Ultrabithorax

Ubx regulation: Table of contents

Transcriptional Regulation

Drosophila Mi-2 protein binds to a domain in the gap protein Hunchback which is specifically required for the repression of HOX genes. Using LexA-Hb as bait, cDNAs were isolated representing six different genes. dMi-2 contains five conserved sequence motifs that are also present in the two human Mi-2 proteins and in two Caenorhabditis elegans ORFs: two chromodomains, a DNA-stimulated adenosine triphosphatase (ATPase) domain, two PHD finger motifs, a truncated helix-turn-helix motif resembling the DNA-binding domain of c-myb, and a motif with similarity to the first two helices of an HMG domain. dMi-2 homozygotes survive until the first or second larval instar. Mutant embryos and larvae show no obvious mutant phenotypes. Specifically, expression of BXC genes such as Ultrabithorax (Ubx) and Abdominal-B (Abd-B) is completely normal in these mutant embryos. This normal expression may be due to maternally deposited dMi-2 RNAs or proteins that persist through subsequent development. Consistent with this, all early embryos from a dMi-2 deletion stock (including those lacking the gene) show the same high levels of dMi-2 RNA. An attempt was made to generate embryos from mutant dMi-2 germ cells. However, germ cells that are mutant for any of the seven tested dMi-2 alleles fail to develop. This failure can be rescued by a dMi-2 transgene, demonstrating that dMi-2 is essential for the development of germ cells (Kehle, 1998).

An attempt was made to detect a genetic interaction between dMi-2 and hb. hb9Q mutants (carrying a premature stop codon upstream of the first finger domain) show only slight anterior derepression of Ubx in embryos because of perdurance of maternal hb products. hb9K57 mutants (carrying a D box lesion) show more extensive anterior derepression of Ubx; this mutant protein is thought to have dominant-negative effects on the persisting maternal wild-type product. dMi-24;hb9K57 double mutants show much more extensive derepression of Ubx than hb9K57 mutants. Similarly, dMi-24;hb9Q double mutants show more extensive derepression than hb9Q mutants alone. These results demonstrate a synergy between hb and dMi-2 that is consistent with the finding that dMi-2 binds to Hb. Furthermore, it provides strong evidence that dMi-2 functions in the repression of BXC genes (Kehle, 1998).

dMi-2 protein was tested to see if it participates in PcG repression. As in the case of dMi-2, maternally deposited PcG product often rescues homozygous mutant PcG embryos to a considerable extent. Extensive derepression of HOX genes can be observed if such homozygous embryos are also mutant for another PcG gene. Thus embryos homozygous for the PcG gene Posterior sex combs (Psc) and dMi-2 were examined and it was found that Ubx and Abd-B are derepressed more extensively in this double mutant than in Psc homozygotes alone. A similar result was found if dMi-2 is combined with other PcG mutations; these double mutants consistently lead to much enhanced homeotic transformations compared with the single PcG mutants. Thus, there is a synergy between dMi-2 and PcG genes. dMi-2 behaves like the PcG mutations Enhancer of Polycomb and Suppressor 2 of zeste, neither of which on their own cause a homeotic phenotype but do so in combination with other PcG mutations. This suggests that dMi-2 functions in PcG repression (Kehle, 1998).

It has been proposed that Hb directly or indirectly recruits PcG proteins to DNA to establish PcG silencing of homeotic genes. The present data suggest that dMi-2 might function as a link between Hb and PcG repressors. Although dMi-2 contains two motifs with similarity to DNA-binding domains (the myb and HMG domains), dMi-2 does not seem to bind to DNA on its own. Therefore, Hb may recruit dMi-2 to DNA. Xenopus Mi-2 was recently purified as a subunit of a histone deacetylase complex with nucleosome remodeling activity. In yeast and in vertebrates, several transcription factors repress transcription by recruiting histone deacetylases. It is possible that in Drosophila, nucleosome remodeling and deacetylase activities of a dMi-2 complex, recruited to homeotic genes by Hb, may result in local chromatin changes that allow binding of PcG proteins to the nucleosomal template. Alternatively, the proposed Hb-dMi-2 complex might directly bind a PcG protein and recruit it to DNA. Finally, the involvement of dMi-2 in PcG silencing suggests that this process may involve deacetylation of histones (Kehle, 1998 and references).

In its posterior domain of action, Tailless is responsible for the establishment of Abdominal-B expression and demarcating the posterior boundary of the initial domain of expression of Ultrabithorax (Reinitz, 1990).

Ubx autoregulation has been examined by manipulating UBX levels, both genetically and with an inducible transgene, and monitoring the effect of these manipulations on the expression of Ubx reporter genes. Positive autoregulation by Ubx is restricted to the visceral mesoderm, while in other tissues Ubx negatively autoregulates, shutting off its own transcription. In some cases, negative autoregulation stabilizes UBX levels, while in others it modulates the spatial and temporal patterns of UBX expression. This modulation of Ubx expression may enable Ubx to specify distinct identities in different segments. The upstream control region of Ubx contains multiple autoregulatory elements for both positive and negative autoregulation (Irvine, 1993).

Homeotic genes often use autoregulation as a mechanism to maintain their expression. Autoregulation of Ultrabithorax in the visceral mesoderm is at least partly indirect and mediated by extracellular signaling from Wingless and Decapentaplegic. UBX controls the localized expression of these two extracellular proteins. There are separate WG and DPP response elements within upstream sequences of Ubx. There are two distinct response factors: after signal-induced activation, either may mediate transcriptional activation through its cognate element, whereas each element is recognized by a repressor in the absence of the corresponding signal (Thuringer, 1993).

ABD-B represses the expression of other homeotic genes, such as Ubx and abd-A, in PS10-14. Ectopic Abd-B does not prevent transformations induced by ectopic Ubx. Instead, ectopic Ubx and Abd-B compete for the specification of segmental identities in a dose-dependent fashion. These results support a quantitative competition among the homeotic proteins rather than the existence of a strict functional hierarchy (Lamka, 1993).

Ubx is shown to be down-regulated in the posterior compartment of parasegment 6 by Engrailed. The significance of Ubx repression by EN is demonstrated by characterizing the expression of the Ubx target gene, Distal-less. In the posterior compartment of parasegment 6, Dll is normally expressed in a small cluster of cells. If Ubx is expressed uniformly via a heat-shock promoter, Dll is inappropriately repressed in these posterior compartment cells. In the anterior compartment of parasegment 6, Dll is normally repressed by high levels of UBX. However, if en is expressed uniformly via a heat-shock promoter, Ubx is repressed and Dll is derepressed. Because DLL is required for the development of larval sensory structures, these results demonstrate that EN-mediated repression of Ubx in the posterior compartment is necessary for the morphology of parasegment 6 (Mann, 1994).

Is it possible to estimate the number of target genes of the homeoproteins Even-skipped and Fushi tarazu? Eve and Ftz have been shown to bind with similar specificities to many genes, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Eve and Ftz bind at the highest levels to DNA fragments throughout the length of three probable target genes: eve, ftz and Ubx. However, Eve and Ftz also bind at only two- to ten-fold lower levels to four genes chosen in an attempt to find non targets: Adh, hsp70, rosy and actin 5C, suggesting that Eve and Ftz bind at significant levels to a majority of genes. The expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve (Liang, 1998).

In vitro transcription experiments demonstrate that (1) Eve protein can directly repress the Ubx promoter, and (2) endogenous Eve protein binds to the Ubx gene in embryos. Genetic experiments have shown that eve represses Ubx in stage 11 embryos. However, this effect may be indirect and could be mediated via eve’s effect on engrailed. Consequently, the expression of Ubx was examined in wild-type and eve^1.27 embryos at stage 5 -- a time before Engrailed protein is significantly expressed. UBX mRNA is present in four stripes in the posterior half of wild-type stage 5 embryos, with the anterior-most stripe (stripe 1) being the more prominent. In eve^1.27 mutants, Ubx expression is derepressed in a region including stripe 1 and stripe 2, reaching the same level of expression as stripe 1. Ubx expression is not significantly affected in the posterior of the embryo, either because Eve binds, but does not regulate Ubx in posterior cells, or because Eve’s function is redundant with that of other transcription factors in these cells. It is also possible that Eve does not bind to Ubx in the posterior of the embryo (though this explanation is considered less likely). Whatever the reason, the early regulation of Ubx supports the evidence that Eve directly represses Ubx (Liang, 1998).

At stages 10-14, 87% of cDNAs in the 8-12 hour library are likely to be directly or indirectly regulated by Eve, Ftz, Engrailed and all of the Hox proteins. These downstream genes are each expressed in unique, segmentally repeating patterns. Some are expressed at dramatically altered levels between segments. Most vary from segment to segment in the number and position of cells in which they are most prominently expressed. This is not simply because expression follows the distribution of a particular cell type. Between segments, the majority of genes are most highly expressed in differently positioned subsets of the same cell types, indicating that these patterns cannot result solely from the action of cell-type specific transcription factors. Eve, Ftz and Engrailed establish the segmentally repeating structure of the embryo. Therefore, all genes expressed in segmentally repeated patterns by stage 11 should be downstream of these three genes. This has been experimentally confirmed for eve and ftz. The expression of all 14 segmentally expressed genes tested is altered in eve and ftz mutant embryos at stage 11. Equally, the Hox genes establish the differences between segments. Thus, all genes expressed differently in each segment should be downstream of all of the Hox genes. This is indeed the case for the Hox gene Ubx. The expression of all seven segmentally expressed genes tested is regulated by Ubx. These downstream genes can be divided into three classes: genes expressed in strong, moderate or weak segmentally repeated patterns. 33% of cDNAs fall into the strongly repeated class. For this class, staining levels vary five fold or more between cells across a transverse section of a segment along the anterior/posterior axis of the embryo. 24% of clones belong to the moderately regulated class. These genes show two- to five-fold variations in staining across the width of a segment. Finally, the weak segmentally repeated genes vary only 1.2 to 2 fold in staining between cells across a segment. Thus, most downstream genes are expressed in all cells, but each are still subject to specific and precise control by the selector homeoproteins (Liang, 1998).

Decapentaplegic regulates Ubx through its receptor Thick veins. tkv activity is crucial for patterning the visceral mesoderm; in the absence of functional tkv gene product, visceral mesoderm parasegment 7 cells fail to express Ultrabithorax, but instead accumulate Antennapedia protein. The TKV receptor is therefore involved in delimiting the expression domains of homeotic genes in the visceral mesoderm (Affolter, 1994).

DPP is a primary signal in maintaining Ubx expression in the visceral mesoderm in a pattern different from Ubx expression in the embryonic ectoderm and in providing a cell-cell communication mechanism by which Ubx expression influences gene expression across germlayers and across the ps7 to ps8 parasegment boundary in the visceral mesoderm (Staehling-Hampton, 1994).

The fact that Transforming growth factor beta at 60A mutations are dominant enhancers of a sensitized dpp pathway implicates Tgfbeta-60A in potentiating dpp signaling. This is most obvious in the visceral mesoderm of the midgut where dpp signaling is required to regulate homeotic gene expression and to maintain its own expression through a positive feedback mechanism. Although dpp signaling in the visceral mesoderm appears intact in Tgfbeta-60A mutants, a requirement for Tgfbeta-60A is revealed in tkv 6 Tgfbeta-60A double mutants. When dpp signaling is attenuated through a mutant tkv receptor, eliminating Tgfbeta-60A function reduces the signaling to below threshold level. The derepression of Sex combs reduced in the anterior midgut and the loss of expression of dpp target genes (wingless, Ultrabithorax and dpp) in the visceral mesoderm and labial in the endoderm are consistent with inadequate dpp signaling. A similar requirement for Tgfbeta-60A is observed during dorsal closure of the embryonic ectoderm (Chen, 1998).

Ultrabithorax and labial are a target of wingless signaling in the midgut. dishevelled, shaggy/zeste-white 3 and armadillo are required for transmission of the wingless signal in the Drosophila epidermis. These genes act in the same epistatic order in the embryonic midgut to transmit the wingless signal. In addition to mediating transcriptional stimulation of the homeotic genes Ultrabithorax and labial, they are also required for transcriptional repression of labial by high levels of wingless . Efficient labial expression thus only occurs within a window of intermediate wingless pathway activity. The shaggy/zeste-white 3 mutants reveal that wingless signaling can stimulate decapentaplegic transcription in the absence of Ultrabithorax, identifying decapentaplegic as a target gene of wingless. Since decapentaplegic itself is required for wingless expression in the midgut, this represents a positive feed-back loop between two cell groups signaling to each other to stimulate one another's signal production (Yu, 1996).

Genetic studies of the signaling pathway of the Drosophila Wnt homolog, Wingless, have identified a number of genes, including zeste white 3, that function to transduce the Wingless signal. zeste white 3 encodes a serine/threonine kinase. zw3 is expressed maternally and uniformally in the early embryo. It has been proposed that the Wingless signal is mediated by repression of this kinase activity. This hypothesis was tested by overexpressing zeste white 3 in a tissue-specific fashion using the UAS/GAL4 binary expression system. The wild-type zw3 cDNA was placed under transcriptional control of the yeast GAL4 upstream activating sequence (UAS). UAS-zw3 flies were mated to flies that express the yeast transcriptional activator GAL4 in either a cell- or tissue-specific fashion to drive chronic expression of zw3. Elevated levels of zeste white 3 in the ectoderm and mesoderm result in phenotypes that resemble a loss of wingless. Overexpression of zeste white 3 in the mesoderm disrupts several Wingless-dependent processes, including the specification of a unique cell type in the larval midgut (the copper cell), the formation of the second midgut constriction, and the expression of Wingless target genes Ultrabithorax and decapentaplegic in the mesoderm, and labial in the endoderm. Interstitial cells normally found interspersed with the copper cells are still present. This loss of copper cells is similar to the phenotypes observed due to a loss of labial expression or wg expression, both required for the specification of the copper cells. The second midgut constriction is dependent on Wg signaling; in wg, dishevelled, or armadillo mutant embryos, this constriction does not form. Interestingly, in zw3 mutant embryos the second midgut constriction does form, but it is abnomal, appearing to have multiple folds. Zeste white 3 regulates the stability of Armadillo, which is essential for transducing the Wingless signal to the nucleus. zeste white 3 overexpression blocks Wingless signaling through the modulation of Armadillo since expression of a constitutively active form of Armadillo, which is independent of Zeste white 3 regulation, is epistatic to overexpression of zeste white 3 (Seitz,1998).

Two BX-C genes, Ultrabithorax and abdominal-A, require exd activity for their maintenance and function. Using an antibody directed against the Ubx protein, Ubx expression was examined in haltere imaginal discs containing exd clones induced at different times during larval development. In exd clones induced at 48-60 hr after egg laying (AEL), Ubx expression is abolished or greatly reduced in the region of the disc that will develop into metanotum. In exd clones induced later (72-84 hr AEL), Ubx expression is variable; some cells retain the Ubx antigen, whereas it is undetectable in others. As expected, exd clones in the region of the disc giving rise to the haltere pouch, where Exd is cytoplamic, have no effect on Ubx expression. This observation indicates that exd is involved in the maintenance of Ubx activity in the trunk region of the segment (metanotum), but not in the appendage. It is proposed that mutual interactions between Exd and BX-C proteins ensure the correct amounts of interacting molecules. Since the Hoxd10 gene has the same properties as Drosophila BX-C genes, it is suggested that the control mechanism of subcellular distribution of Exd found in Drosophila probably operates in other organisms as well (Azpiazu, 1998).

Mutations in the wingless pathway affect the expression of Ultrabithorax in the visceral mesoderm, disrupting the secondary midgut constriction. Indeed, Ultrabithorax expression is not maintained in pangolin mutants, while the secondary midgut constriction is absent. The primary constriction is not affected and does not move posteriorly. This contrasts with other mutations that disrupt the secondary midgut constriction, indicating that the Ubx regulatory network might be only partially disruption by pangolin mutation (van de Wetering, 1997).

Mothers against dpp (Mad) is the prototype of a family of genes required for signaling by TGF-beta related ligands. In Drosophila, Mad is specifically required in cells responding to Decapentaplegic (Dpp) signals. The role of Mad in Dpp-mediated signaling was examined by utilizing tkvQ199D, an activated form of the Dpp type I receptor serine-threonine kinase thick veins (tkv). In the midgut, dpp is expressed in the visceral mesoderm of parasegments 3 and 7. In response to Dpp signals, cells expressing dpp in parasegment 3 repress the expression of the homeotic gene Sex combs reduced. Dpp signals are also required to maintain dpp expression in parasegment 3 through an autocrine feedback loop. However, cells in parasegment 4 do not appear to be affected by Dpp signals; Scr is expressed while dpp is not. In ps7, the homeotic gene Ultrabithorax initiates dpp expression. Subsequently, Dpp functions in an autocrine manner to maintain Ubx and thus dpp expression. In ps7, Dpp also signals between germ layers to the underlying endoderm. Within the midgut endoderm, which does not express dpp, expression of the homeotic gene labial is dependent on Dpp signals (Newfeld, 1997).

In the embryonic midgut, tkvQ199D mimics Dpp-mediated inductive interactions. There is an anterior expansion of labial midgut endoderm expression in response to ubiquitously expressed tkvQ199D. In early stage tkvQ199D embryos, dpp expressin is expanded to include ps4, ps5 and ps6. In late stage tkvQ199D embryos, the expanded domain of expression is maintained at very high levels, while in late stage Mad mull tkvQ199D embryos, this is not observed. Analysis of Scr expression in Mad null embryos, combined with tkvQ199D, reveals an anterior expansion of Scr, showing that Mad and dpp are required for repressing Scr. Mad function is epistatic to tkvQ199D in the repression of Scr. Thus homozygous Mad mutations block signaling by tkvQ199D and appropriate responses to signaling by tkvQ199D are restored by expression of MAD protein in Dpp-target cells (Newfeld, 1997).

The shortvein (shv) class of cis-regulatory dpp mutants disrupt expression in parasegments 4 and 7 (ps4 and ps7) of the embryonic visceral mesoderm (VM) surrounding the gut and cause abnormalities in gut morphogenesis. Cis-regulatory elements directing expression in ps4 and ps7 are separable and identify DNA fragments that generate ps4 and ps7 expression patterns using reporter gene constructs. dpp reporter gene expression in both ps4 and ps7 is autoregulated as it requires endogenous dpp+ activity. Reporter gene ps7 expression requires the wild-type action of Ubx, and abdominal-A. Thus dpp both responds to and regulates Ubx in ps7 of the visceral mesoderm; Ubx autoregulation within this tissue may be indirect as it requires more components than had previously been thought (Hursh, 1993).

Grainy head/NTF-1 binds to and regulates the proximal promoter of Ubx (Dynlacht, 1989).

The GAGA, NTF-1, and Zeste proteins activate the Ultrabithorax (Ubx) promoter in vitro. Differently mutated Ubx-promoter constructs containing binding sites for none, one, or all three of these transcription factors have been introduced into Drosophila by P-element transformation. Binding sites for each factor activate dramatically different patterns of transcription. In zeste mutant embryos, the activation by zeste protein-binding sites is essentially abolished. These genetic data, when considered with earlier biochemical experiments, demonstrate that zeste directly and potently activates Ubx transcription in vivo (Laney, 1992).

Developmental analysis and squamous morphogenesis of the peripodial epithelium in Drosophila imaginal discs; peripodial cells are displaced into the disc proper during larval development and this movement leads to Ubx repression

Imaginal discs of Drosophila provide an excellent system with which to study morphogenesis, pattern formation and cell proliferation in an epithelium. Discs are sac-like in structure and are composed of two epithelial layers: an upper peripodial epithelium and lower disc proper (DP). Although development of the disc proper has been studied extensively in terms of cell proliferation, cell signaling mechanisms and pattern formation, little is known about these same processes in the peripodial epithelium (PE), the cell layer opposing the disc proper. This topic was addressed by focusing on morphogenesis, compartmental organization, proliferation and cell lineage of the PE in wing, second thoracic leg (T2) and eye discs. A subset of peripodial cells in different imaginal discs undergo a cuboidal-to-squamous cell shape change at distinct larval stages. This shape change requires both Hedgehog and Decapentapelagic, but not Wingless, signaling. Additionally, squamous morphogenesis shifts the anteroposterior (AP) compartment boundary in the peripodial epithelium relative to the stationary AP boundary in the disc proper. Finally, by lineage tracing cells in the PE, it was surprisingly found that peripodial cells are displaced into the disc proper during larval development and this movement leads to Ubx repression (McClure, 2005).

Little is known about when and how disc cells acquire their diverse morphologies. Although Hh and Dpp are well-known for their roles in cell proliferation and patterning it is known that they are also active in epithelial morphogenesis. In eye discs, Hh is both necessary and sufficient to initiate cell shape changes that occur in the morphogenetic furrow. In wing discs, columnar cells require Dpp signaling for normal cytoskeletal organization, shape and pseudostratified organization. This study describes precisely and for the first time when cuboidal, columnar and squamous cell morphologies arise in the epithelia of different imaginal discs. This study examines how the genesis of different morphologies in imaginal discs are affected by loss of a non-autonomous signal (wg, hh and dpp). Hh-dependent Dpp signaling is shown to be required for squamous morphogenesis in the PE of wing and leg discs. Additionally, Dpp signaling is activated as PE cells transition to a squamous morphology. The results indicate that the establishment of columnar morphology in the DP of wing and leg discs is independent of Dpp signaling activity. Clearly, one question still remains: what is the mechanism which causes DP cells to become columnar? The information from these studies provides, at least, an initial framework of how epithelial morphogenesis occurs in imaginal discs (McClure, 2005).

A lineage analysis of cells has been performed in the wing disc using Ubx-Gal4, UAS flp and act5C>stop>nuclacZ (Pallavi, 2003). Since Ubx-Gal4 is initially expressed in both disc epithelia prior to the second larval instar, cells of both the PE and DP were marked. This analysis concluded that cells of the PE and DP share a common origin in the disc primordium but later become separate lineages, although cells that make up the PE and DP lineages are never specified. The current results, based on lineage-tracing cells born in the PE, are in overall agreement with these conclusions; however, there are some differences. Although Pallavi (2003) identified cell clones spanning both disc epithelia, it could not be determined when or where these clones were born. Furthermore, clones that encompassed cells from both PE and DP were interpreted as either fusions between two independent clones or as clones that originated early in the embryo before separation of the two lineages (PE and DP) (McClure, 2005).

Using four different methods, it has now been found that cells that originate within the PE produce progeny that are a part of the DP. The MARCM and estrogen-inducible systems were used to perform a clonal analysis specific to cells within the PE. These two methods indicate that cells born within the PE produce daughter cells that contribute to the DP. Additionally, a twinspot clonal analysis leads to a similar conclusion and has the advantage of marking cells more directly than either the MARCM- or estrogen-inducible systems. Thus, this analysis indicates a lineage relationship between margin cells in both the PE and DP, and squamous cells in the wing disc, and provides evidence that together these cells comprise the peripodial lineage (McClure, 2005).

As cells are displaced from the PE and into the DP they lose Ubx expression. The loss of Ubx may cause cells to acquire a more distal fate, forging a possible link between displacement and cell fate changes. Similar dynamic cell movements, along with changes in gene expression, have been observed in the chick during somite segmentation. In addition, cell movements and changes in gene expression, similar to what is described here, have been reported by Weigmann (1999), who observed that leg disc cells born in the most proximal regions of the disc contribute to more distal leg segments. Finally, it is proposed that once PE cells are displaced into the DP they may change their cell fate by altered cell signaling. Displaced cuboidal cells at the margins of the disc receive not only planar signals from both epithelial layers, which they are a part of at different stages in larval development, but also vertical signals from overlying PE cells after displacement into the DP. These new planar and/or vertical signals may lead to Ubx repression. It is suggested that the mechanisms that play a role in the development of the imaginal discs may be functionally similar to mechanisms that regulate primary neurogenesis in vertebrates. Neural plate formation and patterning cues arise from two sources: a horizontal source within the plane of an epithelium and a vertical source that arises from the underlying mesodermal cells. The current study suggests that patterning of the imaginal discs is a much more dynamic process with cells exposed to not only signals within the plane of an epithelium but also vertical signals between disc epithelia (McClure, 2005).

Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3: Expression of Ultrabithorax and abdominal-A is affected by dLsd1 depletion

Histone-tail modifications play a fundamental role in the processes that establish chromatin structure and determine gene expression. One such modification, histone methylation, was considered irreversible until the recent discovery of histone demethylases. Lsd1 was the first histone demethylase to be identified. Lsd1 is highly conserved, from yeast to humans, but its function has primarily been studied through biochemical approaches. The mammalian ortholog has been shown to demethylate monomethyl- and dimethyl-K4 and -K9 residues of histone H3. This study, along with a second study by Rudolph (2007) describes the effects of Lsd1 (Suppressor of variegation 3-3) mutation in Drosophila. The inactivation of dLsd1 strongly affects the global level of monomethyl- and dimethyl-H3-K4 methylation and results in elevated expression of a subset of genes. dLsd1 is not an essential gene, but animal viability is strongly reduced in mutant animals in a gender-specific manner. Interestingly, dLsd1 mutants are sterile and possess defects in ovary development, indicating that dLsd1 has tissue-specific functions. Mutant alleles of dLsd1 suppress positional-effect variegation, suggesting a disruption of the balance between euchromatin and heterochromatin. Taken together, these results show that dLsd1-mediated H3-K4 demethylation has a significant and specific role in Drosophila development (Di Stefano, 2007).

Su(var)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 dictates the distinction between euchromatic and heterochromatic domains during early embryogenesis. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. Su(var)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. These data indicate an early developmental function for the Su(var)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which Su(var)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation (Rudolph, 2007).

The homeobox (Hox) gene locus is subject to extensive H3-K4 methylation by trithorax-group proteins. It was therefore asked whether the expression level of the Hox genes Ultrabithorax (Ubx) and abdominal-A (abdA) is affected by dLsd1 depletion. Ubx- and abdA-mRNA levels increased 2-fold in SL2 cells treated with dLsd1 double-stranded RNA (dsRNA). These changes were specific and were not seen with other control genes (dDP and Hid). To verify the relevance of these observations in vivo, the expression of these genes was compared in wild-type and dLsd1^ΔN mutant flies. A significant upregulation of each of these targets was found in dLsd1^ΔN mutant flies, confirming the importance of dLsd1-mediated repression in vivo. Intriguingly, it was observe that this upregulation is age dependent: The difference in gene expression is minimal in larval stages, and, consistent with this, the Hox gene-expression pattern in imaginal discs from dLsd1^ΔN mutant larvae and in embryos is largely unaltered. However, the level of nAcrβ, Ubx, and Abd-B gradually and significantly increases with age after eclosion, suggesting that dLsd1 function is especially important for the regulation of gene expression in adult tissues (Di Stefano, 2007).

The data support a model in which heterochromatin formation and gene silencing in PEV are defined during early embryonic development of Drosophila. A dynamic balance between HMTases and demethylases controls establishment of the functionally antagonistic histone H3K4 and H3K9 methylation marks at the border region of euchromatin and heterochromatin. In transcriptionally silent cleavage nuclei, chromatin is in a naive state with only little H3K9me2 and with H3K4 methylation completely missing. A dramatic transition of chromatin structure occurs during blastoderm formation and cellularization by establishing H3K4 and H3K9 methylation. In contrast to H3K9 acetylation, which is already found in cleavage chromatin, H3K4 methylation at prospective euchromatin appears first at the end of cleavage in cycle 12. In parallel, di- and trimethylation of H3K9 and HP1 binding establish heterochromatin. Pole cells, which are the primordial germ cells of Drosophila, are in a transcriptionally silent state and show extensive H3K9me2 and H3K9me3. During the definition of the euchromatin-heterochromatin boundaries in blastoderm cells and for the establishment of repressive H3K9 methylation marks in primordial germ cells, the SU(VAR)3-3 demethylase plays an early and inductive regulatory role. SU(VAR)3-3 might also be involved in control of early transcriptional activities within Drosophila pericentromeric sequences preceding heterochromatin formation, as suggested by a model of heterochromatin formation that depends on the RNAi pathway (Rudolph, 2007).

Genetic analysis revealed that SU(VAR)3-3 functions upstream of the H3K9 HMTase SU(VAR)3-9 and the heterochromatin-associated proteins HP1 and SU(VAR)3-7 in control of gene silencing in PEV. Combined with earlier studies of epigenetic interactions, heterochromatic gene silencing is established by a sequential action of SU(VAR)3-3, SU(VAR)3-9, the amount of Y heterochromatin, HP1, and SU(VAR)3-7. RPD3 also acts upstream of SU(VAR)3-9, because Rpd3 mutations dominate the dose-dependent PEV enhancer effect of SU(VAR)3-9. Additional genomic copies of Su(var)3-3 are epistatic to a Rpd3 mutation placing the H3K4 demethylase SU(VAR)3-3 together with RPD3 at the top of a mechanistic hierarchy controlling heterochromatic gene silencing in Drosophila. Such a role is in agreement with the enriched association of SU(VAR)3-3 to prospective heterochromatin in early blastoderm nuclei. In Su(var)3-3 null embryos, there is an extension of H3K4me2 and concomitant reduction of H3K9me3 at prospective heterochromatin, suggesting that SU(VAR)3-3 has a protective function at heterochromatic regions to restrict expansion of H3K4 methylation. Similarly, H3K9 acetylation becomes expanded toward heterochromatin. H3K4 methylation precedes H3K9 methylation in blastoderm nuclei, and both SU(VAR)3-3 and SU(VAR)3-9 are abundant proteins within cleavage chromatin. A developmentally regulated silencing complex between SU(VAR)3-3, RPD3, and SU(VAR)3-9 is therefore likely to dictate the distinction between euchromatic and heterochromatic domains during early embryogenesis. A comparable functional crosstalk between human LSD1 and HDAC1/2, which depends on nucleosomal substrates and the CoREST (see Drosophila CoREST) protein, has been demonstrated in vertebrates. The interaction between SU(VAR)3-3 and RPD3 could also explain butyrate sensitivity of Su(var)3-3 mutations. The effect of SU(VAR)3-3 on heterochromatin formation during blastoderm could involve both maternal and zygotic protein. Association of SU(VAR)3-3 with cleavage chromatin is dependent on maternal sources. In contrast, all other effects on gene silencing are zygotically determined, and no maternal effects on PEV were found in any of the Su(var)3-3 mutations. This is also supported by clonal analysis showing early onset and stable maintenance of gene silencing in PEV (Rudolph, 2007).

Molecular dissection of cis-regulatory modules at the Drosophila bithorax complex reveals critical transcription factor signature motifs

At the Drosophila bithorax complex (BX-C) over 330kb of intergenic DNA is responsible for directing the transcription of just three homeotic (Hox) genes during embryonic development. A number of distinct enhancer cis-regulatory modules (CRMs) are responsible for controlling the specific expression patterns of the Hox genes in the BX-C. While it has proven possible to identify orthologs of known BX-C CRMs in different Drosophila species using overall sequence conservation, this approach has not proven sufficiently effective for identifying novel CRMs or defining the key functional sequences within enhancer CRMs. This study demonstrates that the specific spatial clustering of transcription factor (TF) binding sites is important for BX-C enhancer activity. A bioinformatic search for combinations of putative TF binding sites in the BX-C suggests that simple clustering of binding sites is frequently not indicative of enhancer activity. However, through molecular dissection and evolutionary comparison across the Drosophila genus it was discovered that specific TF binding site clustering patterns are an important feature of three known BX-C enhancers. Sub-regions of the defined IAB5 and IAB7b enhancers were both found to contain an evolutionarily conserved signature motif of clustered TF binding sites which is critical for the functional activity of the enhancers. Together, these results indicate that the spatial organization of specific activator and repressor binding sites within BX-C enhancers is of greater importance than overall sequence conservation and is indicative of enhancer functional activity (Starr, 2011).

The clustered organization of TF binding sites has been shown to be crucially important to the functional activity of enhancers. However, despite detailed studies of a small set of enhancers in Drosophila, including the eve stripe 2 (S2E) enhancer, the precise rules of cis-regulatory grammar have yet to be fully elucidated. In an effort to investigate the role of clustering of predicted TF binding sites for the identification of enhancers in the 330 kb Drosophila BX-C, a search for simple clusters of HB and KR binding sites was performed. The search algorithm returned 26 putative enhancers (PCRMs), of which 6 (23%) overlapped previously identified enhancers. The overlapping regions for four of these confirmed enhancers (BRE, IAB2, IAB5 and IAB8) were tested in transgenic reporter gene assay and recapitulated the known domains of regulatory activity in the embryo. Furthermore, the 1037 bp R10 region that was tested, that is able to recapitulate IAB2 enhancer functional activity, refines the boundaries of the previously characterized 1970 bp IAB2 sequence. The search also identified 20 additional PCRM sequences. Twelve of these previously uncharacterized genomic regions were analyzed for enhancer activity and only one (R8 from the bxd/pbx region) was found to be a novel embryonic enhancer capable of driving expression in a pattern indicative of Ubx gene expression. This result indicates that the approach of searching for novel enhancers in the BX-C using simple clustering may have significant limitations (Starr, 2011).

A key question is why 11 of the 16 PCRMs tested (69%) are false positives. Two possibilities include; a) that the PCRMs may in fact be actively regulating expression of the BX-C genes at later stages of development or in very specific patterns in post-embryonic tissues, and b) that in testing a specific ~ 1 kb genomic fragment from each PCRM critical regulatory sequences in neighboring regions may have been removed. However, the recent availability of in vivo TF binding data may also offer some potential answers. The binding of anterio-posterior restricted gap/terminal and pair-rule transcription factors in stages 4-5 embryos appears to correlate strongly with the functional activity of the PCRMs. When scored for ten specific TFs which are potential regulators of the BX-C enhancers, all the PCRMs tested in the transgenic assay that had chromatin immunoprecipitation (ChIP) binding peaks for 6 or more of the protein factors function as embryonic enhancers. For each of these confirmed enhancers, both KR and HB demonstrate in vivo binding at the endogenous genomic region corresponding to the enhancer. In contrast, all the false positive PCRMs do not have binding peaks for more than 5 of the TFs and most have less than 3, often reflecting an absence of binding for KR or HB (Starr, 2011).

One interpretation of this data is that the predicted TF binding sites in many of the false positive PCRMs do not represent actual in vivo embryonic binding sites and, as a result, the PCRM is not functional. In addition to KR and HB repressor binding sites, it is also important to consider the presence of potential binding sites for an appropriate activator (FTZ or EVE) necessary for the functional activity of the enhancer. Analysis of the 5 PCRMs that demonstrate in vivo activity reveals that each contains at least 3 strong predicted binding sites for the appropriate pair-rule activator. However, in many cases the false positive PCRMs tested also appear to contain putative activator binding sites. In these cases it is possible that additional architectural requirements (for example, close spacing between multiple activator and/or repressor binding sites) may be necessary for in vivo embryonic activity to occur. In support of this idea, analysis of the genomic fragments that were tested from the iab-2 to iab-8 genomic regions (R10, 11, 12, 13, 14, 15, 17, 20, and 21), predicts that R15 (overlapping IAB5) has a closely-spaced cluster of FTZ-KR sites and that R10 (overlapping IAB2) and R20 (overlapping IAB8) possess a closely spaced cluster of EVE-KR sites within 150 bp of one another, whereas the other regions do not appear to harbor pair-rule activator (FTZ or EVE) and repressor (KR) clusters in such close proximity. A third possibility is that additional protein factors may be involved which may affect the ability of TFs to access the binding sites within the predicted enhancer sequence. Such proteins, which control the recruitment of chromatin components and nucleosome positioning, are thought to be critical to the regulation of embryonic gene expression through the modulation of TF binding affinity at enhancers (Starr, 2011).

The presence of a simple cluster of KR and HB binding sites in many of the enhancers of the BX-C argues that certain precise patterns of TF binding site clusters may be responsible for functional activity among similarly-regulated enhancers. In the IAB8 enhancer, a distinct cluster of EVE-KR binding sites (one KR, two EVE sites) is highly conserved across different Drosophila species. The 3' third of IAB8 harboring the EVE-KR motif (minIAB8) is able to drive reporter gene expression in the characteristic IAB8-pattern in the presumptive A8 segment of transgenic Drosophila. Deletion of the pair of EVE binding sites (∆EVE) significantly weakens enhancer activity in A8, suggesting that while the these two EVE sites are important, cryptic weak EVE binding sites in the remaining sequence of the enhancer (which are sufficiently low scoring to escape computational prediction at the ln(p-value) cutoff of - 6.0) are capable of partially compensating for the loss of the two strong predicted EVE sites. In support of this idea is the recent discovery that even weak affinity binding sites contribute to TF occupancy at regulatory regions in Drososphila embryos. In that study it was found that the level of factor occupancy in vivo correlates more strongly with the degree of chromatin accessibility at a given site, rather than in vitro measurements of the affinity of a factor for a particular DNA sequence (Li, 2011). This observation may be especially relevant in the case of pair-rule factors (such as EVE), where a high localized concentration of the protein in each stripe may also facilitate the increased occupancy of low affinity binding sites (Starr, 2011).

A 141 bp fragment (EK) from within the minIAB8 region containing only the EVE-KR cluster drives strong reporter gene expression in A8, but also ectopic expression immediately posterior of A8 and weaker expression immediately anterior of A8. Ectopic reporter gene expression is also observed in the anterior head domain of the embryo. This result indicates that the EK fragment by itself lacks important binding sites responsible for repression in the anterior head domain of the embryo (such as HB) and for the region immediately anterior of A8 (such as KNI). Several predicted HB and KNI repressor sites capable of performing this role are present within the 602 bp minIAB8 enhancer. Importantly, in the C3-A4 domain of the embryo where the KR repressor protein is expressed, there is a lack of enhancer-driven reporter gene expression from the EK fragment, suggesting that the single KR site within the EVE-KR cluster is sufficient to allow KR-mediated repression in that domain of the embryo. The continued presence of the EVE-KR cluster within the IAB8 enhancer, despite extensive reorganization of TF binding sites across the Drosophila orthologs, is reminiscent of the architectural constraints in the Drosophila and Sepsid eve S2E orthologs, which possess a highly conserved cluster of overlapping BCD activator and KR repressor binding sites necessary for enhancer function (Starr, 2011).

To extend the analysis of the functional role of clustered TF binding sites the IAB5 and IAB7b enhancers from the Drosophila BX-C were also analyzed. Chimeric enhancers assembled from the D. melanogaster and D. pseudoobscura IAB5 orthologs appear to have their functional activity entirely preserved and drive reporter gene expression in presumptive abdominal segments A5, A7 and A9. This result contrasts with an earlier study in which chimeric enhancers assembled from reciprocal halves of D. melanogaster and D. pseudoobscura S2E orthologs did not accurately recapitulate enhancer activity. It is possible that the regulatory output for the chimeric IAB5 enhancers may be subject to very subtle modifications. Such modifications may result in changes to expression patterns that are beyond the detection of the reporter gene assay. However, one explanation for the difference in functional output between these two examples is that in the case of the S2E the organization of TF binding sites within the chimeric enhancer was sufficiently modified so as to destroy the functional activity of the enhancer, whereas for IAB5 this was not the case (Starr, 2011).

To further dissect the organization of TF binding sites in IAB5 the predicted TF binding sites in the sequence were examined. This approach reveals a highly evolutionarily conserved signature TF binding site motif consisting of two strong FTZ activator sites close to two strong KR repressor sites in the center of the defined 1 kb enhancer. The FTZ-KR signature motif is present and intact in both the functional IAB5 chimeric enhancers, cMP and cPM. In the case of the cMP enhancer, the signature motif is present in the IAB5.2 half from D. pseudoobscura, while in the case of the reciprocal cPM enhancer, the signature motif is present in the IAB5.2 half from D. melanogaster. Molecular dissection of IAB5 shows that the individual IAB5.2 halves from Drosophila and D. pseudoobscura each show functional enhancer activity, while the corresponding IAB5.1 halves that lack the FTZ-KR signature motif do not. Furthermore, the 424 bp region containing the center peak of sequence conservation of IAB5 (cIAB5) and the FTZ-KR signature motif drives reporter gene expression in the characteristic three-stripe IAB5-pattern in transgenic Drosophila. In support of the critical functional role of this region, previous functional studies showed that the strongest predicted KR binding site within this signature motif is in fact critical to regulate the spatially restricted expression directed by IAB5 to the posterior presumptive A5, A7, and A9 segments in the Drosophila embryo. In the context of the endogenous gene complex a single point mutation in this KR repressor binding site (Superabdominal mutation) causes an anterior expansion of the embryonic domain of Abd-B expression and results in a homeotic transformation of the A3 segment into the more posterior A5 segment. This result confirms that the strong KR binding site in the signature motif is essential for the in vivo functional activity of the IAB5 enhancer (Starr, 2011).

The IAB7b enhancer, which is expressed in the presumptive A7 segment of the Drosophila embryo, is thought to be regulated by many of the same activators and repressors as IAB5. Bioinformatic analysis reveals that a highly conserved FTZ-KR signature motif, very similar to the one identified in IAB5, is also present in the IAB7b enhancer. Molecular dissection of IAB7b to test the role of the signature motif in the activity of the enhancer demonstrates that a 154 bp region containing the motif (2F2K, with two FTZ and two KR sites) from within the Drosophila IAB7b enhancer is able to drive reporter gene expression in the presumptive A5, A7 and A9 segments of transgenic Drosophila, with notably stronger expression in A7. This expression pattern is very similar to that driven by the IAB5 enhancer. A 114 bp region (2F1K, containing two FTZ and one KR site) from within the Drosophila IAB7b enhancer also drives this same pattern of reporter gene expression, suggesting that the 3' KR site is dispensable for repression of enhancer activity in the central domains of the embryo. Despite the fact that the 3' KR site also overlaps predicted BCD and HB repressor binding sites, no ectopic anterior enhancer-driven expression is observed in the 2F1K construct when tested in transgenic embryos, suggesting that the single remaining 5' KR binding site is sufficient for repression. In fact, in more distantly related Drosophila species, the presence of two KR sites positioned near the pair of FTZ sites is lost, and only a single KR site remains (Starr, 2011).

A 110 bp region (1F2K, containing 1 FTZ and two KR sites) from IAB7b does not drive gene expression, demonstrating that the outer FTZ site is required for activation of the enhancer. One possible molecular explanation for the necessity of the outer FTZ binding site is that FTZ may be acting as a dimer in order to activate IAB5 and IAB7b. In both enhancers a pair of strong FTZ sites are present in the FTZ-KR signature motif. While the ability of FTZ to dimerize has not been reported in the literature, other homeodomain-leucine zipper proteins have been shown to function as dimers. In many such cases the protein factors are also able to bind DNA target sequences as monomers, albeit with comparatively lower affinity. There is also evidence that FTZ is capable of interaction with other proteins, namely the orphan nuclear receptor FTZ-F1 through its LXXLL leucine zipper motif. In this case the heterodimer is capable of co-activation of target genes. Given that the consensus binding sites for the two factors are very different; FTZ (NNYAATTR), FTZ-F1 (BSAAGGDKRDD, it is perhaps to be expected that none of the predicted FTZ and FTZ-F1 binding sites in the IAB5 or IAB7b enhancers directly overlap. However, in future studies it will be of interest to explore the role of FTZ homo- and hetero-dimerization in regulating IAB5 and IAB7b activity (Starr, 2011).

The ability of the 2F2K and 2F1K regions to drive reporter gene expression in an IAB5-like manner in the presumptive A5, A7 and A9 segments of transgenic Drosophila suggests that additional inputs into IAB7b are required to spatially restrict endogenous enhancer-driven gene expression to only the A7 segment. A likely candidate for repression of IAB7b activity in the A5 segment of the embryo is KNI, which is expressed in the presumptive A1-A6 segments. Bioinformatic analysis predicts several candidate KNI binding sites in the full length 728 bp IAB7b enhancer, whereas the 2F2K and 2F1K regions lack any such predicted KNI sites. Previous studies revealed that the repression of the IAB7b enhancer in A5 is mediated by sequences in the 728 bp fragment and does not require additional flanking 5' or 3' regions. In addition, while disruption of the two KR sites in the signature motif does result in reporter gene activation by IAB7b in anterior regions of the embryo, repression persists in the A5 segment. This result indicates that a factor other than KR is responsible for repression in A5. In the entire 728 bp enhancer only three strong KNI sites are predicted, all located in the ~ 300 bp region on the abd-A side of the signature motif. These sites all lie within an evolutionarily conserved genomic region and some of the sites are conserved in distantly related Drosophila species. The significance of these KNI sites in restricting the IAB7b mediated-expression pattern is currently under investigation (Starr, 2011).

A key question in understanding cis-regulatory grammar is why certain arrangements of TF binding sites confer functional enhancer activity while others fail to do so. The turnover of binding sites is common during the evolution of enhancers in different species, yet the functional activity of rapidly-evolving enhancer orthologs from different species is often robust, even across several hundred million years of evolutionary divergence. In the case of the BX-C, bioinformatic analysis demonstrates that there is extensive binding site turnover in the IAB5, IAB7b, and IAB8 enhancers across the Drosophila genus, particularly in more distantly related species. Despite this turnover of TF binding sites, the newly identified FTZ-KR signature motif present in both IAB5 and IAB7b and the functionally important EVE-KR cluster within IAB8 are composed of similar patterns of conserved binding site architecture. Specifically, the organization of sites is such that a pair of strong activator (FTZ or EVE) binding sites and at least one strong repressor (KR) site are in close proximity (< 116 bp) to each other. Notably, the spacing between the FTZ and KR sites in the signature motif is largely unchanged across IAB5 and IAB7b enhancer orthologs in distantly related Drosophila species, although in the case of IAB7b there is the loss of the secondary KR binding site in species more distantly related to Drosophila. Conservation of genomic architecture of these TFBSs in the BX-C enhancers does not directly indicate that the specific spacing between sites is essential. However, the functional activity of genomic regions containing these motifs supports previous findings that closely spaced activator and repressor binding sites are critical for enhancer function and suggests that the architecture of binding sites within an enhancer is subject to significant evolutionary constraint (Starr, 2011).

It has recently been suggested through computational synthetic evolution studies that the inherent bias for deletions over insertions in the genome of Drosophila (and many other species) may result in the gradual loss of nucleotide space between TF binding sites. In effect, this deletion bias helps to artificially cluster binding sites together. In this case, although clustering of TF binding sites may not itself be a feature originally selected for in evolution on the basis of its functional significance, once established in the genome it may still play a functional role in enhancer activity. Molecular dissection of IAB5, IAB7b, and IAB8 enhancer function argues that specific clusters of activator and repressor binding sites do play a key role in enhancer activity. As a result, such clusters, once present in enhancers, may well be under positive evolutionary selective pressure, as evidenced by the largely invariant organization of the binding sites in the IAB5 and IAB7b FTZ-KR signature motif. This selection does not preclude the possibility that if binding sites arise nearby in the genome de novo, these new binding sites may also contribute to enhancer functional activity. In this scenario, the original TF binding site cluster may no longer be necessary for enhancer function. Indeed, in the case of the IAB8 enhancer, the ∆EVE region tested in s transgenic assay may be an example of this phenomenon. This fragment is able to exhibit a weak IAB8-like enhancer function even with the deletion of the pair of strong predicted EVE binding sites, potentially through the activity of weaker EVE binding sites that are present in the remaining sequence (Starr, 2011).

Although the precise spatial arrangement of TF binding sites within an enhancer may not exactly mirror the ancestral arrangement, computational predictions suggest that functional clusters of TF binding sites are likely to result from the spatial re-organization of older pre-existing sites during evolution. Such clusters therefore also likely indicate genomic regions with robust enhancer activity. The fact that enhancer activity in the BX-C appears to be dependent on signature motifs that represent specific spatial arrangements of TF binding sites in minimal modular regions, indicates that the physical patterns of binding site clustering are functionally significant in terms of enhancer architecture (Starr, 2011).

Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis by transcriptional interference

Much of the genome is transcribed into long non-coding RNAs (ncRNAs). Previous data have suggested that bithoraxoid (bxd) ncRNAs of the Drosophila bithorax complex prevent silencing of Ultrabithorax (Ubx), and recruit activating proteins of the trithorax group to their maintenance elements. This study found that, surprisingly, Ubx and several bxd ncRNAs are expressed in non-overlapping patterns in both embryos and imaginal discs, suggesting that transcription of these ncRNAs is associated with repression, not activation, of Ubx. The data rule out siRNA or miRNA-based mechanisms for repression by bxd ncRNAs. Rather, ncRNA transcription itself, acting in cis, represses Ubx. The Trithorax complex TAC1 (containing Trx, Sbf1 and dCBP) binds the Ubx coding region in nuclei expressing Ubx, and the bxd region in nuclei not expressing Ubx. It is proposed that TAC1 promotes the mosaic pattern of Ubx expression by facilitating transcriptional elongation of bxd ncRNAs, which represses Ubx transcription (Petruk, 2006).

The Hox genes of the bithorax complex (BX-C) have spatially restricted expression patterns that vary within and between segments and tissues. Transcription factors encoded by segmentation genes establish the patterns of the Hox genes Ubx, abd-A, and Abd-B of the BX-C in embryos. After the segmentation proteins decay, Hox expression patterns are maintained epigenetically by proteins of the trithorax group (trxG) and the Polycomb group (PcG). PcG genes maintain the silent state, whereas trxG genes maintain the active state of Hox genes. PcG and trxG proteins act through partially overlapping sets of response elements known as maintenance elements (Petruk, 2006).

One of the most startling discoveries of the genomic era has been that much of the genome is transcribed into non-coding RNAs (ncRNAs). Recent attention has focused on small interfering RNAs (siRNAs) and micro-RNAs (miRNAs) that modulate gene activity by an antisense mechanism termed RNA interference (RNAi), which interferes with mRNA stability or translation. However, the most abundant and least characterized class of ncRNAs are long and have mostly unknown functions (Petruk, 2006 and references therein).

The intergenic regions of the Hox genes in D. melanogaster produce many long ncRNAs that may regulate Hox gene coding sequences. Increasing attention has been directed to the role of transcription of MEs in the regulation of BX-C genes. Several ncRNAs are transcribed through a well-studied ME in the bxd regulatory region that lies between the Ubx and abd-A transcription units. The bxd ME regulates Ubx. Transcription through bxd precedes activation of Ubx coding RNAs (hereafter referred to as 'Ubx RNA', or simply as 'Ubx'), suggesting that ncRNAs might regulate Ubx. Transcription patterns of ncRNAs appear similar to those of the neighboring Hox genes and are collinear with regulatory domains along the chromosome. A synthesis of genetic and transgenic studies led to the idea that transcription of ncRNAs through MEs interferes with PcG-mediated silencing, perhaps by preventing recruitment of PcG proteins. A recent study suggests that transcription of MEs may recruit trxG proteins to maintenance elements. If indeed transcription of MEs simultaneously prevents PcG binding and establishes trxG binding, this could be a key element of Hox regulation. Clearly, this model requires that intergenic bxd RNAs are expressed in the same cells as Ubx. However double-labeling of intergenic and coding RNAs at high resolution has not been performed, so this attractive model has not been rigorously tested (Petruk, 2006 and references therein).

The trxG protein complex TAC1 plays important roles in maintaining expression of homeotic genes throughout embryogenesis. Recent attention has focused on the role of trxG proteins in histone modifications and in altering nucleosome positioning. TAC1 contains three proteins, Trx, Sbf1 and dCBP, and thus acetylates histones and methylates histone H3 at Lys-4 (H3-K4), due to the enzymatic activities of dCBP and the SET domain of Trx, respectively. How binding of Trx to MEs regulates expression of Hox genes is unclear. Because embryos have a mixture of cells expressing and not expressing each Hox gene, it has not been possible to determine precisely how trxG protein binding correlates with transcription of Ubx and bxd ncRNAs (Petruk, 2006).

This study shows that Ubx is repressed by bxd transcription. The bxd ncRNAs do not act by siRNA or miRNA-based mechanisms, but repress Ubx in cis through a transcription-dependent mechanism. Alternative association of TAC1 with either Ubx or bxd correlates with their transcription. TAC1 appears to be part of an interdependent network of general elongation factors that associate with active genes. It is suggested that a key role of TAC1 in establishing the mosaic pattern of Ubx expression is to promote elongation of bxd ncRNAs, which in turn represses expression of Ubx (Petruk, 2006).

An attractive notion has been that transcription of bxd ncRNAs, which precedes that of Ubx in embryos, facilitates correct spatial expression of Ubx. Previous studies showed that transcription through the ME could interfere with silencing, so it was proposed that bxd ncRNA transcription normally prevents recruitment of PcG proteins to the ME. However, the current experiments unambiguously demonstrate that Ubx and bxd ncRNAs are transcribed in different cells in embryos. The results also suggest that bxd ncRNAs do not facilitate Ubx expression in larval imaginal discs, as was recently proposed. Instead, transcription of ncRNAs correlates with repression of Ubx. It is possible that the abnormal transcription induced in previous studies interfered with transcription of ncRNAs in the BX-C, rather than with ME function, a possibility that can be tested experimentally. It will be interesting to use the system of sorting Ubx+ and Ubx− nuclei to examine binding of PcG proteins in nuclei where bxd ncRNAs either are, or are not, transcribed (Petruk, 2006).

The experiments rule out trans-repression by bxd ncRNAs, and instead support repression of Ubx in cis by transcription of these RNAs per se. A likely mechanism of this repression is transcriptional interference, since it was shown that ncRNA transcription extends into the region just upstream of the Ubx initiation site, which may well disrupt protein-DNA interactions required for Ubx initiation. However, this does not rule out promoter competition, and both of these mechanisms may contribute to the observed effects. Previous genetic studies and the results presented in this study show that bxd ncRNAs do not work by RNAi. An RNAi-based repression mechanism has been described for the miRNA produced by the iab-4 transcript, which directly interacts with the 3'-untranslated region of Ubx and prevents translation. This work showed that ectopic expression of iab-4 leads to homeotic phenotypes in the haltere, but do not show that loss of RNAi prevents this effect, nor has the effect of loss of function mutations of the iab-4 transcript been tested, so it remains to be seen if the iab-4 transcript is a bona fide miRNA (Petruk, 2006).

Since significant levels of bxd ncRNAs were not detected in imaginal discs, nor do they persist to late embryonic stages, they are unlikely be responsible for repression of Ubx throughout development. In fact, it has been reported that Trx is bound to the bxd ME in both wing and haltere discs, which have low and high levels of Ubx expression, respectively. The difference between binding of Trx to the bxd ME in embryos and in discs may be a consequence of the absence of transcription of bxd ncRNAs in discs and its presence in embryos, or to other uncharacterized differences between Ubx regulation in embryos and discs. Also, since Trx binds constitutively in some areas of the ME, Papp may have detected such binding in imaginal discs (Petruk, 2006).

Intergenic transcription also cannot explain repression of Ubx in the anterior of the embryo, where it is thought that hunchback and PcG genes set up and maintain the anterior boundary of Ubx expression. However, the pattern of bxd ncRNA transcription, which prefigures, in a complementary fashion, the mosaic pattern of Ubx expression within the parasegments of the embryonic trunk, appears to be essential for proper Ubx initiation. The Ubx pattern may then be maintained or modified at later embryonic stages through repression by other Hox proteins (i.e., abdA and AbdB) and by PcG genes. Thus, maintenance of Ubx expression likely requires multiple mechanisms that are employed at different developmental stages (Petruk, 2006).

The data support a role for Trx in transcriptional elongation as a mechanism for maintenance of a developmentally regulated gene. It has been argued that Trx does not have a direct role in activation of homeotic genes in Drosophila, but instead prevents repression of transcription by PcG proteins. However, the current data suggest that trx is required for recruitment of elongation factors and for efficient completion of transcripts. Therefore, maintenance of transcriptional activity by Trx may be a consequence of its role in elongation, and a block in elongation might lead to the establishment of PcG-mediated repression. Alternatively, Trx may be required only for normal levels of Hox gene expression, and not for maintenance of low levels of expression, a possibility consistent with at least some aspects of the trx mutant phenotype (Petruk, 2006).

This work strongly supports a general role for Trx and TAC1 in transcription, and agrees with previous findings that TAC1 relocates from other genes to the transcribed region of hsp70 following induction of the cellular stress response. The histone methyltransferase activity of Set1, the SET domain protein homologous to Trx, has a role in transcription, and MLL was suggested to play a similar role in mammals. It is suggested that this role is in transcriptional elongation, because Trx and elongation factors are co-ordinately recruited, because Trx binds downstream of the promoter more strongly to the 5’ than the 3’ end, and because transcripts extending to the 3’ end are more strongly affected by trx mutations, for both Ubx and bxd ncRNAs (Petruk, 2006).

TAC1 is also present at the promoter, and this is unaffected by mutations in elongation factors. Therefore, association of TAC1 with the promoter likely precedes the recruitment of elongation factors. Thus, TAC1 may play several distinct roles, one in initiation, another during the recruitment of the elongation complex and perhaps a third during subsequent elongation, where its ability to modify histones may be required for effective completion of long transcripts (Petruk, 2006).

This work provides the first direct evidence of the involvement of long ncRNAs in regulation of homeotic genes of Drosophila. Repression of Ubx is apparently mediated by expression of several intergenic ncRNAs in different germ layers of Ubx-expressing parasegments. TAC1 may be required for efficient read-through by Pol II into the region upstream of the Ubx initiation site, and as a result, for efficient repression of Ubx. Therefore, a direct link is proposed between elongation facilitated by the TAC1 epigenetic complex and repression of Ubx by intergenic transcription. A goal for the future will be to determine if other homeotic genes of Drosophila, and of other organisms, are also regulated by long ncRNAs whose expression is regulated by TAC1 proteins (Petruk, 2006).

Association of trxG and PcG proteins with the bxd maintenance element depends on transcriptional activity

Polycomb group (PcG) and trithorax group (trxG) proteins act in an epigenetic fashion to maintain active and repressive states of expression of the Hox and other target genes by altering their chromatin structure. Genetically, mutations in trxG and PcG genes can antagonize each other's function, whereas mutations of genes within each group have synergistic effects. This study showd in Drosophila that multiple trxG and PcG proteins act through the same or juxtaposed sequences in the maintenance element (ME) of the homeotic gene Ultrabithorax. Surprisingly, trxG or PcG proteins, but not both, associate in vivo in any one cell in a salivary gland with the ME of an activated or repressed Ultrabithorax transgene, respectively. Among several trxG and PcG proteins, only Ash1 and Asx require Trithorax in order to bind to their target genes. Together, these data argue that at the single-cell level, association of repressors and activators correlates with gene silencing and activation, respectively. There is, however, no overall synergism or antagonism between and within the trxG and PcG proteins and, instead, only subsets of trxG proteins act synergistically (Petruk, 2008).

Despite much interest, there is little understanding of how the epigenetic TRE/PRE-containing MEs function. One key unresolved issue pertains to the organization of these complex transcription regulatory elements with regard to the response elements/binding sites of particular trxG and PcG proteins. Response elements for several PcG proteins were mapped in the bxd ME previously, and some PcG proteins were detected at this DNA element in ChIP assays. However, information about the association of trxG proteins in the bxd ME is very limited. Several Trx-dependent TREs have been mapped in the bxd ME. In addition, Trx and Ash1 proteins have been detected at the bxd ME in ChIP assays. Given the apparent functional heterogeneity of the trxG proteins, it is revealing that besides Trx, many other trxG genes are essential for functioning of the bxd ME. Two of the interacting genes, skd and kto, encode components of the Drosophila Mediator complex, so it is possible that their role in the functioning of the bxd ME relates to the transcription of some of the non-coding RNAs that are known to be transcribed through this element. Ash2 is a component of several purified MLL (a human homolog of Trx) protein complexes. The identification of an ash2 response element in the bxd ME suggests that a second putative Trx-containing MLL-like complex might reside at the bxd ME. The genes urd and sls have only been minimally characterized, mainly as suppressors of Pc phenotypes. Therefore, it is premature to speculate about their function at this element, although they clearly interact there in some capacity (Petruk, 2008).

Identification of multiple TREs and PREs within the same ME raises an important question with regard to potential interdependency or competition in the association of proteins from the same and different protein families. To address this, focus was placed on the fine mapping of response elements for several major trxG genes that are essential for functioning of the bxd ME: ash1, the brm component of the BRM chromatin remodeling complex, and the ETP gene Asx. These proteins or components of their protein complexes (i.e. Snr1, a component of BRM) can physically associate with Trx. Thus, finding their response elements either in DNA fragments that are juxtaposed to (brm and ash1) or the same as (Asx) the previously mapped trx response element is consistent with direct interactions of these proteins with Trx. It should be noted, however, that all these proteins are components of protein complexes other than the Trx complex TAC1. Nevertheless, this suggests that there might be interdependency in recruitment and/or association of these protein complexes at the bxd ME. However, the results indicate that this suggestion is only partially true. Binding of the components of the BRM complex and of another trxG protein, Kis, were not affected by elimination of Trx. However, the association of Ash1 and Asx at all their sites on the salivary gland polytene chromosomes is completely dependent on the presence of Trx. Previous results of the reciprocal experiments indicated that binding of Trx is strongly decreased in ash1 mutant animals. This suggests that Trx, Ash1 and Asx represent a special, and at least partially interdependent, set of trxG proteins. This also suggests, in contrast to the previously mentioned genetic studies, that not all trxG proteins are mutually dependent in their functioning (Petruk, 2008).

Close proximity or even overlap between some TREs and PREs in the bxd ME suggests the existence of potential competitive relationships with regard to the binding of these functionally opposing groups of proteins. Furthermore, some ChIP assays indicate that some trxG and PcG proteins can bind to the bxd ME of both the activated and silenced gene, suggesting a potential interaction of these proteins on DNA. This was tested by asking whether binding of the components of two major PcG complexes, PRC1 and PRC2, is affected by elimination of Trx. No significant change was detected in the number or intensity of immunostained bands for all tested PcG proteins on the polytene chromosomes of trx mutant larvae. This suggests that not only is the association of PcG proteins independent of Trx, but also that Trx is not essential for preventing binding of the PcG proteins to their response elements. This is an important conclusion because some genetic studies have proposed that the main function of Trx and Ash1 is to prevent silencing by the PcG proteins (Petruk, 2008).

An important issue in understanding the molecular mechanism of trxG/PcG functioning is to correlate their association at MEs with the state of expression of their target genes. Although most of the existing data were obtained in cultured cells, two studies addressed this issue in Drosophila larval tissues. ChIP analysis in larval imaginal discs suggests that some trxG and PcG proteins are associated with the bxd ME irrespective of the status of gene expression. However, the results of another study suggest alternative association of Trx and Pc at the site of the endogenous BX-C on polytene chromosomes from both fat body and salivary glands, where BX-C is correspondingly activated or repressed. Ideally, to resolve this issue it is essential to investigate the association of PcG and trxG proteins with the ME in the same tissue at the single-cell level and at a gene of defined expression status. Such a test system was established. In this system the bxd-ME-containing transgene is either activated or repressed in cells within the same salivary gland. Direct visualization of the association of different proteins to the site of insertion of this transgene clearly indicates that major trxG and PcG proteins bind to the bxd ME in an alternative fashion. Importantly, using markers for activated and repressed transcription, it was possible to correlate binding of trxG and PcG proteins in a single cell with either the activated or repressed bxd transgene, respectively. The differences between these results and those of Papp (2006) might be explained by technical differences and by the fact that trxG and PcG proteins may behave differently in different tissues and/or in polyploid versus diploid cells. It is important to note that although the current analysis is limited to studies of a transgene, the detected alternative association of Trx and Pc on the bxd ME transgene correlates well with the results obtained at the endogenous BX-C on polytene chromosomes. It is concluded, therefore, that on a cell-by-cell basis, binding of trxG and PcG proteins is strictly dependent on the status of gene expression, in that they bind alternatively to the epigenetic regulatory elements of either activated or repressed target genes, respectively (Petruk, 2008).

In summary, this is the first work on the fine mapping of multiple TREs at any target gene. This is also the first assessment of mutual dependencies within the trxG group of activators and between the trxG and PcG of antagonistic proteins. It provides a glance of the enormously complex regulatory element that binds proteins with opposite transcriptional regulatory activities. The main conclusions of this study are that two major trxG proteins, Trx and Ash1, and the ETP protein Asx, constitute a specific subgroup of interacting proteins that depend on each other in their functioning at the bxd ME and throughout the genome. Although multiple trxG proteins are essential for epigenetic functioning of the bxd ME, their association with this element and other binding sites in the genome might not necessarily require Trx and associated proteins, as exemplified by the components of the BRM complex and Kis. The components of the major PcG complexes, PRC1 and PRC2, also associate with target genes independently of Trx, Ash1 and Asx. Another important conclusion of this work is that trxG and PcG proteins are associated with the bxd ME only at activated and repressed genes, respectively. It will be important to determine whether the choice between the establishment of trxG-mediated activation or PcG-mediated repression occurs only at very specific early stages of development, or whether it can also occur at later developmental stages (Petruk, 2008).

Regulation of Hox gene activity by transcriptional elongation in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Ubx regulation: Table of contents

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