

The regulation of ato by the Hh-signaling pathway was studied further by generating clones of marked cells expressing a membrane-tethered Hh protein tagged with CD2 (Hh-CD2). ato expression in cells that have gained hh was examined. Misexpression of hh-CD2 can either activate (when clones are lying anteriorly) or repress (when they lie adjacent to the furrow) the expression of ato. Repression of ato is autonomous in the hh-CD2 cells, suggesting that Hh may repress ato directly. These observations suggest that Hh is secreted near the advancing furrow: close to the source ato expression is inhibited, further away it is induced. If hh-CD2 is misexpressed, naive cells begin to express ato prematurely and this ectopic ato initiates precocious ommatidial formation. However, slightly later (and within the region of influence of the endogenous hh), misexpression of hh-CD2 results in the premature repression of ato. Thus, cells experiencing the extra Hh exhibit no ato expression while the wild-type neighbors just begin to express ato. This model has been tested by manipulating the reception of the Hh signal using in vivo assays. Genetic evidence shows that Hh is required for both promoting and inhibiting ato expression (Dominguez, 1999).

In the proposed model, the induction of Hh has two effects in the responding cells: (1) as an ato inducing signal, through the activation (by upregulation) of the Zn-finger transcription factor Ci, and (2) as an inhibitory signal, through activation of Rough, to inhibit ato expression in the cells in and behind the furrow. The two responses occur in a cell sequentially, as monitored by ato and rough expression in the wild-type pattern and by analysis of their expression in marked clones. The expression domains of ato, Ci protein and rough and their relationship with Hh supports the model. Ci and rough are activated and expressed, respectively, by Hh in restricted spatial domains across the furrow and their expression either overlaps (in the case of Ci) or is complementary (in the case of rough) with ato, consistent with their respective roles in promoting or inhibiting ato expression (Dominguez, 1999).

ato expression is controlled by two enhancer elements located 5' or 3' to the coding sequences (Sun, 1998). A 3' enhancer directs initial expression in a stripe anterior to the furrow and a distinct 5' enhancer drives expression in the proneural clusters and R8 cells within and posterior to the furrow. The 5' enhancer, but not the 3' enhancer, depends on endogenous ato function. The identification of the factors that activate the 5' enhancer element will require refining the ato regulatory sequences followed by binding studies in vitro and in vivo. One of the factors binding to these ato promoters might be Ci. Preliminary results for the loss of ci in mitotic clones are consistent with Ci acting as a positive transcriptional regulator of ato (M. D. and E. Hafen, unpublished, cited in Dominguez, 1999). During furrow progression, Ci is upregulated in the cells anterior to the furrow and in groups of cells in the furrow that coincide with cells expressing ato. These high levels of Ci are then later downregulated to a low level behind the furrow. Ci is thought to act as a transcriptional factor activating or repressing target genes in a concentration-dependent manner. The transcriptional activator form of Ci is thought to correlate with high levels of full-length Ci protein induced by Hh. This upregulation of Ci proteins by Hh is a conserved feature of Hh signaling in all systems. Therefore it is surprising that in the eye Ci is not upregulated near to the Hh source but only in cells far away. The analysis of Ci distribution in smo³, hh ^AC and viable fused alleles (where the reception and transduction of the Hh signal is blocked or very reduced) suggests that high levels of Hh protein may inhibit Ci protein levels. Probably this regulation is required to restrict the domain of Ci activation and therefore, the cells that are competent to express ato. Thus, by combining a positive long-range inductive signal with short-range inhibition of Ci, Hh may act to pattern ato expression along the anteroposterior axis and refine the array of R8 cells (Dominguez, 1999 and references).

The localized expression of Hedgehog (Hh) at the extreme anterior of Drosophila ovarioles suggests that it might provide an asymmetric cue that patterns developing egg chambers along the anteroposterior axis. Ectopic or excessive Hh signaling disrupts egg chamber patterning dramatically through primary effects at two developmental stages. (1) Excess Hh signaling in somatic stem cells stimulates somatic cell over-proliferation. This likely disrupts the earliest interactions between somatic and germline cells and may account for the frequent mis-positioning of oocytes within egg chambers. (2) The initiation of the developmental programs of follicle cell lineages appears to be delayed by ectopic Hh signaling. This may account for the formation of ectopic polar cells, the extended proliferation of follicle cells and the defective differentiation of posterior follicle cells, which, in turn, disrupts polarity within the oocyte. Somatic cells in the ovary cannot proliferate normally in the absence of Hh or Smoothened activity. Loss of protein kinase A activity restores the proliferation of somatic cells in the absence of Hh activity and allows the formation of normally patterned ovarioles. Hence, localized Hh is not essential to direct egg chamber patterning (Zhang, 2000).

Hh signaling in Drosophila generally regulates the abundance and activity of Ci proteins without altering CI mRNA levels. By contrast, vertebrate Hh homologs frequently regulate transcription of the Ci-related GLI family of transcriptional effectors. The induction of CI RNA in ptc mutant follicle cells provides the first evidence that this circuitry can also be found in Drosophila. Other consequences of altering the activity of Hh signaling components in ovarian somatic cells substantiate the hypothesis that Hh signaling activates at least two distinct intracellular pathways. One pathway, involving protection of Ci-155 from proteolysis and perhaps also release from cytoplasmic anchoring, is phenocopied by PKA and cos2 mutations. In the ovary, cos2 mutations elicit stronger phenotypes than PKA mutations, perhaps because cos2 mutations preferentially disrupt cytoplasmic anchoring of Ci-155. The second pathway increases the specific activity of Ci-155 in opposition to the inhibitory effects of Su(fu). This pathway is elicited by ptc, but not by PKA mutations and requires Fu kinase activity. In accordance with this model, PKA Su(fu) double mutant cells produce phenotypes almost as strong as for ptc mutants in ovaries, whereas ptc fu double mutant cells exhibit minimal phenotypes and PKA mutant phenotypes are not greatly altered by additional loss of Fu kinase activity. In imaginal discs high level Hh signaling to nearby cells is phenocopied by ptc mutations and requires Fu kinase activity, whereas only low level Hh signaling to more distant cells can be phenocopied by PKA mutations and does not require Fu kinase activity. PKA mutations in somatic ovarian cells can effectively substitute for Hh activity: Fu kinase activity is not essential for somatic cell proliferation and ptc mutations engender excessive Hh signaling phenotypes even in the absence of Hh activity. Hence, it is surmised that ovarian somatic cells normally undergo only low levels of Hh signaling, in keeping with the observation that the source of Hh in the germarium is separated from its target cells by several cell diameters (Zhang, 2000).

Hindsight mediates the role of Notch in suppressing Hedgehog signaling and cell proliferation

Temporal and spatial regulation of proliferation and differentiation by signaling pathways is essential for animal development. Drosophila follicular epithelial cells provide an excellent model system for the study of temporal regulation of cell proliferation. In follicle cells, the Notch pathway stops proliferation and promotes a switch from the mitotic cycle to the endocycle (M/E switch). This study shows that zinc-finger transcription factor Hindsight mediates the role of Notch in regulating cell differentiation and the switch of cell-cycle programs. Hindsight is required and sufficient to stop proliferation and induce the transition to the endocycle. To do so, it represses string, Cut, and Hedgehog signaling, which promote proliferation during early oogenesis. Hindsight, along with another zinc-finger protein, Tramtrack, downregulates Hedgehog signaling through transcriptional repression of cubitus interruptus. These studies suggest that Hindsight bridges the two antagonistic pathways, Notch and Hedgehog, in the temporal regulation of follicle-cell proliferation and differentiation (Sun, 2007).

How developmental signals coordinate to control cell proliferation and differentiation remains largely unknown. These data reveal a molecular mechanism that links signal-transduction pathways and the cell-cycle machinery. Hnt is induced by Notch signaling and mediates most, if not all, Notch functions in the downregulation of Hh signaling and the M/E switch in follicle cells during midoogenesis. Loss of hnt function in follicle cells results in an extra round of the mitotic cycle after stage 6 and a delayed entry into the endocycle. In contrast, misexpression of Hnt at an earlier stage causes the follicle cells to differentiate prematurely and enter the endocycle. Hnt suppresses both stg and Cut, whose expression must be downregulated to ensure the M/E switch. In addition, Notch signaling appears to act through Hnt to downregulate Hh signaling by suppressing ci transcription, so Hnt links the two antagonistic signaling pathways in follicle-cell development. The transcriptional repression of ci is probably not mediated by Hnt alone, because ttk exhibited a similar defect in transcriptional regulation of ci and stg (Sun, 2007).

Studies have shown that downregulation of Cut mediates part of Notch function during the M/E switch. Specifically, Cut promotes cell proliferation and maintains an immature-cell fate, but Stg, the Cdc25 homolog, is not regulated by Cut. To induce the mitotic division ectopically during midoogenesis in follicle cells, both Cut and Stg must be misexpressed. The current study suggests that both Cut and Stg are suppressed by Hnt. Without Stg activity, a major regulator of G2/M transition, follicle cells are arrested before they enter the M phase, and downregulation of Cut allows accumulation of Fzr, causing degradation of CycA and CycB by the UPS, thus lowering CDK activity. This process allows endocycling follicle cells to by-pass the M phase and enter the next S phase. Repeated gap phases and S phases constitute the endocycle (Sun, 2007).

The finding that hnt follicle cells enter the endocycle after one additional round of the mitotic cycle suggests that hnt mutation causes a delay in the M/E switch. Mutations of the Notch pathway may also result in only a delay in entering the endocycle. In Notch mosaics, the cell number in mutant clones is approximately twice that of the twin spots, suggesting that an additional cell cycle also takes place. Further testing of this hypothesis requires a detailed analysis of the DNA content and clone size in Notch pathway mutants. Alternatively, Hnt may not be the sole mediator of the Notch effect; for example, Su(H)-independent Notch signaling may also be required in the M/E switch. Although hnt mutant cells can enter the endocycle late, they could not enter the chorion-gene-amplification program even much later, suggesting that Hnt function is also required for chorion-gene amplification (Sun, 2007).

The removal of negative components of the Hh pathway such as ptc causes overproliferation in follicle cells. Loss-of-function analyses of fu, a positive regulator of the pathway, revealed fewer cells in the mutant clones than in twin spots. The nuclear sizes of fu mutant cells were similar to those of the wild-type at the same developmental stage, and no fragmentation of the chromosomes was observed. Hh signaling therefore promotes cell proliferation in follicle cells during early oogenesis. Thus, Hnt-mediated downregulation of Hh signaling through suppression of ci transcription plays an important role in the M/E switch. Hh signaling is probably not involved in regulating Cut or Stg expression, because ectopic expression of Ci-155 in follicle cells during midoogenesis did not extend Stg-lacZ or Cut expression beyond stage 6, and fu mutant follicle cells showed normal Cut expression during early oogenesis. Other factors may therefore mediate the role of Hh signaling to modulate proliferation of follicle cells (Sun, 2007).

Hnt is not only required to mediate the role of Notch in regulating the M/E switch in follicle cells, but it is also sufficient to drive premature entry into the endocycle. Only a few cells misexpressing Hnt at the early stages of oogenesis were recovered, consistent with the role of Hnt in terminating the mitotic phase. In an extreme case, a stage-4 egg chamber contained only ~20 follicle cells, most of which misexpressed Hnt. Hnt misexpression suppresses Cut and stg-lacZ expression, suggesting that Hnt acts as a transcriptional repressor. Consistent with this interpretation, the mammalian homolog of Hnt, RREB1, also acts as a transcriptional repressor in several cellular contexts (Sun, 2007).

An interesting observation from these studies is that ttk clones have a phenotype similar to that of hnt clones. As in Notch regulation of Hnt, ttk is possibly downstream of Notch, but the current analysis of Notch mutants in stage-1 to stage-10 egg chambers showed no obvious change in Ttk expression. It was also found that Hnt has no role in regulating ttk expression. The findings that ttk expression is not regulated by Hnt or Notch during midoogenesis is perhaps not surprising given that Ttk69 is evenly expressed throughout early and midoogenesis. The phenotypic similarity between hnt and ttk mutants suggests that ttk and hnt act cooperatively to suppress gene expression at the M/E transition. Ttk may act as a permissive signal for Hnt to regulate Ci expression and the M/E switch. In the absence of either one, the M/E switch cannot take place properly. Consistent with this hypothesis, Ttk is known to act as a transcriptional repressor in the Drosophila eye. Whether Hnt and Ttk bind directly to the regulatory sequence of the cell-cycle genes and/or ci remains unclear (Sun, 2007).

Several lines of evidence suggest that the role of Hnt in promoting the M/E switch is not universal. First, during embryogenesis, a hnt-deficiency line enters the G1 arrest normally after cycle 16 in epidermal cells and undergoes normal M/E switch in the salivary gland, although Fzr is required for this process. Second, nurse-cell endoreplication does not require Hnt; no obvious defect was detected in hnt germline clones. The specific role of Hnt in follicle-cell-cycle regulation may stem from its role in regulating cell differentiation. For example, Hnt expression may cause upregulation of Fzr through the downregulation of Cut. This indirect role of Hnt suggests that the cell-cycle regulation may be a by-product of cell differentiation (Sun, 2007).

Both Notch and Hh signaling pathways are implicated in the regulation of differentiation and proliferation, but precisely how the two interact in regulating cellular processes is poorly understood. Depending on the cellular environment, their effects on proliferation and differentiation differ. In Drosophila eye imaginal discs, Notch triggers the onset of proliferation during the second mitotic wave (SMW), the opposite of its role in follicle-cell development. In the SMW, Notch positively affects dE2F1 and CycA expression and promotes S phase entry. In these cells, Hh signaling, along with Dpp, activates Dl expression, thereby activating the Notch pathway. Hh and Notch therefore act sequentially and positively during the SMW, whereas, in follicle cells, they act antagonistically. Hh signaling is active in the mitotic follicle cells in early oogenesis, but it is downregulated during the M/E switch when Notch signaling is activated. Notch appears to be superimposable on Hh signaling; mutation of the negative regulator of the Hh pathway, ptc, in follicle cells cannot interfere with the activation of Notch signaling as long as these cells are in direct contact with the germline cells. These ptc mutant cells show no accumulation of Ci-155, consistent with the finding that Notch signaling suppresses ci transcription through Hnt. The ptc^S2 cells that were out of contact with germline cells remained in the mitotic cycle because they could not receive Dl signaling from them, suggesting that Hh signaling is sufficient to keep these cells in the undifferentiated and mitotically active state (Sun, 2007).

Notch-dependent activation of Hnt and downregulation of Ci may be involved in another follicle-cell process, the migration of a specialized group of anterior follicle cells toward the border between the nurse cells and the oocyte at stage 9. These so-called border cells showed downregulation of ci during migration. When slbo-Gal4 was used to drive Ci overexpression in border cells, ~66% of egg chambers showed defects in border-cell migration. Notch signaling, as well as ttk, has been reported to be required for border-cell migration. Hnt was found to be expressed in the border cells and depended on Notch signaling. The occasional hnt border-cell clones observed also showed defects in border-cell migration, so the crosstalk between Hh and Notch through Hnt may go beyond the regulation of the M/E switch in follicle cells (Sun, 2007).

Drosophila ptip is essential for anterior/posterior patterning in development and interacts with the PcG and trxG pathways

Development of the fruit fly Drosophila depends in part on epigenetic regulation carried out by the concerted actions of the Polycomb and Trithorax group of proteins, many of which are associated with histone methyltransferase activity. Mouse PTIP is part of a histone H3K4 methyltransferase complex and contains six BRCT domains and a glutamine-rich region. This study describes an essential role for the Drosophila ortholog of the mammalian Ptip (Paxip1) gene in early development and imaginal disc patterning. Both maternal and zygotic ptip are required for segmentation and axis patterning during larval development. Loss of ptip results in a decrease in global levels of H3K4 methylation and an increase in the levels of H3K27 methylation. In cell culture, Drosophila ptip is required to activate homeotic gene expression in response to the derepression of Polycomb group genes. Activation of developmental genes is coincident with PTIP protein binding to promoter sequences and increased H3K4 trimethylation. These data suggest a highly conserved function for ptip in epigenetic control of development and differentiation (Fang, 2009).

The establishment and maintenance of gene expression patterns in development is regulated in part at the level of chromatin modification through the concerted actions of the Polycomb and trithorax family of genes (PcG/trxG). In Drosophila, Polycomb and Trithorax response elements (PRE/TREs) are cis-acting DNA sequences that bind to Trithorax or Polycomb protein complexes and maintain active or silent states, presumably in a heritable manner. In mammalian cells however, such PRE/TREs have not been conclusively identified. Polycomb and Trithorax gene products function by methylating specific histone lysine residues, yet how these complexes recognize individual loci in a temporal and tissue specific manner during development is unclear. Recently, a novel protein, PTIP (also known as PAXIP1), was identified that is part of a histone H3K4 methyltransferase complex and binds to the Pax family of DNA-binding proteins (Patel, 2007). PTIP is essential for assembly of the histone methyltransferase (HMT) complex at a Pax DNA-binding site. These data suggest that Pax proteins, and other similar DNA-binding proteins, can provide the locus and tissue specificity for HMT complexes during mammalian development (Fang, 2009).

In mammals, the PTIP protein is found within an HMT complex that includes the SET domain proteins ALR (GFER) and MLL3, and the accessory proteins WDR5, RBBP5 and ASH2. This PTIP containing complex can methylate lysine 4 (K4) of histone H3, a modification implicated in epigenetic activation and maintenance of gene expression patterns. Furthermore, conventional Ptip^-/- mouse embryos and conditionally inactivated Ptip^-/- neural stem cell derivatives show a marked decrease in the levels of global H3K4 methylation, suggesting that PTIP is required for some subset of H3K4 methylation events (Patel, 2007). The PTIP protein contains six BRCT (BRCA1 carboxy terminal) domains that can bind to phosphorylated serine residues. This is consistent with the observation that PAX2 is serine-phosphorylated in response to inductive signals. In mammals, PAX2 specifies a region of mesoderm fated to become urogenital epithelia at a time when the mesoderm becomes compartmentalized into axial, intermediate and lateral plate. These data suggest that PTIP provides a link between tissue specific DNA-binding proteins that specify cell lineages and the H3K4 methylation machinery (Fang, 2009).

To extend these finding to a non-mammalian organism and address the evolutionary conservation of Ptip, it was asked whether a Drosophila ptip homolog could be identified and if so, whether it is also an essential developmental regulator and part of the epigenetic machinery. The mammalian Ptip gene encodes a novel nuclear protein with two amino-terminal and four carboxy-terminal BRCT domains, flanking a glutamine-rich sequence. Based on the number and position of the BRCT domains and the glutamine-rich domain, the Drosophila genome contains a single ptip homolog. To understand the function of Drosophila ptip in development, a ptip mutant allele was characterized that contained a piggyBac transposon insertion between BRCT domains three and four. Maternal and zygotic ptip mutant embryos exhibited severe patterning defects and developmental arrest, whereas zygotic null mutants developed to the third instar larval stage but also exhibited anterior/posterior (A/P) patterning defects. In cell culture, depletion of Polycomb-mediated repression activates developmental regulatory genes, such as the homeotic gene Ultrabithorax (Ubx). This derepression is dependent on trxG activity and also requires PTIP. Microarray analyses in cell culture of Polycomb and polyhomeotic target genes indicate that many, but not all, require PTIP for activation once repression is removed. The activation of PcG target genes is coincident with PTIP binding to promoter sequences and increased H3K4 trimethylation. These data argue for a conserved role for PTIP in Trithorax-mediated epigenetic imprinting during development (Fang, 2009).

Embryonic development requires epigenetic imprinting of active and inactive chromatin in a spatially and temporally regulated manner, such that correct gene expression patterns are established and maintained. This study shows that Drosophila ptip is essential for early embryonic development. In larval development, ptip coordinately regulates the methylation of histone H3K4 and demethylation of H3K27, consistent with the reports that mammalian PTIP complexes with HMT proteins ALR and MLL3, and the histone demethylase UTX. In wing discs, ptip is required for appropriate A/P patterning by affecting morphogenesis determinant genes, such as en and ci. These data demonstrate in vivo that dynamic histone modifications play crucial roles in animal development and PTIP might be necessary for coherent histone coding. In addition, ptip is required for the activation of a broad array of PcG target genes in response to derepression in cultured fly cells. These data are consistent with a role for ptip in trxG-mediated activation of gene expression patterns (Fang, 2009).

Early development requires ptip for the appropriate expression of the pair rule genes eve and ftz. The characteristic seven-stripe eve expression pattern is regulated by separate enhancer sequences, which are not all equally affected by the loss of ptip. The complete absence of en expression at the extended germband stage also indicates the dramatic effect of ptip mutations on transcription. The characteristic 14 stripes of en expression depends on the correct expression of pair rule genes, which are clearly affected in ptip mutants. However, the maintenance of en expression at later stages and in imaginal discs is regulated by PREs and PcG proteins. If ptip functions as a trxG cofactor, then expression of en along the entire A/P axis in the imaginal discs of ptip mutants might be due to the absence of a repressor. This might explain the surprising presence of ectopic en in the anterior halves of imaginal discs from zygotic ptip mutants. This ectopic en expression is likely to result in suppression of ci through a PcG-mediated mechanism. Yet, it is not clear how en is normally repressed in the anterior half, nor which genes are responsible for derepression of en in the ptip mutant wing and leg discs (Fang, 2009).

The direct interaction of PTIP protein with developmental regulatory genes is supported by ChIP studies in cell culture. Given the structural and functional conservation of mouse and fly PTIP, mPTIP was expressed in fly cells; it can localize to the 5' regulatory regions of many PcG target genes that are activated upon loss of PC and PH activity. Consistent with the interpretation that a PTIP trxG complex is necessary for activation of repressed genes, mPTIP only bound to DNA upon loss of Pc and ph function. In the Kc cells, suppression of both Pc and ph results in the activation of many important developmental regulators, including homeotic genes. A recent report details the genome-wide binding of PcG complexes at different developmental stages in Drosophila and reveals hundreds of PREs located near transcription start sites. Strikingly, most of the genes found to be activated in the Kc cells after PcG knockdown also contain PRE elements near the transcription start site (Fang, 2009).

In vertebrates, PTIP interacts with the Trithorax homologs ALR/MLL3 to promote assembly of an H3K4 methyltransferase complex. The tissue and locus specificity for assembly may be mediated by DNA-binding proteins such as PAX2 (Patel, 2007) or SMAD2 (Shimizu, 2001), which regulate cell fate and cell lineages in response to positional information in the embryo. In flies, recruitment of PcG or trxG complexes to specific sites also can require DNA-binding proteins such as Zeste, DSP1, Pleiohomeotic and Pipsqueak. Whereas PcG complexes have been purified and described in detail, much less is known about the Drosophila trxG complexes. Purification of a trxG complex capable of histone acetylation (TAC1) revealed the proteins CBP and SBF1 in addition to TRX. By contrast, the mammalian MLL/ALL proteins are components of large multi-protein complexes capable of histone H3K4 methylation. Although the mutant analysis, the reduction of H3K4 methylation and the dsRNA knockdowns in Kc cells all suggest that Drosophila ptip has trxG-like activity and hence might be a suppressor of PcG proteins, a more definitve biochemical analysis awaits the generation of antibodies and the delineation of in vivo DNA-binding sites for PTIP and its associated proteins at specific target genes (Fang, 2009).

Mammalian PTIP is also thought to play a role in the DNA damage response based on its ability to bind to phosphorylated p53BP1. PTIP also binds preferentially to the P-SQ motif, which is a good substrate for the ATR/ATM cell cycle checkpoint regulating kinases. Several reports demonstrate that PTIP is part of a RAD50/p53BP1 DNA damage response complex, which can be separated from the MLL2 histone H3K4 methyltransferase complex. Both budding and fission yeast contain multiple BRCT domain proteins that are involved in the DNA damage response, including Esc4, Crb2, Rad9 and Cut5. All of these yeast proteins have mammalian counterparts. However, neither the fission nor budding yeast genomes encodes a protein with six BRCT domains and a glutamine-rich region between domains two and three, whereas such characteristic PTIP proteins are found in Drosophila, the honey bee, C. elegans and all vertebrate genomes. These comparative genome analyses suggest that ptip evolved in metazoans, consistent with an important role in development and differentiation (Fang, 2009).

In summary, Drosophila ptip is an essential gene for early embryonic development and pattern formation. Maternal ptip null embryos show early patterning defects including altered and reduced levels of pair rule gene expression prior to gastrulation. In cultured cells PTIP activity is required for the activation of Polycomb target genes upon derepression, suggesting an important role for the PTIP protein in trxG-mediated activation of developmental regulatory genes. The conservation of gene structure and function, from flies to mammals, suggests an essential epigenetic role for ptip in metazoans that has remained unchanged (Fang, 2009).

Drosophila eyes absent is required for normal cone and pigment cell development

In Drosophila, development of the compound eye is orchestrated by a network of highly conserved transcriptional regulators known as the retinal determination (RD) network. The retinal determination gene eyes absent (eya) is expressed in most cells within the developing eye field, from undifferentiated retinal progenitors to photoreceptor cells whose differentiation begins at the morphogenetic furrow (MF). Loss of eya expression leads to an early block in retinal development, making it impossible to study the role of eya expression during later steps of retinal differentiation. Two new regulatory regions have been developed that control eya expression during retinal development. These two enhancers are necessary to maintain eya expression anterior to the MF (eya-IAM) and in photoreceptors (eya-PSE), respectively. Deleting these enhancers affects developmental events anterior to the MF as well as retinal differentiation posterior to the MF. In line with previous results, reducing eya expression anterior to the MF was found to affect several early steps during early retinal differentiation, including cell cycle arrest and expression of the proneural gene ato. Consistent with previous observations that suggest a role for eya in cell proliferation during early development, deletion of eya-IAM was found to lead to a marked reduction in the size of the adult retinal field. On the other hand, deletion of eya-PSE leads to defects in cone and pigment cell development. In addition it was found that eya expression is necessary to activate expression of the cone cell marker Cut and to regulate levels of the Hedgehog pathway effector Ci. In summary, this study uncovers novel aspects of eya-mediated regulation of eye development. The genetic tools generated in this study will allow for a detailed study of how the RD network regulates key steps in eye formation (Karandikar, 2014, PubMed ID: 25057928).

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).