Regulation of engrailed by Trithorax and Trithorax-like

Maintenance but not initiation of engrailed expression requires trithorax (trx), which is also required to maintain stable long-term expression of the homeotic genes throughout development. en expression is dependent on trx in only a subset of embryonic cells normally expressing en, including specific cells in the nervous system and the dorsal fat body cells surrounding the gonad. Loss of en expression in the dorsal fat body is correlated with the sterility of en females that are also mutant in trx . In addition, trx is required for normal en expression in the posterior compartment of the developing wing, as reflected in enhancement of en phenotypes in en adults which also carry trx mutations. trx appears to be dispensable for maintenance of en expression in other embryonic cells (Breen, 1995).

The expression of engrailed and other homeotic selector genes in imaginal discs was experimentally manipulated using mutations in trithorax group genes ash1 and ash2. Variegated expression of engrailed as well as Antennapedia, Sex combs reduced, and Ultrabithoraxis caused by hypomorphic ash1 mutations. These experiments demonstrate that both ash1 and ash2 are trans-regulatory elements of homeotic selector gene regulation (LaJeunesse, 1995).

The proximal promoter of engrailed does not direct expression in a tissue or stage-specific manner, but contains promoter activity which can be activated by nearby genomic enhancers. Three so-called pairing sensitive sites (PS) demonstrate sensitivity to the actions of Polycomb and trithorax group genes (Kassis, 1994).

moira (mor) is a member of the trithorax group of homeotic gene regulators in Drosophila. moira is required for the function of multiple homeotic genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal tissues. Heterozygous mor mutations suppress the following Polycomb-induced phenotypes:

  1. Derepression of the Antp gene in the eye-antennal disc causes replacement of adult antennal structures with leg structures.
  2. Derepression of the Scr gene in the second and third leg discs causes the appearance of first leg structures in the second and third legs of the adults.
  3. Derepression of the Ubx gene in the wing discs causes the appearance of haltere tissue in the adult wing.
  4. Derepression of the genes in the BXC (abd-A and Abd-B) causes cells of the fourth abdominal segment of the adult to differentiate structures of a more posterior identity.
moira mutations suppress the derepression phenotypes caused by mutations in another Pc group gene, Polycomblike. moira mutant clones in the haltere differentiate large bristles, characteristic of the anterior wing margin, and often lead to absence or duplication of halteres. Homozygous mor mutations in the posterior wing result in a distorted wing shape; the venation is disrupted and large socketed bristles appear along the posterior wing margin. Leg clones result in the femur and tibia being short and twisted and enlargement of the tarsal segment. Clones of the head cause the shape of the head to be abnormal in the dorsal region and sometimes cause the ocellus to be abnormal or absent. Embryos homozygous for moira mutations have defects in head structures, including truncated lateralgraten and defects in the mouth hooks and dorsal bridge. The first and second midgut constrictions are shifted posterior to their wild-type positions (Brizuela, 1997).

The requirement for moira function is at the level of transcription. The ability of moira mutations to supppress Antp homeotic phenotypes is dependent on the promoter. moira is also required for transcription of the engrailed segmentation gene in the imaginal wing disc. Because homozygous mor clones have phenotypes similar to those seen in clones of cells that have lost en function, en transcription was examined in clones of cells in the posterior wing. In the absence of transcriptional activation by mor, the pattern of en is altered. Greatly reduced en expression is found in wing clones. The abnormalities caused by the loss of moira function in germ cells suggest that at least one other target gene requires moira for normal oogenesis (Brizuela, 1997).

Drosophila Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To clarify the biological functions of Iswi, null and dominant-negative Iswi mutations were generated and characterized. Iswi mutations affect both cell viability and gene expression during Drosophila development. Iswi mutations also cause striking alterations in the structure of the male X chromosome. The Iswi protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for Iswi in transcriptional repression. These findings reveal novel functions for the Iswi ATPase and underscore its importance in chromatin remodeling in vivo (Deuring, 2000).

To determine when Iswi is required during development, the lethal phase and phenotype of Iswi null mutants were examined. Individuals heterozygous for ISWI1 or ISWI2 are viable and phenotypically normal. ISWI1/Df(2R)vg-C individuals die during late larval or early pupal development and display no obvious homeotic transformations or other pattern defects. Similar results were obtained for both ISWI2/Df(2R)vg-C and ISWI1/ISWI2 individuals (Deuring, 2000).

In vitro studies have suggested that Iswi plays an important role in transcription by facilitating the interaction of transcription factors with chromatin. One of the best candidates for a transcription factor that requires Iswi for its activity is the GAGA factor. GAGA factor binds to GA-rich sequences near the promoters of a wide variety of Drosophila genes and is thought to activate transcription by altering local chromatin structure. As the ATPase subunit of NURF, Iswi assists the GAGA factor to remodel chromatin in vitro, suggesting that the two proteins may act in concert to modulate chromatin structure in vivo as well. To examine possible interactions between Iswi and GAGA factor in vivo, the phenotypes of mutations in the two genes have been compared. GAGA factor is encoded by Trithorax-like (Trl), a member of the trithorax group of homeotic gene activators. Trl mutations enhance mutations in trithorax and cause homeotic transformations resulting from the decreased transcription of homeotic genes. Trl mutations also enhance position effect variegation, suggesting that GAGA factor antagonizes the assembly or function of heterochromatin. Unlike Trl mutations, Iswi mutations fail to enhance or suppress position effect variegation. No dominant interactions could be detected between mutations in Iswi and other genes, including Trl, other trithorax group genes (trithorax and brm), and Polycomb, a repressor of homeotic genes that is thought to act at the level of chromatin structure. These data suggest that Iswi and GAGA factor play distinct roles in chromatin remodeling in vivo (Deuring, 2000).

To investigate the role of Iswi in transcriptional activation in vivo, the effect of Iswi mutations on the expression of two targets of the GAGA factor were examined: the segmentation gene engrailed (en) and the homeotic gene Ultrabithorax (Ubx). The expression of En protein is reduced dramatically in imaginal discs of ISWI1/ISWI2 mutant larvae. Similar results are observed for Ubx. These data suggest that Iswi is essential for the expression of both en and Ubx in imaginal discs, although the possibility that this interaction is indirect cannot be ruled out (Deuring, 2000).

To directly observe interactions between Iswi and chromatin in vivo, the distribution of Iswi protein on salivary gland polytene chromosomes in third instar larvae was examined by immunofluorescence microscopy. Consistent with a fairly general role in transcription or other processes, Iswi protein is present at a large number of euchromatic sites in the polytene chromosomes. The same pattern was observed using whole sera and affinity-purified antibodies. The chromosomal distribution of Iswi protein is not appreciably altered following heat shock (Deuring, 2000).

Iswi protein is also associated with a subset of heterochromatin, as evidenced by punctate staining at the chromocenter. It is difficult to analyze the distribution of heterochromatic proteins on salivary gland chromosomes, since heterochromatic sequences are underreplicated in polytene tissues. To more accurately map the regions of heterochromatin with which Iswi interacts, the distribution of Iswi protein on mitotic chromosomes from larval neuroblasts was examined. On mitotic chromosomes, Iswi protein is abundantly present on the euchromatic arms of all chromosomes and is concentrated in regions of heterochromatin enriched with middle-repetitive sequences. For example, on the heterochromatic Y chromosome, Iswi is concentrated in the h11–13 region, which is composed almost entirely of middle repetitive DNA families. By contrast, little Iswi protein is detected in regions containing predominantly satellite DNA. The distributions of Iswi and GAGA factor on polytene and mitotic chromosomes were determined by double-label immunofluorescence microscopy. Both GAGA factor and Iswi are associated with hundreds of sites in the euchromatin of polytene chromosomes, but the distributions of the two proteins do not overlap extensively. Even greater differences in the distributions of the two proteins were observed in mitotic chromosomes where the GAGA factor, but not Iswi, is associated with GAGA-satellite sequences. The lack of extensive colocalization does not rule out an interaction between Iswi and GAGA at specific loci, but it does suggest that Iswi and GAGA are not obligatory partners (Deuring, 2000).

The decrease in en and Ubx expression in Iswi mutant larvae is consistent with reports that Iswi is involved in transcriptional activation in vitro. Consequently, it was not anticipated that the distributions of Iswi and RNA Pol II on salivary gland polytene chromosomes would be mutually exclusive. The preferential association of Iswi with transcriptionally inactive regions suggests that Iswi may create changes in chromatin structure that are not conducive to RNA Pol II transcription in vivo. Although there is no direct evidence that Iswi represses transcription, such a function would be consistent with the proposal that Iswi acts antagonistically toward histone acetyltransferases to compact chromatin structure. Based on these observations, further investigation of the role of Iswi in transcriptional repression is clearly warranted (Deuring, 2000).

How can the distributions of Iswi and RNA Pol II on polytene chromosomes be reconciled with the effect of Iswi mutations on gene expression in imaginal discs and the ability of Iswi complexes to activate transcription in vitro? One possibility is that Iswi has roles in both transcriptional repression and activation. NURF, ACF, and CHRAC were purified from Drosophila embryo extracts, and nothing is known about the nature or relative abundance of Iswi complexes in larvae. Perhaps only one Iswi complex is associated with transcriptionally inactive chromatin in the larval salivary gland, while others are either less abundant or transiently interact with chromatin to activate transcription. It is also possible that the interaction of Iswi with en and Ubx is indirect. For instance, the decreased expression of the two genes may be a secondary consequence of reduced cell viability in Iswi mutant larvae (Deuring, 2000).

Regulation of engrailed Polyhomeotic

Polycomb group (PcG) genes maintain cell identities during development in insects and mammals and their products are required in many developmental pathways. These include limb morphogenesis in Drosophila, since PcG genes interact with identity and pattern specifying genes in imaginal discs and clones of polyhomeotic (ph) null cells induce abnormal limb patterning. Such clones are associated with ectopic expression of engrailed, hedgehog, patched, cubitus interruptus and decapentaplegic, in a compartment specific manner. The results also reveal negative engrailed regulation by ph in both disc compartments: ph silences engrailed in anterior cells and maintains the level of engrailed expression in posterior ones. It is suggested that PcG targets are not exclusively regulated by an on/off mechanism, but that the PcG also exerts negative transcriptional control on active genes (Randsholt, 2000).

polyhomeotic is expressed in all imaginal disc territories. ph is required for limb patterning. Induction of ph null clones in larvae, by irradiating ph heterozygotes, leads to appendage pattern defects resembling those caused by ectopic expression of the hedgehog pathway. Adults that are heterozygous for ph null (pho) mutations and that have been irradiated as larvae, show only small ph null clones, no larger than 16 cells, that are not homeotically transformed. Irradiation of such animals performed during late second or early third larval instars (L2 or L3) causes the appearance of small vesicles of tissue trapped inside the body cavity and easily visualized in the wing. Animals irradiated earlier during larval development can show pattern abnormalities of all their appendages: this is particularly striking in the anterior wing compartment. Wing phenotypes range from slight margin or vein deformations to blisters and symmetrical mirror duplications of anterior compartment elements (Randsholt, 2000).

One copy of P[phd+] rescues lethality and homeotic transformations of pho. Furthermore, in the presence of a P[phd+] transgene, pho/1 irradiated flies never exhibit limb pattern defects, indicating that these are indeed induced by the ph null clones. To understand the origin of these abnormalities, the expression patterns of a series of lacZ reporters were examined in wing discs where ph null clones had been induced. The anterior-specific pattern disruptions in irradiated pho/1 animals mimic exactly those caused by ectopic expression of either engrailed, hedgehog or decapentaplegic, all genes that control A/P identity specification and limb morphogenesis. These developmental processes depend on interactions between posterior cells (which express en, the en-related gene invected and hh), and anterior cells, which respond to the Hedgehog signal by activating dpp through Ci in a stripe of cells at the A/P compartment border (Randsholt, 2000).

Starting with en-lacZ, ectopic en-lacZ expression is rapidly detected (three to four cell divisions after irradiation) in the shape of a spotted pattern in anterior disc compartments. Larger spherical anterior en-lacZ expressing domains are seen in late L3 discs that had been irradiated during L1. The shape of these ectopic en-lacZ domains suggests that they correspond to cells that are minimizing their contact surface with the rest of the disc. When ph null cells are induced in a hh-lacZ background, ectopic hh-lacZ expression is detected with the same time-lag and in a similar pattern in the anterior compartment. A ptc-lacZ reporter, whose peak expression is normally restricted to anterior cells along the compartment boundary, reveals ectopic ptc-lacZ expression in irradiated pho/1 discs in both compartments. A similar experiment performed in a ci-lacZ background detected ectopic ci-lacZ expression in the posterior compartment, despite the presence of Engrailed in these cells. Activation of dpp along the A/P boundary is the normal result of hh signaling, so dpp expression was monitored with a dpp-lacZ reporter. Ectopic dpp-lacZ expression is consistently seen in the anterior wing compartment of irradiated pho/1;dpp-lacZ/1 discs. dpp-lacZ expression was never detected in the posterior compartment. This anterior-specific induction of dpp-lacZ makes dpp a likely candidate for causing the anterior appendage defects of irradiated pho/1 animals. From these data, it has been concluded that loss of ph product leads to misregulation of hh pathway genes in both compartments. Additional experiments have shown that maintenance of A/P cellular identities in the developing appendages requires not only ph, but also several PcG products (Randsholt, 2000).

It was important to determine whether anterior activation of hh is a consequence of anterior engrailed activation or reflects an independent effect of loss of ph on the hh gene. The latter situation would agree with the fact that hh is upregulated in a trans-heterozygous mutant context for both ph and en. The data collectively suggest that anterior deregulation of hh in a ph mutant background can be independent of engrailed. It is suggested that Ci expression is increased when the ph level decreases (Randsholt, 2000).

Double-immunostaining of irradiated discs from different genetic backgrounds allows a precise determination of which genes are deregulated by the absence of functional Ph. pho cells were followed by Myc expression after irradiation of pho/Myc larvae. Staining of discs from pho/Myc;en-lacZ/1 larvae show that the anterior ph null cells form bubble-like shapes that are in a different plane from the rest of the disc. This shows that the pho cells are sorting-out from the disc surface and explains the origin of the vesicles of tissue trapped between the wing surfaces or in the body cavities of irradiated pho/1 adults. Furthermore, all cells that do not express Myc in the anterior compartment, and thus are ph null, express beta- Galactosidase, hence ectopic Engrailed. This shows that ph negatively regulates engrailed in the anterior compartment and that the induction of En is cell autonomous in all anterior compartment ph null clones. Ectopic engrailed expression allowed for the identification of the ph null cells in the anterior compartment (Randsholt, 2000).

Similar results were obtained when clones were induced in a hh-lacZ or ptc-lacZ background, showing that both hh and ptc are autonomously induced in anterior compartment ph null cells. In discs from pho/1 larvae stained with anti-En and anti-Ci antibodies, ci expression is present in some but absent in other anterior compartment ph clones. The presence of En in pho cells might secondarily lead to repression of ci when sufficient En has accumulated. Finally, in discs from pho/1;dpp-lacZ/1 larvae, all the ph null clones in the anterior compartment are associated with non-autonomous dpp-lacZ expression in the surrounding cells. Furthermore, similar to what has been observed for ci, ectopic dpp-lacZ expression is detected in some of the anterior ph null clones. These results suggest that the effect of ph on dpp expression is indirect, and that the relative levels of En and Hh within the clones are likely responsible for the expression or non-expression of dpp. Indeed, En is a strong repressor of dpp, whereas Hh stabilizes the activator form of Ci, which promotes dpp expression (Randsholt, 2000).

To conclude, loss of ph in the anterior compartment leads to sorting-out of the ph null cells and to misregulation of en and hh. This changes the identity of the cells toward cells that are not quite posterior either since they strongly express ptc, and sometimes even ci and dpp (Randsholt, 2000).

Discs from irradiated pho/phlac+2 larvae stained with antibodies against Ci and beta-Galactosidase exhibit cell autonomous expression of Ci in all posterior ph null cells, recognized by the fact that they do not express beta-Galactosidase. ph null cells also sort-out in the posterior compartment. Similarly, posterior ph null clones are found to be associated with ectopic ptc expression. Irradiated discs from pho/phlac+2 larvae labeled with Ptc and beta-Galactosidase antibodies exhibit Ptc in all the ph null cells. Ectopic expression of either ci or ptc allows the posterior ph null clones to be identifed by the presence of these markers. Discs from pho/1 larvae stained with antibodies against En and Ci suggest that loss of ph (detected in this case by ectopic Ci expression) is likely to have an effect on engrailed expression in posterior clones. Indeed, the posterior ci-expressing cells apparently express en at a higher level than the surrounding wild-type cells. The clonal cells sort-out, and they are not on the same focal plan as surrounding cells (Randsholt, 2000).

Enhanced En expression is also revealed in clones induced in pho/1 larvae and labeled with antibodies directed against Ptc and En. It is noteworthy that discs from irradiated pho/1;dpp-lacZ/1 larvae labeled with anti-beta-Galactosidase antibody never show dpp-lacZ expression in the posterior compartment. Indeed, pattern defaults of irradiated pho/1 flies occur anteriorly, which is in agreement with the presence of ectopic dpp expression associated only with anterior pho clones. Together these analyses reveal that posterior pho clones also sort-out. Loss of ph in posterior cells induces misregulation of ci and ptc, which are anterior specific genes, suggesting that ph is involved in maintenance of posterior cell identity. Several sets of data indicate that engrailed expression is affected by loss of ph in both compartments, suggesting that ph participates in the repression of engrailed in the anterior compartment, but also in the maintenance of a certain level of engrailed expression in posterior cells (Randsholt, 2000).

Negative regulation of engrailed in the anterior compartment is complex. Indeed, engrailed does not depend on transcriptional repression by the PcG alone; the groucho gene product, for one, also participates in silencing of en in anterior cells. The data presented here indicate that posterior ph null cells that have lost all Ph product express engrailed more strongly than their wild-type neighbors, suggesting that posterior compartment regulation of engrailed also involves more than a simple on/off mechanism. ph could intervene in this regulation either through direct regulation of en or the loss of ph could deregulate other genes that in turn control the expression level of en. Alternatively, ph could, together with other PcG products, maintain a rate of en transcription in posterior cells, possibly by regulating chromatin structure or accessibility. The repressive mechanism controlling en expression in the posterior compartment might, as in mammalian cell systems, change the probability that a given promoter is transcribed. The fact that Ph and Psc can bind transcribed loci in cell cultures suggests that control of gene activity by PcG products could extend to the regulation of active genes. The data from ph null clones provides further evidence that such a regulation does indeed take place during Drosophila development, and suggests that it plays a crucial role in the regulation of selector genes whose wildtype function requires, like engrailed, a strict control of their expression level (Randsholt, 2000).

To maintain cell identity during development and differentiation, mechanisms of cellular memory have evolved that preserve transcription patterns in an epigenetic manner. The proteins of the Polycomb group (PcG) are part of such a mechanism, maintaining gene silencing. They act as repressive multiprotein complexes that may render target genes inaccessible to the transcriptional machinery, inhibit chromatin remodelling, influence chromosome domain topology and recruit histone deacetylases (HDACs). PcG proteins have also been found to bind to core promoter regions, but the mechanism by which they regulate transcription remains unknown. To address this, formaldehyde-crosslinked chromatin immunoprecipitation (X-ChIP) was used to map TATA-binding protein (TBP), transcription initiation factor IIB (TFIIB) and IIF (TFIIF), and dHDAC1 (RPD3) across several Drosophila promoter regions. Binding of PcG proteins to repressed promoters does not exclude general transcription factors (GTFs) and depletion of PcG proteins by double-stranded RNA interference leads to de-repression of developmentally regulated genes. PcG proteins interact in vitro with GTFs. It is suggested that PcG complexes maintain silencing by inhibiting GTF-mediated activation of transcription (Breiling, 2001).

For X-ChIP analysis of promoter regions, the following PcG target genes were chosen: Abdominal-B (Abd-B, B-promoter), iab-4, abdominal-A (abd-A, AI-promoter) and Ultrabithorax (Ubx), all located in the Bithorax complex (BX-C), engrailed (en) and empty spiracles (ems). Also chosen were RpII140 (the subunit of RNA polymerase II with relative molecular mass 140,000 [Mr 140K]) and brown (bw): these last two do not reside in PC binding sites on polytene chromosomes and thus are most probably not PcG regulated. Expression of these genes in Drosophila SL-2 culture cells was assessed by polymerase chain reaction with reverse transcription (RT-PCR) and it was found that Abd-B and RpII140 are transcribed whereas iab-4, abd-A, Ubx, en, ems and bw are inactive (Breiling, 2001).

Acetylation of histones H3 and H4 is considered to be a mark for ongoing transcription. Thus, the promoters of the genes were screened for the presence of amino-terminally acetylated H4 and H3 by X-ChIP. Two antisera were used, one that recognizes H4 acetylated at lysine 12 and one or more other lysines, and one that recognizes H3 acetylated at lysines 9 and/or 18. H4 was found generally acetylated across the promoter regions analysed, in some cases with reduced levels in upstream and downstream regions. H3 is strongly acetylated in the active Abd-B and RpII140 promoters, whereas the inactive loci (iab-4, abd-A, Ubx, en, ems and bw) showed a decrease (5-10 times less than the H3 signal in the active Abd-B and RpII140 promoters) or absence of acetylation both at the core promoters as well as downstream of the initiator. Thus, H3 is acetylated in the active but underacetylated in the inactive promoters, whereas H4 acetylation shows no such changes. Acetylation of histones H3 and H4 seems to be regulated independently across the BX-C, consistent with results in other systems (Breiling, 2001).

The same promoter regions were analyzed by X-ChIP using antibodies against the PcG proteins Polycomb (PC) and Polyhomeotic (PH), dHDAC1, TBP, TFIIB and TFIIF (RAP 30 subunit, associated with RNA polymerase II). All six proteins were found in the core promoter regions (200 base pairs [bp] around the initiator) of the Abd-B, iab-4, abd-A, Ubx, en and ems transcription units. PC was found in most regions both upstream and downstream of the transcription start site (Breiling, 2001).

The major conclusion from this work is that promoters constitute a key target of PcG function. Evidence is provided that, unexpectedly, GTFs are retained at PcG-repressed promoters and that PcG proteins may function through direct physical interactions with GTFs. This mechanism of transcriptional regulation may provide both transcriptional competence and the flexibility necessary for the rapid re-arrangement of patterns of gene expression in response to developmental signals. Thus, the presence of GTFs and some trxG proteins at PcG-repressed promoters would allow a relatively fast re-activation of these genes, as differentiation processes require. In this context, PcG proteins would need to be continuously present at target gene promoters to constitutively inhibit transcription, a prediction supported by the finding that PcG-repressed genes are re-expressed in cells depleted of PcG proteins by dsRNA interference (Breiling, 2001).

Spps, a Drosophila Sp1/KLF family member, binds to PREs and is required for PRE activity late in development

The Polycomb group of proteins (PcG) is important for transcriptional repression and silencing in all higher eukaryotes. In Drosophila, PcG proteins are recruited to the DNA by Polycomb-group response elements (PREs), regulatory sequences whose activity depends on the binding of many different sequence-specific DNA-binding proteins. Previous studies have shown that that a binding site for the Sp1/KLF family of zinc-finger proteins is required for PRE activity (Brown, 2005). This study reports that the Sp1/KLF family member Spps binds specifically to Ubx and engrailed PREs, and that Spps binds to polytene chromosomes in a pattern virtually identical to that of the PcG protein, Psc. A deletion of the Spps gene causes lethality late in development and a loss in pairing-sensitive silencing, an activity associated with PREs. Finally, the Spps mutation enhances the phenotype of pho mutants. It is suggested that Spps may work with, or in parallel to, Pho to recruit PcG protein complexes to PREs (Brown, 2010).

Spps binds polytene chromosomes in a pattern comparable with the PcG protein Psc and, as shown by chromatin immunoprecipitation, is bound to both the en and Ubx PREs in S2 cells and in larvae. Furthermore, a mutation in Spps abrogates PRE activity in a mini-white assay, and enhances the phenotypes seen in a pho mutant. It is suggested that Spps acts either with or in parallel to Pho to recruit PcG protein complexes to the DNA. This result is particularly interesting in lieu of the recent report that the PcG protein Scm is recruited to the DNA independently of Pho (Wang, 2010). It has been speculated that Scm is in a complex with another PRE-DNA-binding protein, and it was shown that, like Pho, Scm plays a role in recruitment of PRC1 and PRC2 to the PRE. It will be interesting to explore whether Spps or another Sp1/KLF family member recruits Scm to the DNA (Brown, 2010).

The mammalian homologues of Pho and Spps, YY1 and Sp1 are extremely versatile proteins. Their activities can be changed from repressor to activator or vice versa depending on the cellular and binding site context. The activity of both these proteins is sensitive to the influence of many different co-repressors and co-activators. Both factors have been shown to bend DNA. Finally, YY1 and Sp1 have also been shown to interact directly (see Li, 2008). It is intriguing that such proteins bind to the PREs of Drosophila genes. Given that PREs may mediate the action of both the Polycomb and Trithorax group proteins, DNA-binding/recruitment proteins with such versatility and adaptability could be one way to facilitate the change from repression to activation. In fact, there is a report that Pho and Phol, in addition to their association with PREs, are bound to regions of chromatin with active histone modifications (Schuettengruber, 2009). Finally, a single Pho-binding site in a PRE in the even-skipped gene has been shown to be important for both activation and repression, dependent on the context (Fujioka, 2008). It will be interesting to explore whether Spps also has a dual role in gene regulation (Brown, 2010).

Regulation of engrailed by Eyelid and Dead ringer

In maternally mutant eyelid/osa gene embryos, although the initial engrailed expression is initiated in a relatively normal fashion, its later expression is abnormal: several stripes appear broadened, others are partially missing, and their spacing is disrupted. Nevertheless, wingless stripes are not expanded. These results are consistent with eld acting to counteract Wingless signaling in regions posterior to the engrailed stripes, in addition to responding to earlier patterning signals that affect the positioning of pair-rule gene stripes (Treisman, 1997).

It has been suggested that eld acts downstream rather than upstream of wingless for the following reasons:

  1. wingless expression is present in stripes of approximately normal width in embryos containing no Eld protein.
  2. Ectopic wingless expression is not found in clones of eld mutant cells, indicating that eld does not repress wingless expression.
  3. En stripes are present in eld, wg double mutants, suggesting that eld is not upstream of wingless.
Eld meets the criteria for a direct nuclear effector: its expression is ubiquitous in the early embryo and imaginal discs, and therefore cannot be dependent on localized Wingless signaling. Eld appears to function as a repressor of engrailed expression although this effect need not be direct. Intriguingly, a homolog of Eld, Drosophila Dead ringer, binds to target sequences of Engrailed protein, which are derived from possible autoregulatory sites within the engrailed genomic region (Gregory, 1996). If Eld also binds to these sites, it might compete with En to prevent the establishment of autoregulation. This would imply that Eld acts as a repressor; several active repression domains have been shown to have a similarly high proline content (Treisman, 1997).

The Drosophila eyelid/osa gene, like yeast SWI1, encodes an AT-rich interaction (ARID) domain protein. Genetic and biochemical evidence is presented that Osa is a component of the Brahma complex, the Drosophila homolog of SWI/SNF. To determine whether Osa is associated with the high molecular weight Brm complex, Schneider cell nuclear extracts were fractionated through a glycerol gradient and immunoblotted with antibodies against the various proteins. Osa, Brm and Snr1 co-sediment in the bottom third of the gradient, suggesting that they are part of a large protein complex. Thus, in vivo, Osa is found in a large complex with Brm and Snr1, but does not bind to proteins in other chromatin remodeling complexes. The ARID domain of Osa binds DNA without sequence specificity in vitro, but it is sufficient to direct transcriptional regulatory domains to specific target genes in vivo. Endogenous Osa appears to promote the activation of some of these genes. Brm-related complexes are thought to promote transcription by altering the architecture of nucleosomal DNA, thus generating a conformation that is more favorable to binding by transcription factors and the basal transcriptional machinery. Some genes, such as even-skipped, show reduced levels of expression in osa mutant embryos, supporting the role of Osa as an activator of gene expression. However, other genes, such as engrailed, show expanded domains of expression in osa mutants. These genes could be directly activated or repressed by Osa, or their changes in expression level could be secondarily due to the regulation of other transcription factors by Osa. Some Brahma-containing complexes do not contain Osa and Osa is not required to localize Brahma to chromatin. These data suggest that Osa modulates the function of the Brahma complex (Collins, 1999).

Regulation of engrailed by Single minded

The single-minded (sim) gene of Drosophila encodes a nuclear protein that plays a critical role in the development of the neurons, glia, and other nonneuronal cells that lie along the midline of the embryonic CNS. In sim mutant embryos the midline cells fail to differentiate properly into their mature CNS cell types and do not take their appropriate positions within the developing CNS. sim is required for midline expression of a group of genes, engrailed among them. Others include slit, Toll, rhomboid, and a gene at 91F. The sim mutant CNS defect may be largely due to loss of midline slit expression. The snail gene is required to repress sim and other midline genes in the presumptive mesoderm (Nambu, 1990).

Regulation of engrailed by Egfr signaling

Arthropod and vertebrate limbs develop from secondary embryonic fields. In insects, the wing imaginal disk is subdivided early in development into the wing and notum subfields. The activity of the Wingless protein is fundamental for this subdivision and seems to be the first element of the hierarchy of regulatory genes promoting wing formation. Drosophila epidermal growth factor receptor (Egfr) signaling has many functions in fly development. Antagonizing Egfr signaling during the second larval instar leads to notum to wing transformations and wing mirror-image duplications. Egfr signaling is necessary for confining the wing subregion in the developing wing disk and for the specification of posterior identity. To do so, Egfr signaling acts by restricting the expression of Wingless to the dorsal-posterior quadrant of wing discs, suppressing wing-organizing activities, and by cooperating in the maintenance of Engrailed expression in posterior compartment cells (Baonza, 2000).

To study Egfr function during early wing development, Egfr signaling was reduced at different times by using thermosensitive alleles of Egfr or by overexpression of dominant negative Raf (DNRaf). Hypomorphic vein (vn) and connector enhancer of ksr (cnk) [a regulatory member of the Ras signaling cascade] alleles were also analyzed. Under these conditions, posterior to anterior transformations, proximal (notum) to distal (wing) transformations, and a reduction (or absence) of the notum region were observed with high frequency. When DNRaf is expressed in clones induced during the second instar, different kinds of phenotypes are found. Large clones in the posterior notum/hinge anlage lead to notum to wing transformations, whereas large clones covering the posterior of the wing give rise to posterior to anterior transformations. These phenotypes were found only after inducing a large amount of confluent clones. Clones of cells overexpressing DNRaf in other regions at this age, or anywhere at later stages, give rise to different defects, such as those previously described on cell proliferation and vein cell fates (Baonza, 2000).

Posterior to anterior transformations are associated with mirror-image duplications that are reminiscent of those observed after reducing the expression of engrailed, a gene that confers posterior identity in posterior wing cells. En represses ci and limits the expression of dpp to anterior compartment cells adjacent to En-expressing cells. Dpp acts as a long-range morphogen emanating from the compartment border and it directs the growth and patterning of the wing. Local loss of en function is sufficient to generate a complete transformation of posterior cells to anterior, and as a consequence, to induce the ectopic expression of dpp and an ectopic anterior compartment (Baonza, 2000).

The mirror-image wing duplication resulting from the reduction of Egfr signaling in the posterior compartment correlates with a down-regulation of En protein expression and the up-regulation of Ci. Accordingly, ectopic dpp expression is activated and a new A/P border is implemented. It should be mentioned that dpp is activated within the posterior compartment, suggesting that the posterior En-nonexpressing cells are not recruited from anterior regions of the wing disk (Baonza, 2000).

Cells that neighbor those that express DNRaf in clones are recruited to generate the new A/P border. This is a nonautonomous effect that also has been described for en clones, leading to mirror-image duplications. Moreover, when DNRaf is expressed in clones, not all cells within the clone down-regulate En. Egfr signaling is therefore important for the maintenance of En expression in the posterior cells of the wing pouch and the enactment of posterior cell fates, although its effects appear to be nonautonomous (Baonza, 2000).

The ISWI chromatin remodeling complex NURF targets engrailed

The nucleosome remodeling factor (NURF) is one of several ISWI-containing protein complexes that catalyze ATP-dependent nucleosome sliding and facilitate transcription of chromatin in vitro. To establish the physiological requirements of NURF, and to distinguish NURF genetically from other ISWI-containing complexes, mutations were isolated in the gene encoding the large NURF subunit, nurf301. NURF is shown to be required for transcription activation in vivo. In animals lacking NURF301, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, NURF is shown to be required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate the phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that causes melanotic tumors to form (Badenhorst, 2002).

ISWI, the catalytic subunit of NURF, is required for expression of the homeotic gene engrailed (en). However, ISWI is also a component of two other chromatin remodeling complexes, ACF and CHRAC. To resolve which ISWI-containing complex is required for homeotic gene expression, expression of Ultrabithorax (Ubx) and engrailed (en) were examined in nurf301 mutant animals. When both copies of nurf301 are mutated, in homozygous mutant nurf3011 larvae, expression of the Ubx protein becomes undetectable. The normal expression of Ubx in the haltere and third leg discs of wild-type third instar larvae is absent in nurf301 mutant animals. Expression of the homeotic gene en requires nurf301. The normal expression of En in the posterior compartment of imaginal discs is abolished in nurf3012 mutants. Semiquantitative RT-PCR analysis confirms that Ubx and en transcript levels are reduced in nurf301 mutant animals. These results confirm that the defects in homeotic transcription seen in iswi mutants are caused by abrogated NURF function (Badenhorst, 2002).

A positive regulator of the Bithorax-Complex, E(bx), has been localized genetically to 61A, the same cytological interval as nurf301. However, unlike numerous regulators of the BX-C, E(bx) had not been cloned. Since NURF is required for expression of Ubx, whether nurf301 corresponds to E(bx) was tested. Both alleles of E(bx) were no longer extant, so whether the mutations that were isolated in nurf301 recapitulated the published morphological properties of E(bx) mutants was tested (Badenhorst, 2002).

nurf301 mutants, like E(bx), increase the severity of bithorax (bx) mutant phenotypes. bx is a DNA regulatory element required for correct expression of Ubx in regions that give rise to the third (T3), but not second thoracic segment (T2) of the adult fly. This expression distinguishes T3 from T2 identity. Loss or reduction of Ubx levels in bx mutant animals (Ubx6.28/bx34e and Ubx6.28/bx8 mutant combinations causes a homeotic transformation of the third thoracic segment to the anterior second thoracic segment. Thus, the third thoracic segment, which is normally vestigial and naked, is transformed into the second thoracic segment, increasing its size and causing sensory bristles to develop. Moreover, the haltere (T3) is transformed toward wing fate (T2), manifested by increases in size and the development of bristles. The strength of these transformations is increased when one copy of E(bx) also is removed. Mutation of one copy of nurf301 similarly enhances bx phenotypes. With one copy of either the nurf3011, nurf3012 or a deficiency that removes nurf301 -- Df(3L)3643 -- the strength of the transformation is enhanced. nurf301 enhances both bx34e and bx8 mutations (Badenhorst, 2002).

Although NURF is required for expression of the homeotic genes in imaginal discs, neither E(bx) nor nurf301 homozygous mutant larvae display obvious homeotic transformations of the larval cuticle. The absence of mutant larval cuticle phenotypes is likely due to the large maternal dowry of nurf301 transcript contributed to embryos. Larval cuticular patterning is established before these transcripts have dissipated. Attempts were made to generate embryos lacking the maternal nurf301 contribution through use of the dominant female sterile technique. Although germ-line clones were produced using the parental chromosome, it was not possible to recover germ-line clones using nurf3011. Like ISWI, NURF301 is required for ovary development (Badenhorst, 2002).

An important question is how NURF is recruited to target sites in vivo. Four genes were shown in this study to be dependent on nurf301 for expression: Ubx, en, hsp26, and hsp70. All contain multiple binding sites for the GAGA factor, which is genetically required for their correct expression. On the Drosophila hsp70 and hsp26 promoters, (GA.CT)n cognate elements (to which the GAGA factor binds) are required for HSF-binding. When these sequences are deleted, HSF-binding to transgenes in polytene chromosomes is impaired, consistent with the defects seen in nurf301 mutant animals. It is therefore compelling that recent biochemical studies show that NURF and the GAGA factor bind to each other in crude extracts, and that purified NURF301 and GAGA factor interact directly in vitro. The principal interacting domains map to an N-terminal region of NURF301 and a stretch flanking the Zn finger DNA-binding motif of GAGA factor. These data suggest that NURF is recruited by the GAGA factor through specific, direct interactions with the NURF301 subunit, to catalyze local sliding of nucleosomes at bx, en, hsp26, and hsp70 promoters, increasing accessibility to sequence-specific transcription factors and RNA polymerase II. Curiously, though, reduction of nurf301 levels fails to enhance phenotypes of mutations in Trithorax-like, the gene that encodes the GAGA factor (Badenhorst, 2002).

pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and has been proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibits several mutant phenotypes such as the transformation of antennae to mesothoracic legs. This study examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, is ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation are also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, is also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes are enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are also PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis (Kim, 2008).

Many PcG genes act as zygotic as well as maternal effect genes during whole Drosophila development, but it is not well known when and how they function. Pho is known to work with its redundant DNA-binding protein, Phol and recruits other PcG complexes by binding its binding sites on PREs. pho functions as a maternal effect gene. Its maternal effect mutant embryos show several segment defects and weak homeotic transformation. When pho functions as a zygotic gene, its zygotic mutant adults show homeotic transformation of antennae and legs. In accord to these results, pho functions in identification of ventral appendage were investigated (Kim, 2008).

Mutations in a few PcG genes result in the transformation of antennae to legs. Mutation in esc induces the ectopic expression of Antp and Ubx in the antennal disc, thus transforming antennae to legs. This indicates that esc represses Antp and Ubx expression in the antennal disc during antennal development. Therefore, the possibility was investigated that pho mutation, like esc mutation, would affect the expression of the selector genes that determine the identity of antenna or leg. In the wild type antennal disc, Antp is not expressed, but hth is expressed in almost all cells except for the presumptive arista, allowing for the development of antenna. However, in the leg disc, Antp is expressed and restricts hth expression to the proximal cells, which permits leg development (Kim, 2008).

Antp is ectopically expressed in the antennal disc of the pho mutant, and its expression subsequently but partially represses hth expression in the presumptive a2 or a3. Moreover, in the pho mutant, dac, which is expressed in the presumptive a3 of wild type antennal discs, is overexpressed in the presumptive a2 or a3 where hth expression is reduced. Ectopic expression of Antp in the presumptive a2 represses hth expression, which subsequently results in the transformation from antenna to leg. Ectopic Antp expression in the presumptive a1 permits expression of hth. In addition, when dac is ectopically expressed in a3 using the UAS/GAL4 system, leg-like bristles are newly formed in a3, indicating transformation of a3 to femur. However, the antennal disc of pho mutant shows that hth expression does not completely disappear in all regions of the presumptive a2 and a3 where Antp is ectopically expressed. These indicate that a pho single mutation partially affects expression of Antp, which leads to the incomplete repression of hth. Moreover, as the increased dosage of PcG mutants causes stronger mutant phenotypes than each single mutant, double mutation of pho and Psc strongly affects the expression of Antp, which leads to the complete repression of hth. Therefore, these results indicate that a pho mutation results in the ectopic expression of Antp, which directly represses hth expression in antennal disc and indirectly regulates dac expression through hth expression, which consequently transforms antennae to legs (Kim, 2008).

In the wing imaginal disc, Polycomb (Pc) and Suppressor of zeste (Su(z)) regulate the expression of teashirt (tsh), which specifies the proximal domain with hth. The polyhomeotic (ph) gene regulates the expression of en and the hedgehog (hh) signaling pathway in the wing imaginal disc. Pc also regulates eye specification genes such as tsh and eyeless (ey). PcG genes have recently been found to regulate organ specification genes in addition to homeotic genes, segmentation genes and cell cycle genes (Kim, 2008).

Therefore, it was proposed that pho might regulate the expression of organ specification genes for several reasons. First, Dll is ectopically expressed in the proximal region of the posterior compartment in the antennal disc of the pho mutant. Additionally, Dll is ectopically expressed in the more proximal region of the leg disc in the pho mutant, while dac is ectopically expressed in both the proximal and distal regions. These ectopic expressions do not antagonize each other in their normal region of expression, and result in duplication of distal tibia. Finally, en expression extends to the anterior compartment of both the antennal and leg discs of the pho mutant (Kim, 2008).

According to these reasons the following is proposed; first, pho regulates the expression of Antp in the antennal disc, which in turn might activate Dll. It has been shown that Dll is activated in AntpNS discs, which is similar in younger and older pho discs. Second, pho regulates the expression of en, which affects the expression of Dll. As a gene determining the A/P axis during antenna and leg development, en affects expression of wg and dpp, which determine the D/V axis via Hh signaling. Wg and Dpp act as morphogens, restricting the expression domain of hth, dac and Dll. This study has demonstrated that en is misexpressed in the anterior compartment in the antennal and leg discs of the pho mutant, which leads to misexpression of wg in the anterior-dorsal compartment. Although it has been shown that in the pho zygotic mutant embryos en is hardly derepressed, the current study showed that it is depressed in the pho zygotic mutant adults, suggesting that pho is involved in regulation of en expression and indirect regulation of Dll expression. Finally, pho might directly regulate expression of Dll, because recent studies using X-ChIP analysis have shown that PcG proteins bind PREs of appendage genes including Dll and hth. Hence, pho may directly or indirectly maintain the expression of Antp and en and regulates P/D patterning genes during ventral appendage formation (Kim, 2008).

Pho and Phol are the only PcG proteins that have a zinc finger domain. A mutation in pho results in weaker phenotypes than other PcG mutations despite the functioning of Pho as a DNA-binding protein. Therefore, Pho may interact with other corepressors and repress the homeotic selector genes. In fact, Pho binds to PRE, which is facilitated by GAGA. PRE-bound Pho and Phol directly recruit PRC2, which leads to the anchoring of PRC1. Pho interacts with PRC1 as well as with the BRM complex. Pho has recently been used to construct a novel complex, called the Pho-repressive complex (PhoRC), which has selective methyl-lysine-binding activity. It is currently known that pho interacts with two other PcG genes, Pc and Pcl, in vivo (Kim, 2008 and references therein).

Pho binds to approximately 100 sites on the polytene chromosome and colocalizes with PSC in about 65% of these binding sites. PSC is a component of PRC1 and inhibits chromatin remodeling. In the third instar larvae, PSC is found in the nuclei in all regions of all imaginal discs. Therefore, it is possible that pho and Psc interact with each other during the adult structure formation from the imaginal discs. pho and Psc interact in ventral appendage formation. While the Psc heterozygote was normal, it enhanced the adult mutant phenotypes exhibited by the pho homozygous mutant. Antp is more widely expressed in the antennal disc of the double mutant of pho and Psc than in that of the pho single mutant, while Psc mutant clones induced by FRT/FLP system showed normal expression of Antp, which indicated that Psc does not directly act by itself in regulating expression of Antp, but it certainly interacts with pho (Kim, 2008 and references therein).

hth is expressed in the distal region regardless of Antp expression so that dac was expressed not only in presumptive a3 but also in other segments, which results in the formation of a new P/D axis. According to recent study showing that hth may have a PRE, these results suggest that pho and Psc might interact to maintain hth expression during antennal development. Moreover, Dll expression in the antennal disc might be repressed by an unknown factor that was affected by the double mutation of pho and Psc, suggesting that the factor might be regulated by pho interaction with Psc during antennal development. In addition, legs of the double mutant had fused segments and weakly jointed tarsi, which may be because extension of Hh signal lead to the abnormal expression of the P/D patterning genes. In sum, pho functions as a regulator of selector genes for the identification of ventral appendages and axis formation by interaction with Psc during postembryogenesis (Kim, 2008).

In addition, Pho interacts with Phol in ventral appendage formation. Adults of double mutants showed more severe defects in appendage formation than those of single mutant. The stronger ectopic expression of Antp in the antennal disc of phol; pho double mutant seems to be one of reasons for severe defects. While Antp is not expressed in phol mutant clones of the wild type antennal discs, it is more strongly ectopically expressed in phol mutant clones of the pho mutant antennal discs than in their surrounding phol/+; pho/pho cells, indicating that Phol may not regulate the expression of Antp alone, but it may do that by interaction with Pho, suggesting that this may lead to recruit PRC1 including PSC to PRE sites of Antp and other appendage genes (Kim, 2008).

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

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

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

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

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

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

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

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

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

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

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

Regulation of the Drosophila engrailed gene by Polycomb repressor complex 2

Suppressor-of-zeste-12 (Su(z)12) is a core component of the Polycomb repressive complex 2 (PRC2), which has a methyltransferase activity directed towards lysine residues of histone 3. Mutations in Polycomb group (PcG) genes cause de-repression of homeotic genes and subsequent homeotic transformations. Another target for Polycomb silencing is the engrailed gene, which encodes a key regulator of segmentation in the early Drosophila embryo. In close proximity to the en gene is a Polycomb Response Element, but whether en is regulated by Su(z)12 is not known. This report shows that en is not de-repressed in Su(z)12 or Enhancer-of-zeste mutant clones in the anterior compartment of wing discs. Instead, en expression is down-regulated in the posterior portion of wing discs, indicating that the PRC2 complex acts as an activator of en. These results indicate that this is due to secondary effects, probably caused by ectopic expression of Ubx and Abd-B (Chen, 2009).

This investigation did not find any de-repression of en in the anterior pouch compartment of the wing discs when PRC2 function was removed. Somatic loss of PRC1 subunits (Pc and Ph) on the other hand results in de-repression of en in the anterior compartment of the wing discs. The Pcl protein has been shown to be associated with the PRC2 complex in embryos, but a recent study argues that Pcl is not included in either PRC1 or PRC2 larval complexes. Similarly to Pc and Ph, somatic loss of Pcl results in a de-repression of en in the anterior compartment of wing discs. However, the analysis finds that this de-repression is variable between clones. This study re-analyzed the results in this reference and found that en up-regulation is indeed found in the periphery of the anterior compartment of the wing disc, but not in the pouch region and thus the Pcl results agree well with the PRC2 pattern. Also the PhoRC complex seems to exhibit a similar behaviour since en is mis-expressed in many Sfmbt mutant clones in the anterior compartment of the wing disc, but consistently remains repressed in clones in the pouch compartment (Chen, 2009).

In the posterior part of the wing disc it was unexpectedly found that en expression is down-regulated in Su(z)12 and E(z) mutant clones. It is also noted that the Su(z)12 mutant clones induced in the posterior compartment of wing discs have a rounded shape commonly observed in cells that have lost their En function. This property of cells to become adhesive and minimize contact with surrounding En-expressing cells is caused by the Hedgehog signaling pathway, that induces Decapentaplegic protein that normally maintains the A/P boundary, aiming at exclusion of cell clones with a more anterior fate (Chen, 2009).

It is hypothesize that the down-regulation of en, occurring when PRC2 function is lost in the posterior compartment of wing discs, is caused by ectopic induction of an en repressor, and it was shown that ectopic expression of Ubx or Abd-B indeed blocks en expression. The PRC2 complex is necessary for silencing of Ubx or Abd-B in wing discs, since de-repression in both compartments is readily seen 48- 96 h after induction of Su(z)12 mutant clones. When scrutinizing these results again it was observed that the up-regulation of Ubx and Abd-B is exclusively occurring in the pouch region of wing imaginal discs, for strong as well as weak Su(z)12 alleles. Since en repression was found only within the pouch region in the posterior compartment this further strengthens the hypothesis that Ubx and Abd-B are direct repressors of en. This also poses an explanation for the lack of en mis-expression in the anterior half of the wing pouch; ectopic Ubx and Abd-B, as a consequence of loss of PRC2 silencing, directly repress en and thus override the de-repression expected to occur here. A similar finding was recently published by Jürg Müller and co-workers (Oktaba, 2008), where they had to remove both Scm and BxC (Ubx, Abd-A and Abd-B) gene functions in order to obtain de-repression of the Distal-less gene in the pouch region of wing discs (Chen, 2009).

Ubx and Abd-A are known to function as repressors of several genes in both embryos and larvae. For instance, Ubx is a transcriptional repressor of vestigial (vg) and Serrate (Ser) in larvae and the Ser protein is known to activate wg and cut expression in larval tissue. De-repression of Ubx in Su(z)12 mutant clones could thus explain the findings that wg and cut are down-regulated in their respective expression domains in wing discs (Chen, 2009).

Still, up-regulation of en in Pc and Ph clones does occur in the anterior wing pouch region. Furthermore, clonal loss of Pcl has no effect on en expression in the posterior portion of wing disc. This indicates that there is a discrepancy in the impact caused by loss PRC1 and PRC2 silencing functions, respectively. This could be explained by differences in binding and retention of either silencing complex to the PREs in both the Ubx and the en genes (Chen, 2009).

These results emphasize that each gene has to be regarded as a unique entity whose regulation can differ between developmental stages, tissues and between compartments within tissues and that transcriptional regulators together with different types of epigenetic marks collaborate to regulate gene expression. The en gene, that contains a PRE fragment to which PcG complexes bind in embryos, will have further controlling networks for maintenance of its active or silenced status at later stages. Its regulation is further complicated by the fact that the en PRE also possesses an activating function. This activating function might be due to the presence of PRC2 at the PRE, since loss of Su(z)12 results in repression of en expression in the posterior wing blade compartment. However, this possibility was not corroborated in Su(z)12 over-expression experiments, which did not result in a direct activation of en expression. It is concluded that the en gene has a multitude of regulatory elements that control the expression in its various contexts. Further studies are needed to elucidate the role of the different steps in PcG silencing and how these interact with other activating and repressive mechanisms to regulate gene expression (Chen, 2009).

P-element homing is facilitated by engrailed polycomb-group response elements in Drosophila melanogaster

P-element vectors are commonly used to make transgenic Drosophila and generally insert in the genome in a nonselective manner. However, when specific fragments of regulatory DNA from a few Drosophila genes are incorporated into P-transposons, they cause the vectors to be inserted near the gene from which the DNA fragment was derived. This is called P-element homing. The minimal DNA fragment that could mediate homing was mapped to the engrailed/invected region of the genome. A 1.6 kb fragment of engrailed regulatory DNA that contains two Polycomb-group response elements (PREs) was sufficient for homing. Flies that contain a 1.5 kb deletion of engrailed DNA (enδ1.5) in situ, including the PREs and the majority of the fragment that mediates homing. Remarkably, homing still occurs onto the enδ1.5 chromosome. In addition to homing to en, P[en] inserts near Polycomb group target genes at an increased frequency compared to P[EPgy2], a vector used to generate 18,214 insertions for the Drosophila gene disruption project. It is suggested that homing is mediated by interactions between multiple proteins bound to the homing fragment and proteins bound to multiple areas of the engrailed/invected chromatin domain. Chromatin structure may also play a role in homing (Cheng, 2012).

Previous results indicated that a 2.6 kb fragment of en DNA, extending from -2.4 kb upstream through +188bp of the en transcription unit could mediate P-element homing to the en/inv domain. This study showa that a 1.6kb fragment that extends from -2.0 kb through -0.4 kb is sufficient for homing. It is suggested that homing is mediated by a complex array of proteins and/or chromatin structure (Cheng, 2012).

PcG proteins are thought to mediate long-range chromatin interactions at the Bithorax complex, between the Bithorax and Antennapedia complexes, and also between PcG targets on the same chromosome arm. It is noted that one study suggests that the interactions at the Bithorax complex are not mediated by PREs, but by closely associated insulator elements. Biochemical studies show that PcG protein complexes can interact in vitro. The current results suggest that PREs play a role in P[en] homing: 1) deletion of the 181-bp PRE in the transgene decreases the homing frequency and 2) P[enHSP1] insertions occur in PcG-regulated genes at a higher frequency than P{EYgy2} insertions. It is noted that both the eve and Bithorax homing fragments are thought to be insulator elements. The en homing fragment is located just upstream of the en promoter and it is considered unlikely to be an insulator. However, the insulator proteins GAGA Factor, CTCF, and Mdg4 are associated with this DNA in embryos. Therefore, it is possible that the homing fragment has some of the same properties as insulators (Cheng, 2012).

In a previous study it was found that embryonic lacZ expression from P[en3R] (called P[en1] in that study) occurred in stripes at a much higher frequency than with the enhancer trap P[lacW]. It was hypothesized that P[en3R] caused selective insertion of P[en3R], not just to en/inv, but also to many genes expressed in stripes. It is known now that both the en promoter and en PREs (or sequences closely associated with them) mediate interactions with distant enhancers. Thus, one reason for the enriched number of lacZ stripe patterns with P[en3R] could be its ability to work with distant enhancers. In support of this, when P[en3R] is inserted up to 140 kb and 5 transcription units away from the nearest en stripe enhancer (either upstream or downstream), P[en3R]-encoded lacZ is still expressed in en-like stripes. In contrast, when P[lacW] is inserted about 45kb upstream of the nearest en stripe enhancer, into tou, the gene adjacent to en, P[lacW]-encoded lacZ is not expressed in stripes. In fact, the PREs in P[en3] facilitate long-distance interactions with enhancers in many different regions of the genome. It is suggested that the high percentage of striped lacZ expression from P[en3R] insertions is due both to the ability of the en promoter and PREs to act with distant enhancers and also to increased insertion into PcG-regulated genes, many of which are developmental regulators and expressed in stripes (Cheng, 2012).

Surprisingly, enδ1.5 flies are homozygous viable and fertile. En expression appears normal in these flies. It is suggested that these PREs are redundant with inv PREs and that the en/inv H3K27me3 domain is not disrupted in enδ1.5 flies. Interestingly, P[en] homing still occurs in enδ1.5 flies. These data suggest that homing is not mediated solely by self-self interactions between the homing fragment in P[en] and the genomic homing fragment (Cheng, 2012).

It is suggested that P[en] homing is mediated by the interaction of multiple proteins bound to the en fragment within P[en] and proteins bound to the en genomic region, and that these interactions are facilitated by the H3K27me3 mark characteristic of PcG target genes. Note that since P[en] insertions into the en/inv target occur much more frequently than into other PcG-target genes, protein-protein interactions, specific for the en/inv region must be involved in homing. The smallest fragment that could mediate homing was 1.6kb, a size capable of binding many proteins. This suggests that P[en] homing is not caused by a binding of a single protein or protein complex. However, it is also possible that the 1.6 kb fragment is needed to form the chromatin structure that facilitates homing. Finally, it is suggested that P[en] interacts with multiple proteins bound to the en/inv domain, since homing still occurs in enδ1.5, where the majority of the genomic homing fragment has been deleted (Cheng, 2012).

P-element homing occurs in germ cells. En is not expressed in these cells. Recent results indicate that the H3K27me3 modification is present at many developmental loci in germ cells. P[en] homing suggests that, in addition to the H3K27me marks, there are specific proteins bound to en DNA in the germ cells. These proteins could be present to keep en silenced, or perhaps they are there to facilitate rapid initiation of en transcription in the embryo (Cheng, 2012).

Polycomb group proteins bind an engrailed PRE in both the 'ON' and 'OFF' transcriptional states of engrailed

Polycomb group (PcG) and trithorax Group (trxG) proteins maintain the 'OFF' and 'ON' transcriptional states of HOX genes and other targets by modulation of chromatin structure. In Drosophila, PcG proteins are bound to DNA fragments called Polycomb group response elements (PREs). The prevalent model holds that PcG proteins bind PREs only in cells where the target gene is 'OFF'. Another model posits that transcription through PREs disrupts associated PcG complexes, contributing to the establishment of the 'ON' transcriptional state. These two models were tested at the PcG target gene engrailed. engrailed exists in a gene complex with invected, which together have four well-characterized PREs. The data show that these PREs are not transcribed in embryos or larvae. Tests were performed to see Whether PcG proteins are bound to an engrailed PRE in cells where engrailed is transcribed. By FLAG-tagging PcG proteins and expressing them specifically where engrailed is 'ON' or 'OFF', it was determined that components of three major PcG protein complexes are present at an engrailed PRE in both the 'ON' and 'OFF' transcriptional states in larval tissues. These results show that PcG binding per se does not determine the transcriptional state of engrailed (Langlais, 2012).

In this study sought to learn more about PcG protein complex-mediated regulation of en expression, focusing on mechanisms operating through en PREs. First whether the en and inv PREs are transcribed was investigated, and no evidence of transcription of the PREs was found either by in situ hybridization or by analysis of RNAseq data from the region. It is concluded that transcription of inv or en PREs does not play a role in regulation of en/inv by PcG proteins. Second, using FLAG-tagged PcG proteins expressed in either en or ci cells, it was found that PcG proteins are bound to the en PRE2 in both the 'ON' and 'OFF' transcriptional state in imaginal disks. The data suggest that PcG protein binding to PRE2 is constitutive at the en gene in imaginal disks and that PcG repressive activity must be suppressed or bypassed in the cells that express en (Langlais, 2012).

Transcription through a PRE in a transgene has been shown to inactivate it, and, in the case of the Fab7, bxd, and hedgehog PREs turn them into Trithorax-response elements, where they maintain the active chromatin state. However, is this how PREs work in vivo? Available data suggest that this could be the case for the iab7 PRE. Transcription through the PREs of a few non-HOX PcG target genes, including the en, salm, and tll PREs has been shown by in situ hybridization to embryos. However, in contrast to the robust salm and tll staining, the picture of en stripes using the en PRE probe was very weak and corresponded to a stage where transient invaginations occur that could give the appearance of stripes. Further, there was no hybridization of the en PRE probe to regions of the head, where en is also transcribed at this stage. In situ hybridization experiments with probes to detect transcription of the inv or en PREs did not yield specific staining at any embryonic stage, or in imaginal discs. This finding is confirmed by absence of polyA and non-poly RNA signals in this region at any embryonic or larval stage, upon review of RNA-seq data from ModEncode (Langlais, 2012).

The results show that PcG proteins bind to en PRE2 even in cells where en is actively transcribed. In fact, one member of each of the three major PcG protein complexes, Pho from PhoRC, dRing/Sce from PRC1, and Esc from PRC2, as well as Scm, are constitutively bound to en PRE2 in all cells in imaginal discs. It is noted that dRing/Sce is also present in the PcG complex dRAF, which also includes Psc and the demethylase dKDM2. Further experiments would be necessary to see whether Sce-FLAG is bound to en DNA as part of the PRC1 complex, the dRAF complex, or both (Langlais, 2012).

What are the differences between the 'ON' and 'OFF' transcriptional states? The data suggest that there may be some differences in Pho binding to non-PRE fragments. However, this data has to be interpreted with caution. The en-GAL4 driver is an enhancer trap in the inv intron and contains an en fragment extending from -2.4 kb through the en promoter. Thus, it is possible that the en-GAL4 driver alters Pho binding in the en/inv domain. In fact, the increased Pho-binding to non-PRE probes in the 'ON' versus the 'OFF' state in the FLAG-Sce samples suggests that the presence of the en-GAL4 driver alters Pho binding slightly (Langlais, 2012).

One unexpected result from these experiments was that FLAG-Sce binds to PRE2 but not to PRE1. This is an interesting result that needs to be followed up on. Recent ChIP-Seq data in using imaginal disk/brain larval samples and the anti-Pho antibody show five additional Pho binding peaks between en and tou, which could be five additional PREs. Three of these correspond to known Pho binding peaks. ChIP-seq experiments with the FLAG-tagged proteins expressed in the 'ON' and 'OFF' transcriptional states would be necessary to ask whether the distribution of PcG-proteins is altered at any of the PREs or any other region of the en/inv domain (Langlais, 2012).

In conclusion, the data allows two simple models of PcG-regulation of the en/inv genes to be ruled out. First, the en/inv PREs are not transcribed, so this cannot determine their activity state. Second, PcG proteins bind to at least one of the PREs of the en/inv locus in the 'ON' state, therefore a simple model of PcG-binding determining the activity state of en/inv is not correct. Perhaps the proteins that activate en expression modify the PcG-proteins or the 3D structure of the locus and interfere with PcG-silencing. While FLAG-tagged PcG proteins offer a good tool to study PcG-binding particularly in the 'OFF' state, cell-sorting of en positive and negative cells will be necessary to study the 3D structure and chromatin modification of the en/inv locus (Langlais, 2012).

Return: see engrailed Transcription regulation part 1/3 | back to part 2/3

engrailed: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Developmental Biology | Effects of mutation | References

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