Polycomb

Targets of Activity (part 2/2)

In embryos mutant for genes of the Polycomb group, Ultrabithorax expression to all segments. Thus, parasegmental domains are maintained by Polycomb-mediated repression. Such domains may be realized by parasegmental differences in chromatin structure (McCall, 1994).

Transcriptional repression is a key mechanism operating at multiple levels to control Abd-B expression. The anterior Abdominal-B expression limit is apparently determined by Krüppel repression, whereas the knirps repressor may be responsible for the graded Abd-B expression within the Abd-B domain. iab-5 and two other fragments show region-specific silencing activity. Silencing requires hunchback as well as Polycomb function and evidently provides maintenance of Abd-B expression limits throughout embryogenesis (Busturia, 1993).

DNA fragments in the Scr regulatory region are important for regulation of Sex combs reduced by Polycomb and trithorax group gene products. When DNA fragments containing these regulatory sequences are subcloned into P-element vectors containing a white minigene, transformants containing these constructs exhibit mosaic patterns of pigmentation in the adult eye, indicating that white minigene expression is repressed in a clonally heritable manner. The amount of white minigene repression is reduced in some Polycomb group mutants, whereas repression is enhanced in flies mutant for a subset of trithorax group loci (Gindhart, 1995). The extent of SCR expression is influenced by mutations at the Polycomb locus but not by mutant alleles of the zeste gene (Pattatucci, 1990).

A 14.5-kb fragment from the enhancer like postbithorax/bithoraxoid region of Ultrabithorax exhibits proper regulation by both trithorax and Polycomb in the embryonic central nervous system. Trithorax or Polycomb can function independently through this upstream fragment to activate or repress the Ultrabithorax promoter, respectively. Deletion analysis of this fragment demonstrates that a 440-bp fragment contains response elements for both Trithorax and Polycomb. Furthermore, the integrity of the proximal promoter region is essential for trithorax-dependent activation, implicating a long-range interaction for promoter activation (Chang, 1995).

A regulatory element in the Ubx gene responds to Pc-G and trx-G genes. Placing Polycomb regulatory elements (PRE) in new ectopic sites, create new binding sites for Pc-G products at the new sites. PREs and Pc-G proteins establish a repressive complex that keeps itself and other distal enhancers repressed in cells where they were first active and then repressed, and maintains this repressed state for many cell divisions. PRE functions either to silence these remote enhancers or to maintain expression regulated by trx-group products. Hunchback mediates repression at the PRE. The trx-G products stimulate the expression of separate and distinct enhancers that are active in imaginal discs (Chan, 1994).

Ectopically placed Ultrabithorax DNA regulatory sequences responsible for silencing require Hunchback (HB) and Polycomb (PC). Embryonic silencing is initiated by HB protein that binds to the silencers to repress Ubx, thereby defining the normal Ubx domain. Fragments which mediate silencing in anterior regions of imaginal discs contain embryonic silencers and HB target sites. One exception to this is a fragment called BXD which is not under HB control itself, but whose silencing activity depends on combination with fragments containing HB protein binding sites. Since silencing by BXD also requires Pc function, this suggests that BXD contains target sites for PC or for PC-like proteins (Christen, 1994).

Polycomb response elements (PREs) can establish a silenced state that affects the expression of genes over considerable distances. The ability of insulator or boundary elements to block the repression of the miniwhite gene by the Ubx PRE has been tested. The gypsy element and the scs element interposed between PRE and the miniwhite gene protect miniwhite against silencing but the scs element is only weakly effective. Blocking the action of gypsy requires su(Hw). When the PRE-miniwhite gene construct is insulated from flanking chromosomal sequences by gypsy elements at both ends, the construct can still establish efficient silencing in some lines but not others. This silencing can be caused by interactions in trans with PREs at other sites. PRE-containing transposons inserted at different sites or even on different chromosomes can interact, resulting in enhanced silencing. These trans interactions are not blocked by the gypsy insulator and reveal the importance of nonhomologous associations between different regions of the genome for both silencing and activation of genes. The similarity between the behavior of PREs and enhancers suggests a model for their long-distance action. Thus blocking elements can prevent communication along a chromatin fiber, and enhance silencing of PREs in trans (Sigrist, 1997).

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

In Drosophila, the Polycomb group genes are required for the long-term maintenance of the repressed state of many developmentally crucial regulatory genes. Their gene products are thought to function in a common multimeric complex that associates with Polycomb group response elements (PREs) in target genes and regulates higher-order chromatin structure. The chromodomain of Polycomb is necessary for protein-protein interactions within a Polycomb-Polyhomeotic complex. Posterior sexcombs protein coimmunoprecipitates Polycomb and Polyhomeotic, indicating that all three are members of a common multimeric protein complex. Immunoprecipitation experiments using in vivo cross-linked chromatin indicate that these three Polycomb group proteins are associated with identical regulatory elements of the selector gene engrailed in tissue culture cells. Polycomb, Polyhomeotic, and Posterior sexcombs are, however, differentially distributed on regulatory sequences of the engrailed-related gene invected. High-resolution mapping shows that Pc binding is maximal in a 1.0-kb element, 400 bp upstream of the inv start of transcription. Pc binding sites in en are found in a fragment that contains repetitive elements. The Pc binding sites and the repetitive elements are separable. In fact, Pc associates with two distinct elements, one covering the first intron and the other 1 kb upstream from the start of transcription. Both these regions have been implicated in regulation of en expression during embryogenesis. The binding site upstream of en overlaps with a number of pairing-sensitive elements which have been suggested to mediate PcG repression. Ph and Psc are present at both Pc binding sites in the en upstream region and first intron. The common Pc-Ph-Psc complex does not appear to funcion at inv: no Psc is associated with inv and Ph is associated with a much more restricted element than Pc (Strutt, 1997b).

The B promoter of Abdominal-B is devoid of all three PcG proteins. Ph and Psc are not associated with the peak Pc binding element A (overlapping the gamma promoter). However, other fragments in the vicinity of gamma and C promoters are associated with Ph and/or Psc, and it may be that this regulatory region is unusually complex and contains several PREs that regulate the different Abd-B promoters. Both Ph and Psc are enriched for a restriction fragment in the 3' region of Abd-B, which is relatively poorly enriched by Pc. This element is strongly associated with GAGA factor. In the empty spiracles gene Psc is associated with an upstream fragment, covering a previously identified ems enhancer element. Pc and Ph are not found at this transcribed locus. These results suggest that there may be multiple different Polycomb group protein complexes which function at different target sites. Polyhomeotic and Posterior sexcombs are also associated with expressed genes. Polyhomeotic and Posterior sex combs may participate in a more general transcriptional mechanism that causes modulated gene repression, whereas the inclusion of Polycomb protein in the complex at PREs leads to stable silencing (Strutt, 1997b).

The wing imaginal disc is subdivided into dorsal and ventral compartments. A new dominant homeotic mutation, Dorsal wing1 (Dlw1), transforms ventral into dorsal compartment in heterozygotes. This phenotype is similar to one of the dominant phenotypes of Polycomb mutants. Dlw+ is required for the specification of dorsal compartment: some genes of the Polycomb group act as negative regulators of Dlw+, while some genes of the trithorax group act as positive regulators (Tiong, 1995).

To test for a chromatin structure involved in Polycomb group repression, heterologous DNA- binding proteins were used as probes for DNA accessibility in Drosophila embryos. Binding sites for the yeast transcriptional activator GAL4 and for bacteriophage T7 RNA polymerase were inserted into the bithorax (bx) regulatory region of the endogenous Ultrabithorax gene, which is regulated by PcG proteins. Ubiquitously expressed GAL4 protein directs transcription through its binding sites only in the posterior segments where the bithorax region is active. The block to GAL4 activation in the more anterior segments is dependent on Polycomb function. In contrast, T7 RNA polymerase can transcribe from its target promoter in all segments of the embryo. Thus, Pc-mediated repression blocks activated polymerase II transcription, but does not simply exclude all proteins (McCall, 1996).

When two to six copies of a white promoter-Alcohol dehydrogenase (Adh) reporter fusion gene are introduced into the genome, the transgene's expression is progressively reduced, both in larvae and adults, rather than the expected gene dosage effect. In addition, multiple transgenes reduce endogenous Adh transcripts, a result that is strongly analogous to "cosuppression" phenomena described in many plant species but which has not been previously observed in animals. Silencing of the Adh gene is not influenced by zeste-dependent transvection but strongly affected by the Polycomb and Polycomblike mutations. Polycomb and polyhomeotic proteins bind to the chromatin at the sites of the repressed w-Adh transgenes revealing that the cosuppression process initiates accumulation of Pc-G proteins in the absence of a canonical Polycomb response element (Pal-Bhadra, 1997).

Cosuppression refers to the phenomenon in which silencing among dispersed homologous genes occurs. Two nonhomologous reciprocal fusion genes, white-Alcohol dehydrogenase (w-Adh) and Adh-w, exhibit cosuppression using the endogenous Adh sequence as an intermediary. Deletion of the endogenous Adh gene eliminates the interaction, while reintroduction of an 8.6 kb Adh fragment restores the silencing. Using truncated Adh constructs, a nontranscribed segment in the Adh regulatory region was found to be one of the sequences required for homology recognition. The silencing interaction is initiated during early development. The silenced transgenes are associated with the Polycomb group complex of chromatin proteins (Pal-Bhadra, 1999).

Because PcG proteins are involved with cosuppression of w-Adh, the association of the Pc protein with the Adh and Adh-w insertion sites was examined in the normal and deleted Adh flies under cosuppressing conditions. The cytological location of each insert (Adh-w#1 at 16B region and Adh-w#2 in 70C region) was determined by in situ hybridization. Immunolocalization of the Pc protein in the polytene chromosomes from strains with single inserts (Adh-w#1 or Adh-w#2) shows that Pc protein binds to more than 100 sites in the polytene chromosomes, but is not detected at either Adh-w site. However, labeling under cosuppressing conditions reveals that Pc protein is strongly recruited to each Adh-w insert. No other alteration in Pc banding pattern has been found in these genotypes. The Pc binding was examined in the polytene chromosomes of the two Adh-w stocks in which endogenous Adh sequences are deleted but which also carry one or two copies of w-Adh. Under these circumstances, the Pc proteins fail to associate with the Adh-w sites but retain their association near the endogenous Adh. The deletion of the entire Adh sequence does not eliminate Pc binding at 35D. Thus, the major site for normal Pc binding in this region is not in Adh itself but is sufficiently close to obscure a cytological determination of any changes at Adh (Pal-Bhadra, 1999).

These data indicate that binding of high levels of Pc complex can be initiated and maintained at ectopic positions. There is no obvious PRE sequence, as defined by a consensus, present in the region of the Adh promoter that is required for the interaction with Adh-w. One could perhaps argue that weak PREs exist in Adh. If this is the case, then they must exist in both parts of the Adh gene. However, even on this hypothesis the tendency for a high level of binding must spread in the endogenous Adh gene, because the single Adh-w stock without w-Adh has the same genetic configuration at the endogenous Adh locus but does not exhibit detectable Pc binding or silencing. Only when w-Adh is present in the genome together with the endogenous Adh is silencing and high levels of Pc association observed on Adh-w. Therefore, the results suggest Pc complex association can spread in cis and then in trans to previously naive sites based on the underlying DNA homology (or chromatin array dictated by DNA) (Pal-Bhadra, 1999).

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 presence of PC and PH at the active Abd-B-B promoter is striking, although precedented. Co-localization of PcG proteins and trithorax-group (trxG) proteins, which have been identified as suppressors of the PcG, has been reported for most PcG-bound regulatory regions of the BX-C, including promoters. PcG and certain trxG proteins might simultaneously be needed for changing and maintaining opposite transcriptional states. Thus, coincidental association of repressors and activators with active genes might act to guarantee regulated levels of transcription (Breiling, 2001).

RpII140 was expected to be active and not PcG controlled, and indeed GTFs and also dHDAC1, but not PcG proteins, were found bound to the core promoter. As a putative non-PcG-repressed target, the bw gene was chosen. Again, TBP, TFIIB and dHDAC1 but not TFIIF were found. PC, but not PH, binds to the bw promoter. However, no PC was found downstream of the bw promoter, unlike the other PcG-controlled genes. This and the absence of PH suggest that brown is not a canonical PcG-controlled gene, and PC here might have an as-yet-unknown function, not requiring other PcG proteins (Breiling, 2001).

The acetylation state of the promoters investigated could not be correlated with the presence of dHDAC1. dHDAC1 might be present at active promoters together with histone acetyl transferases (HATs) to maintain an appropriate steady-state level of histone acetylation. Five Drosophila HDACs have been identified so far: dHDAC1 (also known as RPD3), dHDAC3, dHDAC4, dHDAC6 and dSIR2 -- and any of these might contribute to the acetylation patterns observed. dHDAC1 might also regulate transcriptional activity by deacetylating transcription factors. GTFs, in particular TFIIE and TFIIF, are substrates for histone acetyl transferases, and transcriptional regulation involves acetylation of developmentally controlled transcription factors. The presence of dHDAC1 at promoters could also be due to the association with Topoisomerase II, which is bound to PcG binding sites (Breiling, 2001).

The close mapping to the same DNA regions suggests a physical interaction of PcG proteins with TBP and other GTFs. Using antibodies against TBP it was shown that the PcG proteins PC, PH and PSC are co-precipitate in a DNA-independent manner with TBP from embryonic nuclear extracts and from nuclear extracts from SL-2 cells. The long proximal isoform of PH (PH 170p) has been found to interact with other PcG proteins and this protein co-purifies with PC, PSC and TBP. Only PC, not PH or PSC, is co-precipitated with TFIIB, whereas no co-purification was observed of PcG proteins with TFIIF (Breiling, 2001).

PcG proteins at repressed promoters may prevent activation of RNA Polymerase II, otherwise committed to transcribe. This hypothesis was tested by dsRNA interference (RNAi), a targeted destruction of messenger RNA, to see whether the inhibition of PcG protein synthesis would lead to de-repression of inactive genes. After prolonged treatment of SL-2 cells with Pc and ph dsRNAs, PC protein is no longer detectable in cellular extracts by Western blotting, and the amount of PH is significantly lower than in non-treated cells. With the same kinetics, PcG-regulated promoters, which are inactive in non-treated cells (iab-4, abd-A, Ubx, en and ems), become de-repressed upon treatment with Pc or ph dsRNAs. In contrast, bw does not show any change of expression state, like Abd-B and RpII140, underlining the specificity of PcG control. Remarkably, an incubation time of 8 days is necessary to observe a significant de-repression of PcG target genes. It appears that PcG proteins are rather stable and cells have to divide several times (eight times assuming a duplication time of roughly 24 h) with inhibited PcG protein synthesis, before an effect on transcription is seen (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).

In Drosophila, the Trithorax-group (trxG) and Polycomb-group (PcG) proteins interact with chromosomal elements, termed Cellular Memory Modules (CMMs). By modifying chromatin, this ensures a stable heritable maintenance of the transcriptional state of developmental regulators, like the homeotic genes, that is defined embryonically. It was asked whether such CMMs could also control expression of genes involved in patterning imaginal discs during larval development. The results demonstrate that expression of the hedgehog gene, once activated, is maintained by a CMM. In addition, the experiments indicate that the switching of such CMMs to an active state during larval stages, in contrast to embryonic stages, may require specific trans-activators. Thes results suggest that the patterning of cells in particular developmental fields in the imaginal discs does not only rely on external cues from morphogens, but also depends on the previous history of the cells, as the control by CMMs ensures a preformatted gene expression pattern (Maurange, 2002).

Immunoprecipitation using cross-linked chromatin (XChIP) allows the mapping of in vivo DNA target sites of chromatin proteins. Because one Polycomb (PC, a member of the PcG) binding site on polytene chromosomes coincides with the cytological position of hh at 94E, this method was applied to ask whether there are PC and GAGA factor (GAF/Trl, a member of the trxG) binding sites in the hh genomic region. These two factors had previously been found to be hallmarks of CMMs, and the GAF has been shown to be associated with some PcG complexes and necessary for the silencing function of PREs. Initially the immunoprecipitated material was hybridized to a genomic stretch of 45 kb encompassing the hh gene. This led to the identification of PC/GAF-binding sites in regions close to the transcription unit. To further fine-map the location of the PC/GAF-binding sites, the region around the hh gene was subdivided into 1-kb-sized PCR fragments (from 4 kb upstream of the hh transcription start site according to the transcript CG4637 from Flybase, to 13.4 kb downstream to the end of the gene). Slot-blot hybridizations of immunoprecipitated material revealed two main sites where PC and GAF are strongly enriched. The first site (A) is located in a region between 0.07 and 1.06 kb upstream of the transcription start site, whereas the second binding site (B) is found in a region spanning the second exon of the hh gene and spreading about 0.4 kb on both sides of the exon. On both sites a substantial overlap was observed between PC- and GAF-binding sites. The presence of this particular arrangement of PC- and GAF-binding sites in the hh genomic region suggests that these PcG and trxG proteins directly control hh expression (Maurange, 2002).

To investigate this at the functional level, the accessibility of the hh promoter region to a trans-activating factor was assessed. It is known that a PRE placed in the vicinity of an Upstream Activating Sequence (UAS) is able to counteract GAL4 binding, preventing expression of the reporter gene (Zink, 1995; Fitzgerald, 2001). Advantage was taken of the availability of an EP line possessing a UAS site close to the endogenous hh transcription start site to test whether the hh-PREs could inhibit the activation of transcription induced by GAL4. The EP3521 line (termed here EP-hh) possesses an EP transposon containing several UAS sites, and is inserted in the hh promoter region (-0.36 kb). The endogenous hh gene is not transcribed in salivary glands. By using an hs-GAL4 line, which is known to be leaky at 25°C, weak expression of GAL4 in larval salivary glands is observed. When hs-GAL4 is crossed to a line containing UAS-hh integrated randomly in the genome, in situ stainings reveal that at 25°C, by the action of GAL4, the hh mRNA is present in high amounts in all the salivary gland cells. However, when hs-GAL4 is crossed to the EP-hh line, in which the UAS sites are juxtaposed to the presumptive PRE, hh transcription was observed in only a very few cells situated mainly at the base of the glands. It was reasoned, because in most cells transcription is inhibited, that the PcG proteins binding the PREs in the vicinity of the hh promoter block the accessibility of GAL4 to the UAS sites. Accordingly, reducing the amount of some of the PcG proteins in the cells by repeating the experiment with flies heterozygous for the Pc³ allele or with males hemizygous for the ph⁴⁰⁹ allele induces partial derepression of transcription of the endogenous hh gene in a substantial number of gland cells. These results indicate that the repression observed in most of the salivary gland cells in the EP line is caused by the action of the PcG proteins through their binding to the identified PREs. As such, these experiments demonstrate that the transcription of hh is directly repressed by the PcG proteins (Maurange, 2002).

When UAS-en is misexpressed at the D-V boundary in a wild-type genetic background using vg-GAL4, it induces hh expression in most of the cells of the wing pouch except in a stripe along the A-P boundary where hh seems to be repressed. Whereas UAS-en is strongly misexpressed at the D-V boundary, the endogenous en gene is weakly misactivated in some cells of the anterior wing pouch (Maurange, 2002).

Repeating the same experiment in a genetic background hemizygous mutant for an hypomorphic allele of polyhomeotic (ph⁴⁰⁹) leads to a broader domain of expression of hh. Remarkably, the region along the A-P boundary seems to be less refractory to activation of hh transcription, given that the territory of the repressed domain is reduced. Endogenous en is itself overexpressed in the anterior compartment. This is consistent with the findings demonstrating that en expression can be derepressed in a PcG gene mutant background. In this case in the anterior wing pouch cells, the activation of en transcription by Hh is probably more efficient than in a wild-type background because en cannot be correctly silenced by PH (Maurange, 2002).

The same experiment repeated in a genetic background now doubly heterozygote for the trxG genes trithorax (trx^E2) and brahma (brm²) consistently shows that hh expression is activated at the D-V boundary, but can hardly be maintained through cell divisions in the anterior compartment, because with in situ staining, the Hh signal progressively fades away from the D-V boundary. As expected, in such a case, en expression in the anterior compartment is restricted to the D-V boundary, because Hh might not be present in a sufficient amount to activate transcription of the endogenous en gene in the subsequent wing pouch cells (Maurange, 2002).

Furthermore, it is known that PcG-mediated silencing is enhanced at higher temperature, and this hyperrepressed state can be inherited through cell divisions. Based on these observations, it was reasoned that raising embryos at 28°C instead of 18°C would make the Pc-mediated silencing more difficult to derepress, and influence the activation of hh transcription by En. vg-GAL4; UAS-en embryos were allowed to develop at 28°C until the beginning of second instar larvae, when the D-V boundary is established in wing discs and UAS-en is expressed there. As expected, stainings on third instar imaginal discs reveal ectopic clones of wing pouch cells expressing hh. However, the frequency of cells expressing hh is lower than in discs of larvae grown at 18°C, indicating that the Pc-mediated silencing was harder to erase at 28°C. Nevertheless, in contrast with trxG mutant flies, once the transcription has initially been activated in this case, it is maintained in the subsequent daughter cells as suggested by the presence of clones spreading from the D-V midline to the limits of the wing pouch (Maurange, 2002).

These experiments demonstrate that once initiated by En, the maintenance of the transcriptional state of hh to the daughter cells can be attributed to the action of the PcG and trxG proteins. It is concluded that the CMM activity of the hh upstream region described in the transgenic assay is also efficient when considered in its natural chromatin environment and is responsible for the inheritance of the initial transcriptional state of hh from the initiation to the completion of the wing pouch development (Maurange, 2002).

The polyhomeotic (ph) gene is a member of the Polycomb group of genes (Pc-G), that are required for the maintenance of the spatial expression pattern of homeotic genes. In contrast to homeotic genes, ph is ubiquitously expressed and it is quantitatively regulated. ph is negatively regulated by the Pc-G genes, except Psc, and positively regulated by the antagonist trithorax group of genes (trx-G), suggesting that Pc-G and trx-G response elements (PREs and TREs) exist at the ph locus. In this study, PREs and TREs at the ph locus that function in transgenic constructs have been functionally characterized. A strong PRE and TRE has been identified in the ph proximal unit as well as a weak one in the ph distal unit. The PRE/TRE of both ph units appear atypical compared with the well-defined homeotic maintenance elements because the minimal ph proximal response element activity requires at least 2 kb of sequence and does not work at long range. Chromatin immunoprecipitation experiments on cultured cells and embryos have been used to show that Pc-G proteins are located in restricted regions, close to the ph promoters, that overlap functionally defined PRE/TREs. The data suggest that ph PRE/TREs are cis-acting DNA elements that modulate rather than silence Pc-G- and trx-G-mediated regulation, enlarging the role of these two groups of genes in transcriptional regulation (Bloyer, 2003).

Detailed analysis of the php PRE/TRE shows that this element is modular, and contains at least three regions of differential sensitivity to Pc-G and trx-G mutations. Recently, it has been proposed that homeotic 'maintenance elements'(MEs) are composed of several small DNA modules that are bound by several subsets of Pc-G and TRX-G proteins. In other PREs and MEs, the PSR is separable from silencing modules. Pc-G-dependent silencing and PSR are always linked in the ph PRE: both silencing and PSR are lost when the minimal 2-kb P{C4-812} PRE/TRE is dissected into several subfragments. It was not possible to separate PSR from silencing of white, but this may be because these studies lacked sufficient resolution (Bloyer, 2003).

There is not a perfect correspondence between sites of Pc-G binding as detected by ChIP, and functional activity of transgenes. Peak-binding for Pc, Ph, Psc, and GAF mapped to a 0.9-kb fragment which showed no silencing activity and no sensitivity to Pc-G and trx-G mutations when tested in isolation in the P{C4-811} transgenic lines. In both cells and embryos, Pc-G and GAGA factor (GAF) binding overlaps the conserved regulatory sequences found in both the php and phd PREs. However, neither of these conserved sequences were sufficient for PRE activity in these assays. The P{C4-827} and P{C4-824} fragments overlap, and both contain the upstream 350-bp conserved region, but neither demonstrates PRE activity. Similarly, P{C4-811} that contains the downstream conserved region also lacks PRE activity. It is not known why these differences exist; but it is suggested that proteins not assayed in the ChIP experiments, and/or unconserved sequences, must contribute to PRE function. In addition, there were differences between binding of Pc-G proteins in cultured cells and in embryos, which may reflect the different physiology of developing embryos versus cultured cells or experimental variation of the technique (Bloyer, 2003).

The isolated ph PRE/TREs do not completely reproduce the ph wild type regulation even if most of the Pc-G and trx-G response elements are conserved in the minimal P{C4-812} PRE/TRE. An opposite eye color phenotype was observed in a Sce and Pc mutant background when the endogenous ph regulation was compared with transgenic lines containing isolated ph PRE/TREs. It may be simply that the effects of Pc-G mutations on ph regulation are indirect. It may be that different Pc-G complexes function differently in the endogenous and exogenous chromosomal locations. Using an in vivo-functional assay it has been shown that ph and Psc may have a different silencing function than Pc and Sce. Consistent with this, in this study, ph and Psc consistently show strong genetic effects on ph regulation. The results are in accordance with the idea that ph/Psc in one case and Pc/Sce in the other may play different roles in Pc-G silencing complexes, and that the function of these complexes may depend on chromosomal context. An alternative, but not necessarily mutually exclusive explanation for the opposing response of php PRE/TRE transgenes and the endogenous ph locus to Pc-G mutations might be that the isolated PRE/TREs lack distant cis-DNA regulatory elements that can act on ph wild type regulation within the endogenous chromosomal context. In this model, activity of the php PRE/TRE would be modulated by the distant cis-regulatory element, so that transgenes, lacking this sequence would behave differently. These hypotheses cannot be distinguished with the current data (Bloyer, 2003).

Transcription through a PRE interferes with maintenance of Polycomb-mediated silencing.

The Fab-7 chromatin domain boundary ensures functional autonomy of the iab-6 and iab-7 cis-regulatory domains in the bithorax complex (BX-C). Chromatin insulators such as gypsy or scs^min are potent insulators that cannot substitute for Fab-7 function within the BX-C. During the early stages of these swapping experiments, a fragment of scs was initially used that was slightly larger than a minimal scs element (scs^min). This scs fragment, unlike scs^min, interferes in an orientation-dependent manner with the output of a regulatory region covering 80 kb of DNA (from iab-4 to iab-8). At the core of this orientation-dependent phenotype is a promoter located immediately adjacent to the scs insulator. In one orientation, the promoter traps the activity of the iab-3 through iab-5 cis-regulatory domains, diverting them from the abd-A gene. In the opposite orientation, the promoter is transcribing the iab-7 cis-regulatory domain, resulting in ectopic activation of the latter. These data suggest that transcription through a Polycomb-Response Element (PRE) interferes with the maintenance of a Polycomb repression complex. Since the large cis-regulatory regions of the bithorax complex are known to be transcribed, transcriptional activity probably reflects a fundamental mechanism to protect an actively transcribed gene from being inactivated by the Pc-G proteins that are present in all cells (Hogga, 2002).

There are precedents where transcription has been suggested to play a role in chromatin remodeling. For example, the human ß-globin locus is subdivided into three chromatin domains, each of which become more accessible to nuclease digestions upon gene activation. Interestingly, large intergenic transcripts delineate each of these domains and chromatin remodeling of each domain is preceded by its transcription. Another example has been reported in which it was found that transcription across a PRE could interfere with silencing. Evidence has been provided that transcription across the iab-2 cis-regulatory domains in PS6/A1 interferes with iab-2 silencing, resulting in the posterior transformation of PS6/A1 into PS7/A2. In this case, the identity of the affected abdominal segment can easily be recognized in embryos and larvae. Despite the existence of intense transcription of iab-2 in embryos, the dominant gain-of-function phenotype associated with iab-2 misexpression is only detectable in the adult. Thus transcription across the iab regulatory regions appear to interfere with silencing during the late maintenance phase, when the adult structures are forming (Hogga, 2002).

If transcription can interfere with Pc-G silencing, what are the mechanisms responsible for this activity? Factors that affect RNA polymerase II (RNAPII) transcript elongation have been shown to have an effect on chromatin. For example, it has been suggested that histone acetyl transferases (HAT) such as PCAF or ELP3 assist RNAPII in relieving inhibition caused by nucleosome arrays. Although active chromatin requires acetylation of specific lysine residues in the H3 and/or H4 histone tails, the recent purification of Pc complexes suggests that histone deacetylation is required for establishing a stable long-term Pc-G silencing complex. In the case of scs_prom (the inserted scs element), perhaps the frequent passage of RNAPII and its associated histone acetylation activities though the PREs interferes with the assembly of the Pc-G silencing. Involvement of acetylated histones in antagonizing PcG-dependent silencing is supported by the findings showing that high levels of acetylated histone H4 are associated with non-repressive PRE sequences. Alternatively, it has been recently found that variant histone H3.3 is deposited on active chromatin during transcription, providing a mechanism for the immediate activation of genes that are silenced by histone modification. It may be possible that transcription across iab-7 (and also iab-8) results in deposition of new nucleosome marked by H3.3, interfering thereby with the maintenance of silencing by the Pc-G complex (Hogga, 2002).

It has been known for a long time that the large cis-regulatory regions of the bithorax complex are transcribed. In blastoderm stage embryos, the iab-2 though iab-8 regions can be divided into three domains, each transcribed in a region that extends from a specific anterior limit to the posterior limit of the segmented part of the embryo. These domains are only broadly defined but their order on the chromosome reflects the anterior limit of expression for each of them. In the light of the current data, it is tempting to speculate that transcription of the iab domains convey a regulatory signal, preventing assembly of the Polycomb-repressing complex on the iab domains that need to remain active. If this is true, transcripts should appear in the anteriormost parasegments/segments where each cis-regulatory domain is activated. However, so far, transcripts in every regulatory region have not been seen; this would account for the sequential activation of each regulatory domain. Moreover, this model predicts that the iab-7 PRE and iab-7 domains should be transcribed from PS12, where iab-7 is first active. So far transcripts across the iab-7 domain have only been detected in PS13 and 14. Thus, it remains unclear whether intergenic transcription plays a role in wild-type animals to create and/or maintain open chromatin, or whether the existence of intergenic transcripts is the consequence of an open structure. However, these experiments strongly suggest that forced transcription through an inactive cis-regulatory domain interferes with the maintenance of silencing, highlighting an incompatibility between transcription and Pc-G mediated silencing. This activity probably reflects a fundamental mechanism to protect an actively transcribed gene from being inactivated by the Pc-G proteins that are present in all cells (Hogga, 2002).

A series of mutations have been recovered in the bithorax complex of Drosophila that transform the first segment of the abdomen into a copy of the second or third abdominal segment. These dominant Ultraabdominal alleles are all associated with P element insertions which are transcribed in the first abdominal segment. The transcripts proceed past the end of the P element for up to 50 kb, extending through the regulatory regions for the second and third abdominal segments. Blocking transcription from the P element promoter reverts the mutant phenotype. Previously identified Ultraabdominal alleles, not associated with P elements, also show abnormal transcription of the same region. The P elements initiate transcripts that proceed through PREs and boundaries, and the phenotypes depend on the production of these transcripts. Other work shows that transcription across the BX-C can relieve silencing, and transcription has been associated with loss of silencing. These observations raise the possibility that non-coding RNAs in wild-type animals may function to activate segmental regulatory regions (Bender, 2002).

Transcription might change the chromosome in several ways. The RNA polymerase II complex involved in elongation includes a histone acetyltransferase that could modify nucleosomes across the transcribed region. The act of transcription might remove bound complexes (such as the Polycomb complex) or prevent their spread along the chromosome. This mechanism was suggested by studies in yeast that have shown that transcription of yeast telomeres relieves the telomere position effect. Transcription might also allow transient access to DNA sequences near the RNA polymerase which might otherwise be covered with nucleosomes or packaged in a 'higher order' structure. A further possibility is that the ectopic RNA product has a function in activation (Bender, 2002).

It is not clear what site or function is affected by the ectopic transcripts. The boundary between the bxd and iab-2 regulatory regions most likely lies just distal to the Uab^HH1 insertion site; perhaps the ectopic transcription disrupts this boundary. Alternatively, the iab-2 region includes at least one PRE; perhaps transcription across this site relieves the repression imposed by the Polycomb Group. Unfortunately, there is no clear indication from the available BX-C mutations what phenotype to expect from the loss of a PRE (Bender, 2002).

The ectopic RNAs appear not to affect the segmental regulation of the complex early in embryonic development, although the transcripts are abundant in PS6 from stage 10 (elongated germ band) onwards. Misexpression of ABD-A in PS6, which is presumably necessary for the observed segmental transformations, is not seen in embryos except in occasional cells in the central nervous system. Perhaps there is a critical time later in development when ectopic RNA matters, such as the time of abdominal histoblast proliferation in the pupa. Alternatively, continuous transcription might activate abd-A stochastically, so that over time the majority of PS6 cells switch to the active state (Bender, 2002).

The RNA transcripts from Uab^HH1 are antisense to the normal transcripts of the abd-A gene in PS7-12, and one might expect abd-A expression to be blocked. Indeed, the level of ABD-A protein in Uab^HH1 embryos is reduced in the PS7 epidermis relative to wild type. ABD-A expression appears normal in the developing central nervous system, and in the epidermis of PS8-PS12, presumably because the Uab^HH1 transcripts in older embryos are primarily in the epidermis of PS6 and PS7. In Uab^HH1 larvae, there is also evidence of loss of abd-A function in PS7; the second abdominal setal belt is weakly transformed towards the first. The Uab^HH1 adults don't show anterior transformation (loss of abd-A function) in PS7, but any such effect would be masked by the strong posterior transformation (gain of abd-A function) (Bender, 2002).

It seems surprising that there are not more gain-of-function alleles in the BX-C or elsewhere due to readthrough from P elements. However, most P element transposons contain selectable marker genes downstream of the P promoter; perhaps these sequences help to terminate transcripts initiated at the P promoter. It is also likely that strong gain-of-function mutations would be dominant lethals. There are a variety of gain-of-function mutations in the BX-C associated with rearrangements, which could mediate their effects by non-coding readthrough transcription from the juxtaposed DNA. Contrabithorax alleles, like Cbx³ and Cbx^Txt, are good candidates (Bender, 2002).

The dramatic effects of ectopic transcription hint at a function for non-coding transcripts in the wild type BX-C. Non-coding transcripts have been documented in the human ß-globin locus, and such transcription has been correlated with changes in DNaseI sensitivity. Several non-coding transcripts have been described in the BX-C, most notably in the bxd and iab-3 regions. These RNA products appear in blastoderm embryos, at or before the onset of segment-specific expression of the homeotic proteins. Other early RNAs, not associated with BX-C protein products, have been detected by RNA in situs in early embryos (Bender, 2002).

There is, so far, no evidence for a function of these RNAs. A deletion (pbx¹) that removes the promoter for the bxd RNA has no effect on the embryonic expression pattern of UBX, although the UBX pattern in imaginal discs is changed. The latter effect of pbx¹ may well be due to loss of imaginal disc enhancers, but the bxd RNA could matter for the development of the adult, just as ectopic RNA does. A difference between embryos and larvae has been reported in their requirements for Polycomb Group repression. Perhaps the later mode of Polycomb Group repression is sensitive to and regulated by non-coding transcripts (Bender, 2002).

Transcription factor YY1 functions as a PcG protein in Drosophila

Polycomb group (PcG) proteins function as high molecular weight complexes that maintain transcriptional repression patterns during embryogenesis. The vertebrate DNA binding protein and transcriptional repressor, YY1, shows sequence homology with the Drosophila PcG protein, Pleiohomeotic. YY1 might therefore be a vertebrate PcG protein. Drosophila embryo and larval/imaginal disc transcriptional repression systems were used to determine whether YY1 represses transcription in a manner consistent with PcG function in vivo. YY1 represses transcription in Drosophila, and this repression is stable on a PcG-responsive promoter, but not on a PcG-non-responsive promoter. PcG mutants ablate YY1 repression, and YY1 can substitute for Pho in repressing transcription in wing imaginal discs. YY1 functionally compensates for loss of PHO in pho mutant flies and partially corrects mutant phenotypes. Taken together, these results indicate that YY1 functions as a PcG protein. Finally, YY1, as well as Polycomb, was found to require the co-repressor protein CtBP for repression in vivo. These results provide a mechanism for recruitment of vertebrate PcG complexes to DNA and demonstrate new functions for YY1 (Atchison, 2003).

The YY1 repression patterns are the same as those obtained previously with a known PcG protein. Therefore, it was asked whether YY1 repression required PcG function. To determine this, an hbGALYY1 BXDGALUbxLacZ (BGUZ) recombinant chromosome line was prepared and this chomosome was crossed into various homozygous PcG mutant backgrounds. Since PcG proteins function as complexes, mutation of a single PcG gene often abrogates PcG-dependent repression. Strikingly, homozygous mutant Polycomb (Pc), Polycomb-like (Pcl) or Sex combs on midleg (Scm) backgrounds led to complete derepression of YY1 function. Even heterozygous Pc and Pcl mutants abolished YY1 repression. Homozygous mutant Sex combs extra (Sce), Additional sex combs (Asx) or Suppressor of zeste [Su(Z)2] plus Posterior sex combs (Psc) backgrounds yield partial derepression of YY1 activity, perhaps due to maternal effects. Therefore, YY1 repression in vivo required PcG function (Atchison, 2003).

Two distinct PcG complexes have been identified. The first complex, termed the PRC1 complex, contains Pc, Scm, Polyhomeotic (Ph) and Psc proteins. This complex is clearly necessary for YY1 repression since Pc and Scm mutants abolish YY1 function. The second complex contains Esc and E(z). YY1 physically interacts with EED, the vertebrate homolog of Drosophila Esc. Therefore, the necessity of Esc for YY1 repression was tested in vivo. Homozygous mutation of the esc gene causes partial loss of YY1 repression. Thus, both complexes are needed for maximal YY1 repression, although mutations of proteins in the PRC1 complex cause more dramatic loss of YY1 repression (Atchison, 2003).

Most biochemical studies have not revealed a physical association of YY1 with the known PcG complexes, although substoichiometric levels are observed in human Pc complexes, and some associations have been documented for Drosophila Pho. The transient nature of the Drosophila associations suggest that an intermediary protein exists. This study demonstrates genetic and physical associations between YY1 and CtBP, which link YY1 to PcG function and provide a mechanism for the recruitment of vertebrate PcG complexes to DNA. Since CtBP is able to homodimerize, it may interact with Pc by one dimer partner and with YY1 by the other dimer partner. These interactions could define the mechanism by which YY1 functions to repress transcription in both a PcG- and CtBP-dependent fashion. In addition, the CtBP and Pc experiments indicate that CtBP plays a more direct role in PcG repression. Thus, CtBP may perform more than one function in the repression mechanism (Atchison, 2003).

The PcG function of YY1 that was identified in this study extends a list of YY1 functions including transcriptional activation and repression via apparently non-PcG pathways. YY1 binds to numerous promoters and can mediate repression by a variety of mechanisms including binding site competition, DNA bending and interference with activator interactions with the basal transcription machinery. YY1 repression can be influenced by interactions with proteins such as adenoviral E1A and the co-activator p300. YY1 can also interact with histone deacetylase proteins and is speculated to play a role in chromatin remodeling. Thus, the PcG function of YY1 identified in this study may be one of numerous functions mediated by this complex transcription factor. It may not be surprising that YY1 carries out multiple functions, because diverse functions of other PcG proteins are now being elucidated. For example, the PcG proteins Bmi-1 and Mel-18 play roles in controlling the cell cycle and their mutation leads to proliferative defects that impact the hematopoietic system. Therefore, PcG proteins play roles in multiple processes in addition to body axis formation (Atchison, 2003).

Stable transcriptional repression by YY1 is observed, but it was also found that YY1 appears to repress expression of a previously active gene. Generally, PcG proteins are believed to be maintenance repressors that do not initiate de novo repression. However, YY1 has the feature that it can repress de novo and may be able to repress transcription by multiple mechanisms that include PcG-dependent and -independent mechanisms. This is in agreement with the multiple YY1 repression mechanisms that have already been identified (Atchison, 2003).

The peri-implantation lethal phenotype of YY1 knock-out mice is similar to the phenotype of eed^–/– mice. In contrast, pho mutant Drosophila show a phenotype much later in development, potentially indicating some differences between YY1 and Pho. Phenotypic rescue experiments demonstrate considerable functional similarity between these proteins, but 75% of vertebrate YY1 and Drosophila Pho protein sequences contain no discernable homology, suggesting some distinct functions. Pho appears insufficient for repression at early embryonic stages in Drosophila, since a LexA-Pho chimeric protein is incapable of repressing transcription of a LexA-Ubx-LacZ reporter, and a GAL-Pho chimeric protein is incapable of repressing the BGUZ construct. Thus, unlike YY1, Pho does not repress transcription in early embryos. However, Pho is necessary for repression at later stages of development, since mutating Pho binding sites in the Ubx PRE results in loss of silencing in wing imaginal discs. YY1 can clearly repress transcription at both early embryonic stages, as well as at later larval stages in wing imaginal discs. The early function of YY1 is consistent with its early lethal phenotype in YY1 mutant mice. This repression indicates that YY1 can mediate embryonic functions lacking in the Pho protein. Specifically, the association of YY1 with CtBP may provide a bridging function not mediated by Pho. Most proteins that bind to CtBP contain a canonical PXDLS motif. While YY1 contains a similar sequence, this motif is absent from Pho (Atchison, 2003).

The precise role of CtBP in PcG repression is unclear. CtBP mutants in flies show segmentation defects, but homeotic derepression has not been observed. Similarly, mouse ctbp1 and ctbp2 null mutants show a variety of defects including skeletal abnormalities, but these defects do not precisely match the skeletal posterior transformations seen with mammalian PcG mutants. It is quite possible that YY1 and CtBP are necessary for a subset of PcG functions. Similarly, it has been proposed that multiple distinct PcG complexes exist to regulate distinct genes. An additional potential link between YY1 and the PcG complex is the protein RYBP. Similar to CtBP, RYBP can physically interact with both YY1 and PcG proteins. The absence of a corresponding mutant in Drosophila precluded testing of the necessity of RYBP for YY1 repression (Atchison, 2003).

The demonstration that YY1 functions as a PcG protein predicts that vertebrate PREs should contain YY1 binding sites. YY1/Pho binding sites (CGCCATNTT) are indeed present within many Drosophila PRE sequences, and are required for function. Since the YY1 binding motif is well characterized, these results should facilitate the identification of vertebrate PRE regions, which thus far have proved elusive. The experiments linking YY1 to PcG function reveal mechanistic features of YY1-mediated transcriptional repression, with implications for PcG activity in mammals. It will be very interesting in the future to determine whether YY1 heterozygous mice augment mutant phenotypes in PcG mutant heterozyotes (Atchison, 2003).

General transcriptional silencing by a Polycomb response element in Drosophila

Polycomb response elements (PREs) are cis-regulatory sequences required for Polycomb repression of Hox genes in Drosophila. PREs function as potent silencers in the context of Hox reporter genes and they have been shown to partially repress a linked miniwhite reporter gene. The silencing capacity of PREs has not been systematically tested and, therefore, it has remained unclear whether only specific enhancers and promoters can respond to Polycomb silencing. Using a reporter gene assay in imaginal discs, it has been shown that a PRE from the Drosophila Hox gene Ultrabithorax potently silences different heterologous enhancers and promoters that are normally not subject to Polycomb repression. Silencing of these reporter genes is abolished in PcG mutants and excision of the PRE from the reporter gene during development results in loss of silencing within one cell generation. Together, these results suggest that PREs function as general silencer elements through which PcG proteins mediate transcriptional repression (Sengupta, 2004).

A 1.6 kb fragment encompassing the PRE from the Ubx upstream control region was tested for its capacity to prevent transcriptional activation by enhancers from genes that are normally not under PcG control. For this purpose, three different enhancers were tested in a lacZ reporter gene assay in imaginal discs: dpp^WE, the imaginal disc enhancer from the decapentaplegic (dpp) gene; vg^QE the quadrant enhancer from the vestigial (vg) gene; and vg^BE, the vg D/V boundary enhancer. If linked to a reporter gene, each of these enhancers directs a distinct pattern of expression in the wing imaginal disc and activation by each enhancer is regulated by transcription factors that are controlled by a different signaling pathway. Specifically, the dpp enhancer contains binding sites for the Ci protein and is activated in response to hedgehog signaling, the vg quadrant enhancer contains binding sites for the Mad transcriptional regulator and is activated in response to dpp signaling, and the vg boundary enhancer contains binding sites for the Su(H) transcription factor and is regulated by Notch signaling. The dpp^WE, vg^QE and vg^BE enhancers were individually inserted into a lacZ reporter gene construct that contained the PRE fragment and either a TATA box minimal promoter from the hsp70 gene (here referred to as TATA), or a 4.1 kb fragment of the proximal Ubx promoter (here referred to as Ubx_P), fused to lacZ. In each construct, the PRE fragment was flanked by FRT sites that permit excision of the PRE fragment by flp recombinase. Several independent transgenic lines for each of the six PRE transgenes were generated. From individual transgene insertions, derivative transgenic lines were then generated by flp-mediated excision of the PRE in the germline. Thus expression of individual transgene insertions could be compared in the presence and absence of the PRE by staining wing imaginal discs for ß-galactosidase (ß-gal) activity. In the absence of the PRE, each of the three enhancers tested directs ß-gal expression in a characteristic previously characterized pattern. Each enhancer activated expression in the same pattern from either the TATA box minimal promoter or the Ubx promoter with some minor, promoter-specific differences with respect to the expression levels. By contrast, in most of the parental transformant lines, i.e., those carrying the corresponding reporter gene with the PRE, ß-gal expression is completely suppressed. These observations suggest that the PRE fragment very potently silences each of the six reporter genes. It is noted, however, that, at some transgene insertion sites, efficiency of silencing by the PRE fragment appeared to be impeded by flanking chromosomal sequences; in these cases, it was found that ß-gal expression is activated even in the presence of the PRE (Sengupta, 2004).

To test whether silencing of the reporter genes by the PRE depends on PcG gene function, the PRE-containing transgenes >PRE>dpp^WE-TATA-lacZ and >PRE>vg^QE-Ubx-lacZ were introduced into larvae that carried mutations in the PcG gene Su(z)12Suppressor of zeste 12 [Su(z)12]. Su(z)12 encodes a core component of the Esc-E(z) histone methyltransferase. Silencing of both transgenes is lost in Su(z)12²/Su(z)12³ mutant larvae, and the transgenes express ß-gal expression at levels comparable with the transgene derivatives that lack the PRE fragment. Taken together, these observations suggest that the 1.6 kb PRE fragment from Ubx is a very potent general transcriptional silencer element that represses transcription in a PcG protein-dependent manner. Thus, it appears that this PRE acts indiscriminately to block transcriptional activation by a variety of different activator proteins (Sengupta, 2004),

To test the long-term requirement for the PRE for silencing of these reporter genes, the PRE was excised during larval development and ß-gal expression was then monitored at different time points after excision. Forty-eight hours after induction of flp expression, all six reporter genes showed robust derepression of ß-gal, suggesting that, in each case, removal of the PRE results in the loss of PcG silencing. Among the different enhancer-promoter combinations used in this study, the dpp^W enhancer fused to the TATA box minimal promoter appears to direct the highest levels of lacZ expression; >PRE>dpp^W–TZ transformant lines consistently show the strongest ß-gal staining after excision of the PRE. Therefore >PRE>dpp^W-TZ transformants were analyzed at 4, 8, 12 and 24 hours after induction of flp expression to study the kinetics of this derepression. No ß-gal signal was detected at 4 hours or even at 8 hours after flp induction, but 12 hours after flp induction, all discs showed robust ß-gal expression. Thus, even in the case of the most potent enhancer-promoter combination used (i.e. dpp^W enhancer and TATA box minimal promoter), a delay of 12 hours between flp induction and ß-gal expression was observed. Since the average cell cycle length of imaginal disc cells in third instar larvae is 12 hours, this implies that most disc cells have undergone a full division cycle within this period. Derepression of the reporter gene in this experiment requires several steps: (1) excision of the PRE by the flp recombinase; (2) dissociation of the PRE and PcG proteins attached to it -- possibly by disrupting PcG protein complexes formed between the PRE and factors bound at the promoter, and (3) transcriptional activation by factors binding to the enhancer in the construct. It is possible that one or several steps in this process require a specific process during the cell cycle (e.g., passage through S phase) (Sengupta, 2004),

These experiments here show that three reporter genes, each containing a different enhancer linked to a canonical TATA box promoter, are completely silenced by a PRE placed upstream of the enhancer. The data suggest that PcG proteins that act through this PRE prevent indiscriminately activation by a variety of different transcription factors. The PcG machinery thus does not seem to require any specific enhancer and/or promoter sequences for repression (Sengupta, 2004),

Two points deserve to be discussed in more detail. The first concerns the stability of silencing imposed by a PRE. Previous studies have suggested that transcriptional activation in the early embryo could prevent the establishment of PcG silencing by PREs. More specifically, early transcriptional activation of Hox genes by blastoderm enhancers may play an important role in preventing the establishment of permanent PcG silencing in segment primordia in which Hox genes need to be expressed at later developmental stages. Importantly, none of the three enhancers used in this study is active in the early embryo. Moreover, these enhancers probably do not contain binding sites for specific transcriptional repressors, such as the gap repressors, which are required for establishment of PcG silencing at some PREs in the early embryo. It is therefore imagined that, in these constructs, PcG silencing complexes assemble by default on the 1.6 kb Ubx PRE in the early embryo and that PcG silencing is thus firmly established by the stage when the imaginal discs enhancers would become active. Silencing by the PRE during larval stages therefore appears to be dominant overactivation and cannot be overcome by any of the enhancers used in this study. There is other evidence in support of the idea that PcG silencing during larval development is more stable than in embryos. In particular, a PRE reporter gene that contains a Gal4-inducible promoter is only transiently activated if a pulse of the transcriptional activator Gal4 is supplied during larval development; by contrast, a pulse of Gal4 during embryogenesis switches the PRE into an 'active mode' that supports transcriptional activation throughout development. Furthermore, recent studies in imaginal discs suggest that there is a distinction between transcriptional repression and the inheritance of the silenced state; the silenced state can be propagated for some period even if repression is lost. Specifically, loss of Hox gene silencing after removal of PcG proteins in proliferating cells can be reversed if the depleted PcG protein is resupplied within a few cell generations. Taken together, it thus appears that PcG silencing during postembryonic development is a remarkably stable process. Finally, the results reported in this study also imply that, once PcG silencing is established, Hox genes can `make use of virtually any type of transcriptional activator to maintain their expression; PcG silencing will ensure that activation by these factors only occurs in cells in which the Hox gene should be active. The analysis of Ubx control sequences supports this view; if individually linked to a reporter gene, most late-acting enhancers direct expression both within as well as outside of the normal Ubx expression domain (Sengupta, 2004),

The second point to discuss concerns the repression mechanism used by PcG proteins. Biochemical purification of PRC1 has revealed that several TFIID components co-purify with the PcG proteins that constitute the core of PRC1. Moreover, formaldehyde crosslinking experiments in tissue culture cells showed that TFIID components are associated with promoters, even if these are repressed by PcG proteins. This suggests that PcG protein complexes anchored at the PRE interact with general transcription factors bound at the promoter. One possibility would be that PcG repressors directly target components of the general transcription machinery to prevent transcriptional activation by enhancer-binding factors. Three distinct activators act through the three enhancers used in this study and, according to these results, none of them is able to overcome the block imposed by the PcG machinery. But how do the known activities of PcG protein complexes [i.e., histone methylation by the Esc-E(z) complex and inhibition of chromatin remodeling by PRC1] fit into this scenario? Both these activities may be required for the repression process by altering the structure of chromatin around the transcription start site and thus prevent the formation of productive RNA Pol II complexes. Other scenarios are possible. For example, histone methylation may primarily serve to mark the chromatin for binding of PRC1 through Pc, and PRC1 components such as Psc then perform the actual repression process. Whatever the exact repression mechanism may be, the PRE-excision experiment shows that this repression is lost within one cell generation after removal of the PRE. This implies that changes in the chromatin generated by the action of PcG proteins cannot be propagated by the flanking chromatin (Sengupta, 2004),

YY1 DNA binding and PcG recruitment requires CtBP

Mammalian Polycomb group (PcG) protein YY1 can bind to Polycomb response elements in Drosophila embryos and can recruit other PcG proteins to DNA. PcG recruitment results in deacetylation and methylation of histone H3. In a CtBP mutant background, recruitment of PcG proteins and concomitant histone modifications do not occur. Surprisingly, YY1 DNA binding in vivo is also ablated. CtBP mutation does not result in YY1 degradation or transport from the nucleus, suggesting a mechanism whereby YY1 DNA binding ability is masked. These results reveal a new role for CtBP in controlling YY1 DNA binding and recruitment of PcG proteins to DNA (Srinivasan, 2004).

To determine whether YY1 can recruit PcG proteins to DNA, chromatin immunoprecipitation (ChIP) assays were performed in a transgenic Drosophila embryo system consisting of hsp70-driven GALYY1 and a reporter construct containing the LacZ gene under control of the Ultrabithorax (Ubx) BXD enhancer and the Ubx promoter adjacent to GAL4-binding sites (BGUZ). The BGUZ reporter is expressed ubiquitously during embryogenesis but is selectively repressed in a PcG-dependent manner by GALYY1 and GALPc. Embryos were either left untreated or heat shocked to induce GALYY1 expression. After immunoprecipitation with various antibodies, the region surrounding the GAL4-binding sites in the BGUZ reporter was detected by PCR. Prior to heat shock, no GALYY1 could be observed at the reporter gene. After heat shock, GALYY1 binding to the reporter gene was easily detected. Interestingly, concomitant with GALYY1 binding, there was an increase in binding of the Polycomb (Pc) and Polyhomeiotic (Ph) proteins. Thus, YY1 DNA binding results in PcG recruitment to DNA (Srinivasan, 2004).

Binding of PcG proteins to PRE sequences is known to cause deacetylation of histone H3 and methylation on Lys 9 and Lys 27. Interestingly, induction of GALYY1 binding to the reporter gene resulted in loss of histone H3 acetylation on K9 and K14. Simultaneously, there was a gain of methylation on histone H3 Lys 9 and Lys 27. Therefore, YY1 binding to the BGUZ reporter results in the recruitment of PcG proteins to DNA and subsequent post-translational modifications of histones characteristic of PcG complexes (Srinivasan, 2004).

The presence of PcG proteins and the status of histone H3 modifications at the Ubx promoter region, which is 4 kb downstream of the GALYY1-binding site, were determined. To avoid amplification of the endogenous Ubx promoter, immunoprecipitated samples were amplified with primers spanning the Ubx-LacZ boundary. Interestingly, the presence of Pc and Ph was detected at the promoter after GALYY1 induction. The presence of GALYY1 at this site was also detected. The GAL4 protein alone does not bind to the Ubx promoter region, indicating specificity for YY1 sequences. The induced GAL4 protein was functional, however, because it efficiently bound to the GAL4-binding site in the BGUZ reporter. Binding by GALYY1 could, therefore, be due to either cryptic YY1-binding sites present at the promoter, physical association of GALYY1 with other proteins bound at the promoter, or interactions via looping of DNA between the GAL4-binding sites and the Ubx promoter. Again, induction of GALYY1 resulted in loss of acetylation of H3K9 and H3K14 and simultaneous gain of methylation on H3K9 and H3K27. These results are consistent with studies that have reported spreading of PcG proteins and histone modifications to flanking DNA (Srinivasan, 2004).

PHO and YY1 bind to the same DNA sequence, and PHO-binding sites have been identified in multiple PREs. Therefore, it was reasoned that YY1 would bind to endogenous PREs and perhaps increase recruitment of PcG proteins. For this, the major Ubx PRE (PRE_D), that contains multiple PHO-binding sites located in the bxd region, was examined. As expected, upon GALYY1 induction, GALYY1 was detected at this endogenous PRE site. In addition, YY1 binding was accompanied by an increase in Pc and Ph signals when compared with no heat shock controls and a loss of H3 K9 and H3 K14 acetylation and gain of H3 K9 and H3 K27 methylation. Thus, YY1 can bind to an endogenous PRE and can augment PcG recruitment (Srinivasan, 2004).

These results clearly indicated that YY1 DNA binding results in recruitment of PcG proteins, histone deacetylases (HDACs), and histone methyltransferases (HMTases) to DNA. To determine whether the Drosophila E(z) protein (which possesses HMTase activity) was involved, whether YY1 transcriptional repression was lost in an E(z) mutant background was examined. The results are consistent with the observation that E(z) specifically methylates histone H3 on Lys 27, which creates a binding site for the chromodomain of Pc. Thus, the repression observed with GALYY1 requires function of the E(z) PcG protein (Srinivasan, 2004).

It has been shown that YY1 interacts with Drosophila CtBP, a well-characterized corepressor molecule. CtBP can also interact with Pc in vivo. These associations led to a proposal that CtBP might play a bridging function between YY1 and PcG proteins. If true, one would expect loss of PcG recruitment to DNA in a CtBP mutant background. Indeed, ChIP experiments in a CtBP⁰³⁴⁶³/+ background showed greatly reduced Pc and Ph recruitment to the BGUZ reporter. In addition, histone H3 remained acetylated and unmethylated. Surprisingly, in a CtBP mutant background, a dramatic loss of GALYY1 DNA binding was observed. However, full-length GAL4 protein was able to bind to DNA equally well in wild-type and CtBP mutant backgrounds, indicating that the effect of CtBP mutation was specific for YY1. This is a very unexpected result because CtBP has never been demonstrated to control DNA binding of another protein. The absence of GALYY1 and PcG proteins bound to the BGUZ reporter in the CtBP mutant background suggested that expression of the LacZ gene should be increased. Indeed, LacZ expression was increased in CtBP mutant as compared with wild-type embryos. Thus, in a CtBP mutant background, GALYY1 does not bind DNA, PcG proteins are not recruited, histones remain acetylated and unmethylated, and transcription is derepressed (Srinivasan, 2004).

To be certain that this effect is not peculiar to the BGUZ reporter, the effect of CtBP mutation on GALYY1 and PcG binding at endogenous PREs was examined. For this, the Ubx PRE_D, engrailed (en) PRE, and sex combs reduced (scr) PRE were chosen. Strikingly, GALYY1 and Pc binding to all three PREs was greatly reduced in the CtBP mutant background. Reduction in GALYY1 and Pc DNA binding correlated with H3 K9 acetylation at the PRE_D and En PREs. In contrast, H3 K9 acetylation at the Scr PRE was lost in a CtBP mutant background. These results clearly indicate an essential role for Drosophila CtBP in PcG recruitment to DNA (Srinivasan, 2004).

Collectively, these studies clearly demonstrate PcG recruitment function by the multifunctional transcription factor YY1. This establishes YY1 DNA binding as a key mechanism for targeting PcG proteins to DNA. The loss of YY1 DNA binding and concomitant loss of PcG recruitment to reporters and endogenous PRE sequences in CtBP mutants underscores this mechanism. A model of YY1 and CtBP function is presented. It is proposed that in a CtBP mutant background, YY1 is sequestered by a protein that inhibits its ability to bind to DNA. In a CtBP wild-type background, YY1 is released from this protein, thus enabling it to bind to DNA. DNA binding by YY1 results in recruitment of PcG complexes that cause deacetylation of histones and methylation of histone H3 at Lys 9 and Lys 27. Deacetylation may also be mediated by HDACs directly recruited by interaction with YY1 (Srinivasan, 2004).

The ablation of YY1 DNA binding in a CtBP mutant background was totally unexpected. This represents a new mechanism for controlling YY1 DNA binding and PcG recruitment. The mechanism appears to be exquisitely sensitive to CtBP dose because YY1 DNA binding and PcG recruitment are greatly reduced in heterozygous mutant backgrounds. Heterozygous effects by CtBP on knirps and hairy mutant phenotypes have been observed in other systems, suggesting that CtBP levels are limiting in vivo (Srinivasan, 2004).

The exact role of CtBP in PcG-mediated repression is yet to be elucidated. The results suggest that CtBP is required for the function of a large subset of PREs that require YY1/PHO for PcG recruitment. Like PcG mutants, CtBP mutants in flies show segmentation defects, but homeotic derepression has not been observed. Heterozygous ctbp mutants can reverse pair-rule phenotypes observed in hairy mutants, and homozygotes show bristle and cuticle defects. Furthermore, embryos that are trans-heterozygous for wimp and the ctpb⁰³⁴⁶³ allele die and their cuticle preparations show severe segmentation defects. Similarly, mouse ctbp1 and ctbp2 null mutants show a variety of defects including skeletal abnormalities, but these defects do not precisely match the skeletal posterior transformations seen with mammalian PcG mutants. Based on the multiple PREs affected by CtBP mutation, it is unclear why a more severe CtBP heterozygous mutant phenotype is not observed. Perhaps a low level of PcG binding to DNA remains that is below detection in immunostains of polytene chromosomes, but which is sufficient to mediate biological effects. In support of this possibility, polytene spreads were occasionally observed that stained with Pc antibodies nearly as well as wild-type spreads. This suggests a possible threshold effect for CtBP involvement in PcG recruitment. ChIP studies on many more PRE sequences will be needed to clarify this issue (Srinivasan, 2004).

These results show that modulation of YY1 DNA binding by CtBP is a critical step in the recruitment of PcG proteins to DNA. This mechanism might be differentially used during development to control PcG assembly on PREs. The demonstration of recruitment of PcG proteins by YY1 should assist in the identification of mammalian PREs since the YY1 recognition sequence is well characterized (Srinivasan, 2004).

Synergistic recognition of an epigenetic DNA element by Pleiohomeotic and a Polycomb core complex

Polycomb response elements (PREs) are cis-acting DNA elements that mediate epigenetic gene silencing by Polycomb group (PcG) proteins. Pleiohomeotic (Pho) and a multiprotein Polycomb core complex (PCC) bind highly cooperatively to PREs. A conserved sequence motif, named PCC-binding element (PBE), has been identified that is required for PcG silencing in vivo. Pho sites and PBEs function as an integrated DNA platform for the synergistic assembly of a repressive Pho/PCC complex. This nucleoprotein complex is termed the silenceosome to reflect that the molecular principles underpinning its assemblage are surprisingly similar to those that make an enhanceosome (Mohd-Sarip, 2005 ).

Because Pho can directly bind two subunits of the PCC complex, Pc and PH (Mohd-Sarip, 2002), it was of interest to test whether Pho could recruit PCC (PRC1 core complex comprising Pc, Ph, Psc, and dRING1) to DNA. As representative PREs the bxd PRE, located, ~25 kb upstream of the Ubx transcription start site, and the iab-7 PRE, located ~60 kb downstream of the Abd-B promoter, were used. For initial binding studies, focus was placed on Pho sites 4 and 5 within the bxd PRE (Pho4/5-PRE), which are required for PcG silencing in vivo. Pho, Pho lacking the 22-amino acid Pc- and PH-binding domain (DeltaPBD), Pc, and PCC were expressed in Sf9 cells using the baculovirus expression system and were immunopurified to near homogeneity from cell extracts (Mohd-Sarip, 2005).

To test DNA binding by Pho and PCC, DNA mobility shift assays were performed. Whereas Pho alone binds weakly to the Pho4/5-PRE, together with PCC, a Pho/PCC/DNA complex was forms very efficiently, resulting in complete saturation of the probe. In contrast, PCC alone is unable to bind DNA sequence-specifically. Deletion of the PBD of Pho impairs the synergistic formation of a higher-order Pho/PCC/DNA complex, revealing the importance of direct protein-protein interactions between Pho and PCC (Mohd-Sarip, 2005).

To identify the DNA sequences contacted by the Pho/PCC complex, primer extension DNaseI footprinting assays were carried out. After addition of PCC to a subsaturating amount of Pho, which by itself does not yield a footprint, DNA binding is readily detected. The Pho/PCC footprinted area is very large, comprising ~120 bp, indicative of extensive protein-DNA contacts. As expected, PCC alone is unable to bind DNA sequence-specifically. In contrast to Pho/PCC, a saturating amount of Pho generates a small footprinted area of ~40 bp, encompassing the two Pho sites. Next, tests were performed to see whether the cooperation between Pho and PCC also occurred on chromatin templates. The Drosophila embryo-derived S190 assembly system was used to package the template into a nucleosomal array. Pho alone failed to bind its chromatinized sites. However, DNA binding was greatly facilitated by the addition of PCC, which by itself is unable to target the PRE sequence. It is noted that no Pho binding to chromatin was detected even at the highest amounts add. Thus, Pho binding to chromatin appears dependent upon PCC. Because nucleosomes are not positioned on these templates, the DNaseI digestion ladder resembles that of naked DNA. Chromatin footprinting requires the use of high amounts of DNaseI, which completely digests any residual naked DNA in the reaction (Mohd-Sarip, 2005).

To identify specific PCC subunits that directly contact the DNA, a DNA cross-linking strategy was used. A radiolabeled Pho4/5-PRE fragment substituted with bromodeoxyuridine (BrdU) was generated. After binding of Pho and PCC, the resulting protein-DNA complexes were subjected to ultraviolet (UV) cross-linking. SDS-PAGE analysis, followed by autoradiography, revealed very strong labeling of Pho and Pc and weaker labeling of Psc or Ph. The cross-linked PSC and PH could not be resolved well. Because on low percentage gels PSC and PH form a radiolabeled doublet, it is assumed that both proteins bind DNA. No labeling of dRING1 was detected, suggesting that it does not directly contact DNA. Because Pc was strongly cross-linked to DNA and can directly bind Pho (Mohd-Sarip, 2002), tests were performed to see whether Pc can bind DNA together with Pho. After addition of Pc to a subsaturating amount of Pho, DNA binding was readily detected. Pc alone is unable to bind DNA sequence-specifically. Also when Pc was added to a saturating amount of Pho, the footprinting pattern changed and was extended, suggesting additional protein-DNA contacts. Although Pc can cooperate with Pho, the level of cooperation and DNA area contacted is modest compared with Pho-PCC, emphasizing the contribution of other PCC subunits (Mohd-Sarip, 2005).

What are the precise DNA sequence requirements for cooperative PRE binding by Pho and PCC? Within many PREs, the Pho core recognition sequence forms part of a larger conserved motif (Mihaly, 1998). To determine the functional significance of these sequence constraints, the effect of mutations on Pho binding by DNase were examined by footprinting and bandshift analysis. Whereas the downstream motif (D.mt) has no effect on Pho binding, mutation of the upstream motif (U.mt) reduced Pho affinity. As expected, mutation of the core Pho site (C.mt) abrogates Pho binding. These results suggested that the sequence constraints directly upstream of the Pho core site reflect an extension of the Pho recognition site. The sequence downstream of the Pho site, however, appeared to play no role in Pho binding. Therefore, an attractive possibility was that this motif might mediate docking of PCC and function as a PCC-binding element (PBE). To determine whether synergistic Pho/PCC complex assembly is dependent on each Pho site or the downstream sequence motifs, each Pho site and putative PBEs was mutated individually. Strikingly, each mutation aborted formation of the Pho-PCC-DNA complex. Likewise, synergistic binding of Pho and Pc was also abrogated by PBE mutations. It is concluded that cooperative DNA binding of Pho and PCC is strictly dependent on the presence of at least two Pho sites and their juxtaposed PBEs (Mohd-Sarip, 2005).

The conservation of the PBE (Mihaly, 1998) and its requirement for cooperative DNA binding by Pho and PCC led to a test if it is also critical for PRE-directed silencing in vivo. The minimal 260-bp iab-7 PRE, for which an extensive collection of control lines has already been established, was examined. The iab-7 PRE harbors three Pho/PBE elements, but their spacing and phasing is very different from that in the bxd PRE. Whether Pho and PCC bind cooperatively to the iab-7 PRE was tested. In agreement with the results on the bxd PRE, Pho and PCC synergistically recognized the iab-7 PRE, resulting in a very large DNaseI footprint, including all three Pho and PBE elements. Cooperative binding of Pho and PCC was completely abolished by mutations in the three PBEs juxtaposing the Pho sites. Thus, the PBEs are required for Pho/PCC complex formation on both the bxd and the iab-7 PRE (Mohd-Sarip, 2005). Next, the effects of PBE mutations on in vivo silencing were tested. Because the site of integration within the genome influences silencing, repression does not occur in all transgenic lines. Therefore, PSS is expressed as the percentage of lines that show repression. Independent lines were establised harboring the mini-white transgene under control of either the minimal 260-bp iab-7 PRE or the PBE mutant PRE (PBE^mt iab-7). 48 homozygous viable lines were raised with the wild-type PRE in front of the mini-white gene. In 46% of these lines, homozygotes (P[w⁺]/P[w⁺]) have much lighter eyes than their heterozygous (P[w⁺]/+) siblings, revealing PSS. In 8% of the lines, the eye color of homozygotes is about the same as that of heterozygotes, reflecting weak PRE-directed silencing. In the remaining 46% of the lines, no PSS was observed and the eyes of homozygotes were darker than that of heterozygotes. In summary, recruitment of a PcG repressing complex is observed in more than half of the generated lines. Strikingly, when the PBEs were mutated, only one line (4.5%) out of a total of 22 analyzed showed strong repression of the mini-white gene in homozygotes, and for five lines (23%), homozygotes had an eye color similar to that of their heterozygous siblings. It is worthwhile noting that in the case of the wild-type iab-7 PRE, the majority of repressed lines showed strong repression (22 of 26). In contrast, the majority of the repressed lines (five of six) harboring the mutant PRE display only weak silencing. Thus, not only is the proportion of repressed lines decreased in the mutant iab-7 PRE lines but the efficiency of repression is also lowered. These results strongly support the notion that the PBE is a critical PRE element, required for the assembly of a functional repressive PcG complex in vivo (Mohd-Sarip, 2005).

A central problem in understanding epigenetic gene regulation is how specialized DNA elements recruit silencing complexes to a linked gene. This study has identified the PBE, a small conserved sequence element required for PcG silencing in vivo. These results suggest that Pho sites and their juxtaposed PBEs function as an integrated DNA platform for the assembly of a repressive Pho/PCC complex. In a previous study, the failure of Pho sequences fused to a heterologous DNA-binding domain to nucleate the assembly of a silencing complex was interpreted as an argument against its role as a tether of other PcG proteins. However, in light of the critical role of the PBE in PcG silencing, it is not to be expected that artificially tethered Pho can support PcG complex assembly (Mohd-Sarip, 2005).

Synergistic Pho/PCC/PRE nucleocomplex formation is strictly dependent on the presence of at least two Pho sites, their accompanying PBEs and protein-protein interactions between Pho and PCC. The observations revealed a striking similarity in the design of PREs and enhancers. The cooperative assembly of unique transcription factor-enhancer complexes, termed enhanceosomes, is also dependent upon a stereospecific arrangement of binding sites and a reciprocal network of protein-protein interactions. Thus, the basic principles governing the assembly of distinct higher-order nucleoprotein assemblages with opposing activities are surprisingly similar. To reflect the generality of these rules, it is proposed that PRE-bound PcG silencing complexes be called silenceosomes (Mohd-Sarip, 2005).

Like enhancers, PREs are complex and their activity involves the combined activity of distinct recognition elements and their cognate factors. In addition to Pho/PBE sites, these modules include the (GA)n-element, recognized by GAGA or Pipsqueak; Zeste sites; and the recently identified GAAA motif bound by DSP1, a fly HMGB2 homolog (Dejardin, 2005). Finally, histone modifications, including H2A and H2B (de)ubiquitylation, and H3-K27 or H3-K9 methylation, play a critical role in PRE functioning. One scenario is that silenceosome formation is nucleated by direct DNA binding and contextual protein-protein and protein-DNA interactions. Next, the silenceosome could be stabilized further through multivalent interactions with the histones guided by selective covalent modifications. The available evidence strongly suggests that a cooperative network of individually weak protein-DNA and protein-protein interactions drive the formation of a PcG silencing complex. It is proposed that the molecular principles governing silenceosome or enhanceosome formation are very similar (Mohd-Sarip, 2005).

Genome-wide analysis of Polycomb targets in Drosophila

Polycomb group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing PcG proteins regulate homeotic genes in flies and vertebrates, but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. This study determined the distribution of the PcG proteins PC, E(Z) and PSC and of trimethylation of histone H3 Lys27 (me3K27) in the Drosophila genome using chromatin immunoprecipitation (ChIP) coupled with analysis of immunoprecipitated DNA with a high-density genomic tiling microarray. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb response elements (PREs). In contrast, H3 me3K27 forms broad domains including the entire transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors, but they also include genes coding for receptors, signaling proteins, morphogens and regulators representing all major developmental pathways (Schwartz, 2006).

The components of PcG complexes are products of PcG genes, first discovered as crucial regulators of homeotic genes in Drosophila. Immunostaining of Drosophila polytene chromosomes, however, showed PcG proteins at about 100 cytological loci, implying a much larger number of target genes. Functional analysis has identified PREs as DNA sequences able to recruit PcG proteins and establish PcG silencing of neighboring genes. Two types of PcG complexes bind to PREs. PRC1-type complexes include a core quartet of proteins: PC, PSC, PH and dRing. PRC2-type complexes include E(Z), which methylates histone H3 Lys27. Mono- and dimethylated Lys27 is widely distributed in the genome, but PcG sites characteristically contain trimethylated Lys27 (me3K27). The activity of the E(Z) complex is essential for stable silencing, and it has been proposed that H3 me3K27 recruits the PRC1 complex through the specific affinity of the PC chromodomain for me3K27. But the relationships between PRC1 and PRC2 complexes, between their binding sites and histone methylation, and between binding, methylation and gene expression are not well understood and remain the subject of debate. The genomic distribution of three PcG proteins [PC, PSC and E(Z)] and of histone H3 me3K27 was examined using using chromatin immunoprecipitation (ChIP). Since PcG target genes may be repressed in some tissues and active in others, a cultured cell line was used to minimize heterogeneity (Schwartz, 2006).

Viewed at the scale of a chromosome arm, the distributions of PC, PSC, E(Z) and me3K27 coincide at a number of distinct binding peaks (which are referred to as 'PcG sites') that correspond to 70% of the bands reported in salivary gland polytene chromosomes stained with the corresponding antibodies. To minimize false positives, the analysis focussed on the PcG sites that showed simultaneous binding of two or more proteins, each above twofold enrichment. Of the 149 PcG sites detected (see the supplemental figure), 95 showed strong binding of all four proteins ('strong' PcG sites), whereas in 54 sites the binding was lower and below threshold for one of the proteins ('weak' PcG sites). At higher resolution, most PcG sites involve two or more genes, often sharing structural or functional similarities. Thus, PcG sites involve the following: engrailed (en) and invected (inv); the PcG genes ph-p and ph-d; the Dorsocross T-box gene cluster; the muscle NK homeobox gene cluster; the wingless cluster; and the two homeotic complexes ANT-C and BX-C (Schwartz, 2006).

The Bithorax complex (BX-C) is a cluster of three homeotic genes (Ubx, abd-A and Abd-B) responsible for segmental identity in the abdomen and posterior thorax. The most prominent features are two sharp binding peaks for all three PcG proteins at the sites of the bx and bxd PREs that control Ubx. No peak was detected over the Ubx proximal promoter, although the entire gene shows a low but significant level of PC. A series of lower peaks emerged in the abd-A region and part of the Abd-B gene. Some of these correspond to the known PREs iab-2. In contrast, the distribution of H3 me3K27 oscillated rapidly above a high plateau that covers Ubx and abd-A but not Abd-B. RT-PCR was used to determine the mRNA levels corresponding to these three genes. Transcription of Ubx and abd-A in these cells was very low but distinctly above background. Abd-B was highly transcribed, at levels 300 times higher than Ubx. This pattern of activity was reflected by the distribution of both PcG proteins and me3K27. It is noted that in the Abd-B regulatory region, the previously characterized Fab-7 and Fab-8 PREs neither bound PcG proteins nor were methylated in these cells. The Abd-B gene has five distinct promoters. A sharp resurgence of both methylation and PcG protein binding in the region of the most upstream Abd-B promoter suggests that, in contrast to the other four promoters, this one might be repressed in the cultured cells. RT-PCR analysis using primers specific for mRNAs initiating from each promoter confirmed that the most upstream promoter is silent and that the other four are active. These results support the view that binding of PcG proteins to PREs is associated with transcriptional quiescence, whereas robust transcriptional activity is accompanied by lack of binding to the PREs and lack of Lys27 methylation over the transcription unit (Schwartz, 2006).

Strong genomic sites bind all three PcG proteins. The PSC and E(Z) peaks generally rise sharply and are contained within less than 2 kb, whereas PC frequently forms a broader peak that may include shoulders or subsidiary peaks absent for E(Z) and PSC and subsides to background more gradually. These peak binding regions are thought of as corresponding to PREs, which they in fact do in the cases where these are known. Additional binding peaks may be found within or downstream of the transcription unit. In contrast, distribution of H3 me3K27 at each site is very broad, forming a domain of tens or even hundreds of kilobases encompassing the transcription unit and regulatory regions of one or more genes but, rather than a level plateau, it consists of a series of deep oscillations (Schwartz, 2006).

The strong binding peaks or putative PREs are often associated with low values or troughs in the methylation profile and at secondary peaks the PC distribution frequently echoes methylation peaks. Overall, their relationship does not support the idea that methylation of Lys27 suffices to recruit binding of PC. It is proposed instead that PC bound to the strong binding peaks, the presumptive PREs, is recruited by proteins that bind specifically to those sequences. The weaker PC binding peaks and tails that mirror the methylation profile near PREs may represent a second mode of PC binding mediated by the interaction of the chromodomain with H3 me3K27 (Schwartz, 2006).

It is supposed that methylation domains initiated by a PRE might spread bidirectionally until they encounter 'active' chromatin, characterized by histone acetylation or methylation of H3 Lys4, marks typical of transcriptionally active genes. Alternatively, specific features might shape the methylation domain either positively, by attracting the methyltransferase complex, or negatively, by blocking productive interactions with the PRE. As in the case of the Abd-B gene or of CG7922 and CG7956 genes, sudden drops in levels of me3K27 are generally associated with transcriptional activity. Are insulators involved in protecting CG7922 and CG7956 from silencing, or is the activity of these two genes simply epigenetically maintained from the time the cell line was originally established? Further work is required to answer this question (Schwartz, 2006).

In many cases, the presumptive PRE lies between divergently transcribed genes such as dco and Sox100B. Which of the two is the PRE target? As PREs can act at distances of 20-30 kb, the proximity of PcG peaks to a promoter is not a reliable guide. It is proposed that the methylation domain is the clue to the target of PcG regulation. A PcG peak is not considered to regulate a promoter if the gene is not included in the methylation domain. When multiple genes are included in the methylation domain, it is likely that they are all affected by PcG regulation. However, this study distinguishes between genes that contain methylation as well as one or more PcG proteins and genes that contain only methylation (Schwartz, 2006).

The 95 'strong' binding sites in the genome encompass a total of 392 genes. Of these 392 genes, 186 contain both PcG binding and methylation, and the remainder are found within broad methylation domains associated with PcG proteins binding but do not bind PcG proteins over their own promoter or transcription unit. They may represent genes not directly targeted but affected by the spread of methylation. An analysis of their ontology indicates that these two classes are in fact very different. Transcription regulators constitute 64.5% of the first set, compared to 4.3% for the full annotation set. Instead they constitute only 4.0% of those genes that contain only me3K27. These comparisons strongly suggest that (1) genes that regulate transcription are preferred PcG targets, and (2) genes that only include the tails of a methylation domain are probably not primary targets of PcG regulation. A similar preference is also seen among the 'weak' binding sites. These include a total of 74 genes containing both PcG proteins and methylation, 28.4% of which encode transcription regulators. Flanking genes containing only methylation include only 5.7% transcription regulators. Although transcription regulators are preferred PcG targets, secreted proteins, growth factors or their receptors, and signaling proteins are also targeted. PcG target genes include components of all the major differentiation and morphogenetic pathways in Drosophila (Schwartz, 2006).

The major features of PcG binding shown by this work are that, although the proteins themselves are highly localized at presumptive PREs, the domain of histone methylation they produce is much broader. If the E(Z) methyltransferase is localized at the PRE, how is the extensive methylation domain produced? A looping mechanism is proposed in which interaction of PRE-bound complexes with flanking chromatin is mediated by the PC chromodomain. The observed broader distribution of PC might result from crosslinking of the chromodomain to methylated H3, reflecting this mechanism (Schwartz, 2006).

Are PREs defined by characteristic sequence motifs? Although the analysis of the sequences underlying the binding peaks will be presented elsewhere, it is noted that Ringrose (2003) devised an algorithm based on GAGA factor, PHO and Zeste binding motifs to identify sequences likely to represent PREs. This algorithm correctly predicts a number of the strong PcG binding sites (27%) and a few of the weaker sites (7%), overall 20%; however, it does not predict the majority of the PcG sites. The reverse is also true: only 22% of the PREs predicted by Ringrose bind PcG proteins in these experiments. Together, these data suggest that additional criteria are necessary to predict most PREs reliably (Schwartz, 2006).

As expected, PcG proteins and me3K27 are associated with transcriptional quiescence, but the data suggest that this is not an absolute condition. Low but significant transcription levels are detected even for the repressed Ubx and abd-A genes. Two target sites, polyhomeotic and the Psc-Su(z)2 site, contain PcG genes, which must be active to ensure the functioning of the PcG mechanism. The polyhomeotic locus is one of two sites in the entire genome that bind PC but lack appreciable levels of E(Z) and of Lys27 methylation. Instead, the Psc-Su(z)2 region is well methylated and binds both PC and E(Z) at multiple peaks. It is concluded that PcG mechanisms do not invariably lead to transcriptional silencing and are compatible with moderate levels of transcription (Schwartz, 2006).

Another point of interest is the number and kind of genes that are PcG targets. Considering the developmental difference between salivary gland cells and the embryo-derived tissue culture cells, the substantial number of shared PcG sites suggests that a majority of target sites are occupied in a large percent of cells. Target genes are in fact predominantly regulatory genes that control major differentiation and morphogenetic pathways. These pathways and their genes are highly conserved, and recent work shows that they are also regulated by PcG in mammals. It might be expected that in a given cell type most alternative genomic programs would be repressed save the subset required in that cell type. The emerging picture from these studies is that PcG regulation is a key mechanism in genomic programming (Schwartz, 2006).

Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins

Polycomb group (PcG) and trithorax group (trxG) proteins act as antagonistic regulators to maintain transcriptional OFF and ON states of HOX and other target genes. To study the molecular basis of PcG/trxG control, the chromatin of the HOX gene Ultrabithorax (Ubx) was analyzed in Ubx^OFF and Ubx^ONcells purified from developing Drosophila. PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all constitutively bound to Polycomb response elements (PREs) in the OFF and ON state. In contrast, the trxG protein Ash1 is only bound in the ON state; not at PREs but downstream of the transcription start site. In the OFF state, extensive trimethylation was found at H3-K27, H3-K9, and H4-K20 across the entire Ubx gene; i.e., throughout the upstream control, promoter, and coding region. In the ON state, the upstream control region is also trimethylated at H3-K27, H3-K9, and H4-K20, but all three modifications are absent in the promoter and 5' coding region. These analyses of mutants that lack the PcG histone methyltransferase (HMTase) E(z) or the trxG HMTase Ash1 provide strong evidence that differential histone lysine trimethylation at the promoter and in the coding region confers transcriptional ON and OFF states of Ubx. In particular, these results suggest that PRE-tethered PcG protein complexes act over long distances to generate Pc-repressed chromatin that is trimethylated at H3-K27, H3-K9, and H4-K20, but that the trxG HMTase Ash1 selectively prevents this trimethylation in the promoter and coding region in the ON state (Papp, 2006; Full text of article).

Previous studies have shown that PhoRC contains the DNA-binding PcG protein Pho that targets the complex to PREs, and dSfmbt, a novel PcG protein that selectively binds to histone H3 and H4 tail peptides that are mono- or dimethylated at H3-K9 or H4-K20 (H3-K9me1/2 and H4-K20me1/2, respectively) (Klymenko, 2006). PRC1 contains the PcG proteins Ph, Psc, Sce/Ring, and Pc. PRC1 inhibits nucleosome remodeling and transcription in in-vitro assays and its subunit Pc specifically binds to trimethylated K27 in histone H3 (H3-K27me3). PRC2 contains the PcG proteins E(z), Su(z)12, and Esc as well as Nurf55, and this complex functions as a histone methyltransferase (HMTase) that specifically methylates K27 in histone H3 (H3-K27) in nucleosomes (Papp, 2006).

This study used quantitative X-ChIP analysis to examine the chromatin of the HOX gene Ubx in its ON and OFF state in developing Drosophila larvae. Previous genetic studies had established that all of the PcG and trxG proteins analyzed in this study are critically needed to maintain Ubx OFF and ON states in the very same imaginal disc cells in which their binding to Ubx was analyzed in this study. The following conclusions can be drawn from the analyses reported in this study. (1) The PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all highly localized at PREs, but they are all constitutively bound at comparable levels in the OFF and ON state. (2) The trxG protein Ash1 is bound only in the ON state, where it is specifically localized ~1 kb downstream of the transcription start site. (3) In the OFF state, PRC2 and other unknown HMTases trimethylate H3-K27, H3-K9, and H4-K20 over an extended 100-kb domain that spans the whole Ubx gene. (4) In the ON state, comparable H3-K27, H3-K9, and H4-K20 trimethylation is restricted to the upstream control regions and Ash1 selectively prevents this trimethylation in the promoter and coding region. (5) Repressed Ubx chromatin is extensively tri- but not di- or monomethylated at H3-K27, H3-K9, and H4-K20. (6) Trimethylation of H3-K27, H3-K9, and H4-K20 at imaginal disc enhancers in the upstream control region does not impair the function of these enhancers in the ON state. (7) TBP and Spt5 are bound at the Ubx transcription start site in the ON and OFF state, but Kis is only bound in the ON state. This suggests that in the OFF state, transcription is blocked at a late step of transcriptional initiation, prior to the transition to elongation. A schematic representation of PcG and trxG protein complex binding and histone methylation at the Ubx gene in the OFF and ON state is presented (Papp, 2006).

Unexpectedly, ChIP analysis by qPCR used in this study and in a similar study by the laboratory of Vincent Pirrotta (V. Pirrotta, pers. comm. to Papp, 2006) reveals that the relationship between PcG and trxG proteins and histone methylation is quite different from the currently held views. Specifically, X-ChIP studies have reported that H3-K27 trimethylation is localized at PREs and this led to the model that recruitment of PRC1 to PREs occurs through H3-K27me3 (i.e., via the Pc chromodomain). In contrast, the current study and that by Vincent Pirrotta found H3-K27 trimethylation to be present across the whole inactive Ubx gene, both in wing discs and in S2 cells (V. Pirrotta, pers. comm. to Papp, 2006). No specific enrichment of H3-K27 trimethylation at PREs has been detected; rather, a reduction of H3-K27me3 signals is observed at PREs, consistent with the reduced signals of H3 that are detected at these sites. Consistent with these results, genome-wide analyses of PcG protein binding and H3-K27me3 profiles in S2 cells revealed that, at most PcG-binding sites in the genome, PcG proteins are tightly localized, whereas H3-K27 trimethylation is typically present across an extended domain that often spans the whole coding region. How could the differences between this study and the earlier studies be explained? It should be noted that in contrast to the qPCR analysis used in the current study, previous studies all relied on nonquantitative end-point PCR after 36 or more cycles to assess the X-ChIP results. It is possible that these experimental differences account for the discrepancies (Papp, 2006).

PhoRC, PRC1, and PRC2 are all tightly localized at PREs but they are all constitutively bound at the inactive and active Ubx gene. This suggests that recruitment of PcG complexes to PREs occurs by default. Although all three complexes are bound at comparable levels to the bxd PRE in the inactive and active state and PhoRC is also bound at comparable levels at the bx PRE, it should be pointed out that the levels of PRC1 and PRC2 binding at the bx PRE are about twofold reduced in the active Ubx gene compared with the inactive Ubx gene. Even though there is still high-level binding of PRC1 and PRC2 at the bx PRE, it cannot be excluded that the observed reduction in binding helps to prevent default PcG repression of the active Ubx gene. It is possible that transcription through the bx PRE reduces PRC1 and PRC2 binding at this PRE. Transcription through PREs has been proposed to serve as an 'anti-silencing' mechanism that prevents default silencing of active genes by PREs (Papp, 2006),

The highly localized binding of all three PcG protein complexes at PREs, together with earlier studies on PRE targeting of PcG protein complexes supports the idea that not only PhoRC but also PRC1 and PRC2 are targeted to PRE DNA through interactions with Pho and/or other sequence-specific DNA-binding proteins. In the case of trxG proteins, the binding modes are more diverse. In particular, recruitment of Trx protein to PREs and to the promoter is also constitutive in both states but recruitment of Ash1 to the coding region is clearly observed only at the active Ubx gene. At present, it is not known how Trx or Ash1 are targeted to these sites. It is possible that a transcription-coupled process recruits Ash1 to the position 1 kb downstream of the transcription start site (Papp, 2006).

In contrast to the localized and constitutive binding of PcG protein complexes and the Trx protein, it was found that the patterns of histone trimethylation are very distinct in the active and inactive Ubx gene. The results also suggest that the locally bound PcG and trxG HMTases act across different distances to methylate chromatin. For example, H3-K4 trimethylation is confined to the first kilobase of the Ubx coding region where Ash1 and Trx are bound, whereas H3-K27 trimethylation is present across an extended 100-kb domain of chromatin that spans the whole Ubx gene. This suggests that PRE-tethered PRC2 is able to trimethylate H3-K27 in nucleosomes that are as far as 30 kb away from the bxd or bx PREs. Unexpectedly, it was found that the H3-K9me3 and H4-K20me3 profiles closely match the H3-K27me3 profile. At present it is not known which HMTases are responsible for H3-K9 and H4-K20 trimethylation, but analysis of E(z) mutants indicate that these modifications may be generated in a sequential manner, following H3-K27 trimethylation by PRC2. The molecular mechanisms that permit locally tethered HMTases such as PRE-bound PRC2 to maintain such extended chromatin stretches in a trimethylated state are only poorly understood. However, a recent study showed that the PhoRC subunit dSfmbt selectively binds to mono- and dimethylated H3-K9 and H4-K20 in peptide-binding assays (Klymenko. 2006). One possibility would be that dSfmbt participates in the process that ensures that repressed Ubx chromatin is trimethylated at H3-K27, H3-K9, and H4-K20. For example, dSfmbt, tethered to PREs by Pho, may interact with nucleosomes of lower methylated states (i.e., H3-K9me1/2 or H4-K20me1/2) in the flanking chromatin and thereby bring them into the vicinity of PRE-anchored HMTases that will hypermethylate them to the trimethylated state (Papp, 2006).

These analyses suggest that H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region is critical for Polycomb repression. (1) Although H3-K27, H3-K9, and H4-K20 trimethylation is present at the inactive and active Ubx gene, it is specifically depleted in the promoter and coding region in the active Ubx gene. (2) Misexpression of Ubx in wing discs with impaired E(z) activity correlates well with loss of H3-K27 and H3-K9 trimethylation at the promoter and 5' coding region. It is possible that the persisting H3-K27 and H3-K9 trimethylation in the 3' coding region is responsible for maintenance of repression in those E(z) mutant wing discs cells that do not show misexpression of Ubx. (3) In haltere and third-leg discs of ash1 mutants, the promoter and coding region become extensively trimethylated at H3-K27 and H3-K9, and this correlates with loss of Ubx expression. Previous studies showed that Ubx expression is restored in ash1 mutants cells that also lack E(z) function. Together, these findings therefore provide strong evidence that Ash1 is required to prevent PRC2 and other HMTases from trimethylating the promoter and coding region at H3-K27 and H3-K9. The loss of H3-K4 trimethylation in ash1 mutants is formally consistent with the idea that Ash1 exerts its antirepressor function by trimethylating H3-K4 in nucleosomes in the promoter and 5' coding region, but other explanations are possible (Papp, 2006).

But how might H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region repress transcription? The observation that TBP and Spt5 are also bound to the promoter in the OFF state suggests that these methylation marks do not prevent assembly of the basic transcription apparatus at the promoter. However, the nucleosome remodeling factor Kis is not recruited in the OFF state, and transcription thus appears to be blocked at a late step of transcriptional intiation prior to elongation. It was found that the low-level binding of Pc in the coding region correlates with the presence of H3-K27 trimethylation; i.e., Pc and H3-K27me3 are both present in the OFF state, but are absent in the ON state. One possible scenario would thus be that H3-K27 trimethylation in the promoter and coding region permits direct recruitment of PRC1. According to this view, locally recruited PRC1 would then repress transcription; e.g., by inhibiting nucleosome remodeling in the promoter region. However, several observations are not easily reconciled with such a simple 'recruitment-by-methylation' model. First, peak levels of all PRC1 components are present at PREs and, apart from Pc, very little binding is observed outside of PREs. Second, excision of PRE sequences from a PRE reporter gene during development leads to a rapid loss of silencing, suggesting that transcriptional repression requires the continuous presence of PREs and the proteins that are bound to them. A second, more plausible scenario would therefore be that DNA-binding factors first target PcG protein complexes to PREs, and that these PRE-tethered complexes then interact with trimethylated nucleosomes in the flanking chromatin in order to repress transcription. For example, it is possible that bridging interactions between the Pc chromodomain in PRE-tethered PRC1 and H3-K27me3-marked chromatin in the promoter or coding region permit other PRE-tethered PcG proteins to recognize the chromatin interval across which they should act, e.g., to inhibit nucleosome remodeling in the case of PRC1 or to trimethylate H3-K27 at hypomethylated nucleosomes in the case of PRC2 (Papp, 2006).

The analysis of a HOX gene in developing Drosophila suggests that histone trimethylation at H3-K27, H3-K9, and H4-K20 in the promoter and coding region plays a central role in generating and maintaining of a PcG-repressed state. Contrary to previous reports, the current findings provide no evidence that H3-K27 trimethylation is specifically localized at PREs and could thus recruit PRC1 to PREs; widespread H3-K27 trimethylation is found across the whole transcription unit. The data presented in this study provide evidence that PREs serve as assembly platforms for PcG protein complexes such as PRC2 that act over considerable distances to trimethylate H3-K27 across long stretches of chromatin. The presence of this trimethylation mark in the chromatin that flanks PREs may in turn serve as a signal to define the chromatin interval that is targeted by other PRE-tethered PcG protein complexes such as PRC1. The results reported here also provide a molecular explanation for the previously reported antirepressor function of trxG HMTases; selective binding of Ash1 to the active HOX gene blocks PcG repression by preventing PRC2 from trimethylating the promoter and coding region. It is possible that the extended domain of combined H3-K27, H3-K9, and H4-K20 trimethylation creates not only the necessary stability for transcriptional repression, but that it also provides the molecular marks that permits PcG repression to be heritably maintained through cell division (Papp, 2006).

Comparing active and repressed expression states of genes controlled by the Polycomb/Trithorax group proteins

Drosophila Polycomb group (PcG) and Trithorax group (TrxG) proteins are responsible for the maintenance of stable transcription patterns of many developmental regulators, such as the homeotic genes. ChIP-on-chip assay was used to compare the distribution of several PcG/TrxG proteins, as well as histone modifications in active and repressed genes across the two homeotic complexes ANT-C and BX-C. The data indicate the colocalization of the Polycomb repressive complex 1 [PRC1; containing the four PcG proteins Polycomb (Pc), Polyhomeotic (Ph), Posterior sex combs (Psc), and dRing/Sex combs extra (Sce)] with Trx and the DNA binding protein Pleiohomeotic (Pho) at discrete sequence elements as well as significant chromatin assembly differences in active and inactive regions. Trx binds to the promoters of active genes and noncoding transcripts. Most strikingly, in the active state, Pho covers extended chromatin domains over many kilobases. This feature of Pho, observed on many polytene chromosome puffs, reflects a previously undescribed function. At the hsp70 gene, it was demonstrated in mutants that Pho is required for transcriptional recovery after heat shock. Besides its presumptive function in recruiting PcG complexes to their site of action, these results now uncover that Pho plays an additional role in the repression of already induced genes (Beisel, 2007).

This work used two Drosophila tissue culture lines to map the distribution of chromatin proteins required for the transcriptional maintenance of the HOX genes. Although compromising on the precise developmental identity, the tissue culture cells provided a biochemically tractable homogeneous material, which currently would be difficult to obtain from whole animals. This choice was important to obtain the sharply delineated ChIP profiles, which show a highly significant correlation to mapped genetic elements in the two homeotic complexes. As such, the protein patterns obtained seem to reflect a valid situation as found in material from whole animals. In addition, the ChIP profiles uncovered a new function of Pho, which could be confirmed in whole animals (Beisel, 2007).

The results for SF4 cells are consistent with data that used a Schneider cell derivative for ChIP studies. PRC1 binds to discrete sequence elements, whereas H3K27me3 covers large genomic domains, including genic and intergenic regions. These observations indicate that H3K27me3 cannot be solely responsible for PRC1 targeting. How these H3K27 methylated domains influence HOX gene expression and whether the broad methylation pattern is the cause or consequence of gene silencing remains unclear. H3K27me3 may prevent the binding of activating protein factors as e.g., chromatin remodeling complexes and/or prevent the establishment of activating histone modifications. To this regard, a complementary pattern of H3K27me3 and H4ac, which is present in active gene regions, was detected (Beisel, 2007).

Several lines of evidence suggest that PcG proteins propagate their silencing effect by the direct interaction with the promoter region, which results in the inhibition of transcription initiation. In agreement with that, all promoter regions of the silent ANT-C HOX genes are occupied by PRC1. However, the Ubx promoter, which is silent in both cell lines, as well as the silent AbdB transcription units in Kc cells, are devoid of PRC1. Here, probably the numerous PREs, which are occupied by PRC1 in the Ubx and AbdB domains, build up a special chromatin structure that maintains the silent transcription state (Beisel, 2007).

In agreement with the observed H3K27me3 pattern in Drosophila cells, in mammalian Hox clusters inactive domains are covered by H3K27 and active domains are found entirely covered by H3K4 methylation. In contrast, the distribution of the enzymes setting the histone marks are completely different. In Drosophila E(Z), Trx, and Ash1 are bound at discrete sequence elements, whereas the mammalian homologues EZH2 and MLL1 localize to extended regions coincident with the methylation signals. MLL1 acts as a functional human equivalent of yeast Set1. Both proteins colocalize with RNA Pol II at the transcription start site of highly expressed genes and catalyze the trimethylation of H3K4 at this location. Only at active Hox genes MLL1 reveals a different binding behavior covering entire active chromatin domains. In contrast, the current data shows that Trx also localizes to promoter regions of silent HOX genes and does not show the spreading behavior of MLL1 but appears at additional discrete sites. A complete colocalization of Trx with PRC1 sites was observed at silent genes, i.e., in this expression state no obvious competition is taking place with regard to binding sites (Beisel, 2007).

The comparison of the AbdB gene with the Dfd gene shows that the maintenance of the active state can be performed in alternative ways. The absence of PcG complexes does not seem to be a prerequisite of the active state as observed at the promoter of Dfd in this study and at regulatory regions of Ubx in imaginal discs (Beisel, 2007).

In the active AbdB domain Ph stays bound in a minor but significant amount, and Psc is present in the active Dfd intron. In this regard, Ph and Psc could serve as recruiting platforms for other PRC1 subunits in case of the gene switching to the off state. However, both proteins have been reported to be associated with active genes. Consistent with this, Ph was also observed in the proximal part of both homeotic complexes binding actively transcribed non-HOX genes. The function of this binding behavior remains elusive (Beisel, 2007).

The transcription of noncoding RNAs (ncRNAs) seem to play an important, although diverse, role in the regulation of the BX-C. Noncoding transcription found through the bxd PRE is crucial for Ubx repression and transcription through Mcp overlaps with AbdB transcription in the embryo. NcRNA transcription in the AbdB domain coincides with an active AbdB gene indicates a nonuniversal, gene specific function for ncRNAs in the BX-C (Beisel, 2007).

In the silent state PRC1 is bound to all PREs in the AbdB domain and might be recruited by the action of sequence-specific factors like Pho and the E(Z) histone methyltransferase activity, which may also mark the entire domain as being inactive. In the active AbdB domain, ncRNA transcription may directly influence the binding of of PRC1 and E(Z) or may trigger the enzymatic activity of Trx. Consistent with this scenario, Trx has been shown to bind single-stranded DNA and RNA in vitro. The switch of Trx into an activating mode could lead to the methylation of histones and/or other proteins setting positive transcriptional marks and modulate their activity, respectively. In this case, the displacement of PcG proteins could be directly caused by the Trx action. The binding of Trx to the promoter regions of the active AbdB transcription units could either be caused by (transient) chromatin looping events bridging Trx-bound PREs with the promoters, or Trx could be recruited independently to the active HOX promoters by interaction with RNA Pol II, similar to MLL1, which is recruited to actively transcribed genes in mammalian cells. Trx- and TAC1-interacting histone acetyltransferases may then be responsible for setting epigenetic marks that maintain the active transcription state. Trx has been shown to be required for transcription elongation and it is localized in the gene body of active Ubx, caused by the interaction with elongation factors. In contrast, other studies that investigated the distribution of PcG and TrxG proteins at the active and repressed Ubx gene in imaginal discs found the same restricted Trx profile did the current study, namely Trx binding at discrete sites. These differences may be explained by the different Trx antibodies used. Trx is most probably proteolytically processed like human MLL which results in two fragments that form a heterodimeric complex. This raises the intriguing question whether the complete heterodimeric Trx complex might get recruited to the promoter and upon gene induction the N-terminal fragment tracked along the gene body together with elongation factors, whereas the C-terminal fragment stayed at the promoter (Beisel, 2007).

Pho maps were generated to investigate its role in the recruitment of PRC1. However, the distribution of Pho suggests that the protein also functions in the gene body of actively transcribed genes. The immunostaining of polytene chromosomes revealed that Pho seems not only to be limited to HOX gene control but plays a general role in gene regulation. The colocalization of Pho with strong signals of active Pol II on polytenes together with the effect of a pho-null mutation on the recovery of induced hsp70 indicates that Pho may be directly involved in the rerepression of highly active genes (Beisel, 2007).

It is difficult to imagine that the spreading of Pho is the result of the ability of this protein to bind sequence specifically to DNA. Instead, a model is proposed in which Pho either acts directly at the Pol II elongation complex or it interacts with a remodeling complex, carrying it along the chromatin fiber. In this line, Pho has been shown to interact with BRM and dINO80, two nucleosome remodeling complexes. Interestingly, heat-shock gene transcription is independent of BRM but involves the recruitment of the TAC1 complex, possibly through multiple interactions with the elongating Pol II complex. The simultaneous action of Trx and Pho at heat-shock genes is striking and might resemble their antagonistic functions at HOX genes. Further studies are necessary to unravel the exact molecular mechanism of Pho in this process (Beisel, 2007).

Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos

Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. The sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(Rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli (Zhang, 2008).

Irradiation responsiveness appears to be a highly conserved feature of reaper-like IAP antagonists. A recently identified functional ortholog of reaper in mosquito genomes, michelob_x (mx), is also responsive to irradiation. These results highlighted that stress responsiveness is an essential aspect of functional regulation of upstream proapoptotic genes such as reaper/hid. It is also worth mentioning that several mammalian BH3 domain-only proteins, the upstream proapoptotic regulators of the Bcl-2/Ced-9 pathway, are also regulated at the transcriptional level (Zhang, 2008).

This study shows that the irradiation responsiveness of reaper and hid is subject to epigenetic regulation during development. The epigenetic regulation of the IRER is fundamentally different from the silencing of homeotic genes in that the change of DNA accessibility is limited to the enhancer region while the promoter of the proapoptotic genes remains open. Thus, it seems more appropriate to refer this as the 'blocking' of the enhancer region instead of the 'silencing' of the gene. This region, containing the putative P53RE and other essential enhancer elements, is required for mediating irradiation responsiveness. ChIP analysis indicates that histones in this enhancer region are quickly trimethylated at both H3K9 and H3K27 at the sensitive-to-resistant transition period, accompanied by a significant decrease in DNA accessibility. DNA accessibility in the putative P53RE locus (18,368k), when measured by the DNase I sensitivity assay, did not show significant decrease until sometime after the transition period. It is possible that other enhancer elements, in the core of IRER_left, are also required for radiation responsiveness. An alternative explanation is that the strong and rapid trimethylation of H3K27 and association of PRC1 at 18,366,000-18, 368,000 are sufficient to disrupt DmP53 binding and/or interaction with the Pol II complex even though the region remains relatively sensitive to DNase I. Eventually, the whole IRER is closed with the exception of an open island around 18,387,000 (Zhang, 2008).

The finding that epigenetic regulation of the enhancer region of proapoptotic genes controls sensitivity to irradiation-induced cell death may have implications in clinical applications involving ionizing irradiation. It suggests that applying drugs that modulate epigenetic silencing may help increase the efficacy of radiation therapy. It also remains to be seen whether the hypersensitivity of some tumors to irradiation is due to the dedifferentiation and reversal of epigenetic blocking in cancer cells. In contrast, loss of proper stress response to cellular damage is implicated in tumorigenesis. The fact that the formation of heterochromatin in the sensitizing enhancer region of proapoptotic genes is sufficient to convey resistance to stress-induced cell death suggests it could contribute to tumorigenesis. In addition, it could also be the underlying mechanism of tumor cells' evading irradiation-induced cell death. This is a likely scenario given that it has been well documented that oncogenes such as Rb and PML-RAR fusion protein cause the formation of heterochromatin through recruiting of a human ortholog of Su(v)3-9. In this regard, the reaper locus, especially the IRER, provides an excellent genetic model system for understanding the cis- and trans-acting mechanisms controlling the formation of heterochromatin associated with cellular differentiation and tumorigenesis (Zhang, 2008).

The developmental consequence of epigenetic regulation of the IRER is the tuning down (off) of the responsiveness of the proapoptotic genes, thus decreasing cellular sensitivity to stresses such as DNA damage. Epigenetic blocking of the IRER corresponds to the end of major mitotic waves when most cells begin to differentiate. Similar transitions were noticed in mammalian systems. For instance, proliferating neural precursor cells are extremely sensitive to irradiation-induced cell death while differentiating/differentiated neurons become resistant to γ-ray irradiation, even though the same level of DNA damage was inflicted by the irradiation. These findings here suggest that such a dramatic transition of radiation sensitivity could be achieved by epigenetic blocking of sensitizing enhancers (Zhang, 2008).

Later in Drosophila development, around the time of pupae formation, the organism becomes sensitive to irradiation again, with LD₅₀ values similar to what was observed for the 4–7 hr AEL embryos. Interestingly, it has also been found that during this period, the highly proliferative imaginal discs are sensitive to irradiation-induced apoptosis, which is mediated by the induction of reaper and hid through P53 and Chk2. However, it remains to be studied whether the reemergence of sensitive tissue is due to reversal of the epigenetic blocking in the IRER or the proliferation of undifferentiated stem cells that have an unblocked IRER (Zhang, 2008).

The blocking of the IRER differs fundamentally from the silencing of homeotic genes in several aspects. (1) The change of DNA accessibility and histone modification is largely limited to the enhancer region. The promoter regions of reaper (and hid) remain open, allowing the gene to be responsive to other stimuli. Indeed, there are a few cells in the central nervous system that could be detected as expressing reaper long after the sensitive-to-resistant transition. Even more cells in the late-stage embryo can be found having hid expression. Yet, the irradiation responsiveness of the two genes is completely suppressed in most if not all cells, transforming the tissues into a radiation-resistant state (Zhang, 2008).

(2) The histone modification of the IRER has a mixture of features associated with pericentromeric heterochromatin formation and canonic PcG-mediated silencing. Both H3K9 and H3K27 are trimethylated in the IRER. Both HP1, the signature binding protein of the pericentromeric heterochromatin, and PRC1 are bound to the IRER. As demonstrated by genetic analysis, the functions of both Su(var)3-9 and Su(z)12/Pc are required for the silencing. Preliminary attempts to verify specific binding of PRC2 proteins to this region were unsuccessful. The fact that none of the mutants tested could completely block the transition seems to suggest that there is a redundancy of the two pathways in modifying/blocking the IRER. It is also possible that the genes tested are not the key regulators of IRER blocking but only have participatory roles in the process (Zhang, 2008).

(3) Within the IRER, there is a small region around 18,386,000 to 18,188,000 that remains relatively open until the end of embryogenesis. Interestingly, this open region is flanked by two putative noncoding RNA transcripts represented by EST sequences. If they are indeed transcribed in the embryo as suggested by the mRNA source of the cDNA library, then the 'open island' within the closed IRER will likely be their shared enhancer/promoter region. Sequences of both cDNAs revealed that there is no intron or reputable open reading frame in either sequence. Despite repeated efforts, their expression was not confirmed via ISH or northern analysis. Overexpression of either cDNA using an expression construct also failed to show any effect on reaper/hid-induced cell death in S2 cells. Yet, sections of the two noncoding RNAs are strongly conserved in divergent Drosophila genomes. The potential role of these two noncoding RNAs in mediating reaper/hid expression and/or blocking of the IRER remains to be studied (Zhang, 2008).

CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. This study shows that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. It is proposed that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing (Tie, 2009).

The major findings of this work are: (1) that Drosophila CBP acetylates H3K27; (2) that this acetylation requires TRX; and (3) that it prevents H3K27 trimethylation by E(Z) at Polycomb target genes and antagonizes Polycomb silencing. The remarkably complementary developmental profiles of H3K27ac and H3K27me3 (but not H3K27me2) during embryogenesis suggest that the deposition of H3K27me3, which increases steadily after ~4 hours with the onset of Polycomb silencing, occurs at the expense of a substantial fraction of the H3K27ac already present. This suggests that the establishment of Polycomb silencing might require active deacetylation of this pre-existing H3K27ac. The reciprocal effects of knockdown and overexpression of CBP and E(Z) on H3K27 trimethylation and acetylation in bulk chromatin further suggest that the two modifications constitute alternative chromatin states associated with active and inactive genes. Consistent with this, ChIP-chip experiments revealed that H3K27me3 and H3K27ac are mutually exclusive genome wide. Moreover, in S2 cells, the inactive abd-A gene does not have the H3K27ac modification in its promoter region, but acquires it upon RNAi knockdown of E(Z). It will be important to determine whether such a modification switch occurs genome wide after loss of E(Z) (Tie, 2009).

The ability of E(Z) overexpression to suppress the small rough eye phenotype of CBP overexpressers further supports the conclusion that H3K27 trimethylation by E(Z) antagonizes H3K27 acetylation by CBP and suggests that deacetylation of H3K27 by RPD3, and possibly other deacetylases, might be a prerequisite for subsequent methylation by E(Z) and therefore important for reversal of an active state. Conversely, the ability of CBP and TRX overexpression to increase the global H3K27ac level at the expense of H3K27me3 suggests that either active demethylation of H3K27me3 by the H3K27-specific demethylase UTX (Agge, 2007; Lee, 2007; Smith, 2008), or histone replacement (Ahmad, 2002), might be a prerequisite to acetylation by CBP. Indeed, depletion of Drosophila UTX in vivo using a GAL4-inducible UTX RNAi transgene line results in an increase in H3K27me3, as previously reported (Smith, 2008), and in a marked decrease in H3K27ac. These data, together with the evidence of developmentally programmed reversal of Polycomb silencing, now suggest that the widely accepted stability of Polycomb silencing during development might be more dynamically regulated than previously appreciated (Tie, 2009).

This is the first report that CBP/p300 acetylates H3K27. Recombinant Drosophila CBP acetylates H3K27 and K18 in vivo and in vitro. The greatly reduced H3K27ac levels in CBP-depleted S2 cells also strongly suggest that CBP is the major H3K27 acetylase in Drosophila. The conservation of H3K27 acetylation by human p300, together with the reported association of CBP with the TRX homolog MLL in humans (Ernst, 2001), suggest that it is likely to play a similar role in antagonizing Polycomb silencing in mammals (Tie, 2009).

The genome-wide distribution of H3K27ac, as estimated from human ChIP-chip experiments, appears very similar to that of H3K4me3. This suggests that H3K27ac is much more widely distributed than just at Polycomb target genes, which are estimated to number several thousand in mammalian cells and hundreds in Drosophila. Although these numbers could grow with the identification of additional Polycomb-silenced genes in additional cell types, the recently reported strong correlation of H3K27ac with active genes suggests that it plays an additional role(s) in promoting the transcription of active genes, including those that are never targets of Polycomb silencing. (Note that the H3K27ac at non-Polycomb target genes will not be directly affected by global changes in H3K27me3.) Interestingly, like H3K27me3, H3K27ac appears on the transcribed regions of Polycomb target genes, which might reflect a role for H3K27ac in facilitating transcriptional elongation, and, conversely, a role for H3K27me3 in inhibiting elongation. In addition to its anti-silencing role in preventing H3K27 trimethylation, H3K27ac may also serve as a signal for recruitment of other proteins with additional enzyme activities that alter local chromatin structure further to facilitate or promote transcription. Prime candidates are those containing a bromodomain, a conserved acetyl-lysine-binding module present in several dozen chromatin-associated proteins, including a number of TrxG proteins that also antagonize Polycomb silencing (Tie, 2009).

The results presented in this study provide new insight into how TRX and CBP function together to antagonize Polycomb silencing. Robust H3K27 acetylation by CBP is dependent on TRX, suggesting that H3K27ac plays a crucial role in the anti-silencing activity of TRX. Consistent with this, preliminary genetic evidence suggests that the Polycomb phenotypes caused by TRX overexpression are dependent on CBP, as they are suppressed by RNAi knockdown of CBP. The nature of this dependence is currently unknown, but could involve targeting of CBP by TRX or regulation of the H3K27 acetylation activity of CBP by TRX (Tie, 2009).

The physical association of TRX and CBP and the widespread coincidence of H3K27ac and H3K4me3 sites in the human ChIP-chip data further suggest that the two modifications might be coordinately executed by TRX and CBP. However, the results also raise the possibility that H3K4 trimethylation by TRX itself might be less important for antagonizing Polycomb silencing than H3K27 acetylation. This possibility is also suggested by the discovery of Polycomb-silenced genes in ES and human T cells that contain 'bivalent' marks (both H3K4me3 and H3K27me3) in their promoter regions (although the H3K4me3 levels at these inactive genes are typically lower, on average, than they are at active genes, hinting at the possible importance of quantitative effects of the two marks) (Tie, 2009).

A speculative model is proposed for the regulation of Polycomb silencing that incorporates the activities of TRX, CBP, E(Z), RPD3 and UTX. Repressed genes are marked with H3K27me3. H3K27 trimethylation by PRC2 (which can also control DNA methylation in mammals) requires RPD3 (and possibly other histone deacetylases) to deacetylate any pre-existing H3K27ac. H3K27me3 promotes binding of PC-containing PRC1 complexes, which may inhibit H3K27 acetylation and maintain silencing through 'downstream' events, including those promoted by the H2AK119 mono-ubiquitylation mediated by its RING subunit. Conversely, active genes are marked with H3K4me3 and H3K27ac. H3K27 acetylation by CBP is dependent on TRX and possibly other TrxG proteins, as suggested by the observation that H3K27me3 levels are significantly increased on salivary gland polytene chromosomes from trx, ash1 and kis mutants. The current results predict that this increase will be accompanied by a decrease in H3K27ac. Interestingly, ash1 encodes another HMTase that also interacts with CBP and antagonizes Polycomb silencing. Acetylation of H3K27 is likely to also require the K27-specific demethylase UTX when removal of pre-existing H3K27me3 is a prerequisite for acetylation, e.g. for developmentally regulated reversal of Polycomb silencing at the onset of differentiation. H3K27ac prevents H3K27 trimethylation and might also serve as a signal for recruitment of other TrxG proteins with additional chromatin-modifying activities that may protect the H3K27ac modification and also alter local chromatin structure to promote transcription and further inhibit Polycomb silencing (Tie, 2009).

A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber

Regulation of gene expression involves long-distance communication between regulatory elements and target promoters, but how this is achieved remains unknown. Insulator elements have been proposed to modulate the communication between regulatory elements and promoters due to their ability to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, this study shows that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Even at a distance of 8 kb from the PRE, the distal insulator imposes a chromatin constraint that is removed upon mutation of the su(Hw) gene. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3 histone mark reflect this insulator-dependent chromatin conformation, suggesting that Polycomb action at a distance can be organized by local chromatin topology (Comet, 2011).

Long-distance communication between cis regulatory elements and their promoters may involve either chromatin looping or the ability of factors recruited to the regulatory element to scan the surrounding chromatin by mechanisms of linear spreading or tracking along the fiber until they reach the target promoter. 3C technology can distinguish between these mechanisms by detailed analysis of the interaction profile of a chromatin fragment with the surrounding chromatin fiber. This interaction profile reflects contact probabilities, thus giving a perception of the conformational constraints imposed on the chromatin fiber in a given locus (Comet, 2011).

Using this technology, preferential contacts between regulatory elements and their target promoters have been shown to correlate with their regulatory function, suggesting that chromatin looping can be involved in long-distance regulatory phenomena. The data demonstrate that the gypsy insulator functions via chromatin looping. However, it should be noted that these results cannot distinguish between mechanisms whereby homotypic interactions between the gypsy elements directly establish chromatin loops or whether transient contacts between elements tracking along the chromatin fiber are stabilized by such interactions (Comet, 2011).

A second feature of this element is that one copy of the insulator is able to prohibit contacts between adjacent chromatin fragments. Due to the remarkable resolution of H3C, inhibition of contacts between fragments spaced by <1 kb could be detected or by greater distances (up to 8kb was detected). This topological constraint delimits the range of action of a PRE on its surrounding regions independently of the relative distances between the insulator and the PRE. How does the insulator function to prohibit interactions? A model, called enhancer decoy, suggests that an insulator would mimic a promoter architecture without actually being able to drive transcription. This outcome would predict that a regulatory element such as an enhancer or a PRE may interact with it but this interaction would not be functional. Whereas strong homotypic interactions were seen between insulators, the data did not show high contact levels between the PRE and the insulator. Instead, contact levels reduced just adjacent to it and were low at the insulator and on a 5-kb region downstream of it. This result suggests that insulator complexes are repulsive with respect to the PRE. Because the configuration in which the PRE can bypass two insulators induced clearly detectable contacts between PRE and promoter, it is believed that the insulator is not mimicking a promoter with respect to long-range chromatin interactions. Instead, the insulator seems to topologically separate upstream chromatin regions from those downstream. This separation may depend either on the organization of a specific 3D chromatin fiber architecture or on the formation of large insulator bodies that would be repulsive to chromatin regions not bound by insulator proteins. It should be noted, however, that an endogenous Su(Hw) binding site was previously mapped ~4 kb upstream of the transgenic PRE site in the (P)(S)YSW-22E line, in which there are not interactions with either of the transgenic insulators. Moreover, this binding site does not affect local topological constraints or the profiles of PC binding or H3K27me3 deposition on the local chromatin fiber, suggesting that not all endogenous Su(Hw) binding sites are insulators or belong to the same class of insulator (Comet, 2011).

How PcG proteins are recruited to chromatin by PREs to repress distal gene promoters is still unknown. ChIP analysis showed that the PC protein is detectable in large domains flanking the position of a PRE. This could be explained by spreading or by looping models. This study shows that a PRE-containing chromatin fragment can establish many contacts with its surrounding chromatin region, with a probability decreasing with linear distance. Moreover, the ChIP profiles of PC and H3K27me3 are very similar to the PRE-chromatin interaction profile. In particular, when apparent PC 'spreading' can be detected several kilobases away from the insulator, this process is reflected in an almost identical contact profile between the PRE and the 'region of spreading.' Polycomb group protein complexes have been shown to compact nucleosomal arrays in vitro, which may be expected to enhance physical contacts between the PRE and surrounding chromatin. However, removal of PC due to PRE excision or its physical block due to insulator function does not affect the transgene chromatin conformations. These data suggest that in vivo PC targeting to downstream chromatin is not the simple result of chromatin compaction. The most parsimonious scenario thus posits that PRE-bound PcG proteins of the PRC2 histone methyltransferase complex loop out from the PRE to contact and deposit the H3K27me3 mark on PRE-flanking chromatin. The PC distribution surrounding the PRE may thus be due to multiple transient contacts between the PRE and its flanking regions, followed by H3K27me3 binding of PC via its chromodomain. Finally, in the presence of two insulators, PC is found at the PRE and at the distally located target promoter but not in the region located between the two insulators, in perfect agreement with the chromatin contact data. As a corollary it is important to note that PC is not bound to the promoter in the absence of a neighboring PRE. This result shows that the mini-white promoter itself is not able to recruit PC. Therefore, the ChIP signal observed at this promoter strictly reflects 3D chromatin fiber interactions rather than DNA sequence-autonomous protein binding to their target chromatin sites. Because Su(Hw), as well as many other insulator proteins, binds more than a thousand genomic sites, this kind of 3D looped chromatin organization may represent an important component modulating PRE-mediated silencing of gene expression (Comet, 2011).

H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex

The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a 'stairstep' pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity (Bowman, 2014).

The Polycomb Group repression system is often described as a cellular memory mechanism, which can impose lifelong silencing of a gene in response to a transitory signal. That view seems valid, but the concept of a PcG regulatory domain is much richer. In the PS6 domain of the BX-C, for example, there are many enhancers to drive Ubx expression in specific cells at specific developmental times, all of which are blocked in parasegments one through five, but active in parasegments 6 through 12. Individual enhancers need not include a segmental address that is specified, for example, by gap and pair-rule DNA-binding factors; their function is segmentally restricted by the domain architecture. Indeed, these enhancers will drive expression in a different parasegment when inserted into a different domain (as in the Cbx transposition). Each domain has a distinctive collection of enhancers; the UBX pattern in PS5 is quite different from that in PS6. Thus, there are two developmental programs for Ubx, one in each of these parasegments, without the need for a duplication of the Ubx gene. Other loci with broad regions of H3K27 methylation may likewise be parsed into multiple domains, once histone marks are examined in specific cell types (Bowman, 2014).

The all-or-nothing H3K27me3 coverage of the BX-C parasegmental domains validates and refines the domain model. In particular, K27me3 is uniformly removed across the PS5 and PS7 domains in PS5 and PS7, even though the activated genes in those parasegments (Ubx and abd-A, respectively) are only transcribed in a subset of cells. It is interesting that both PRC1 and PRC2 components have binding patterns that do not fully reflect function (repression and K27 methylation, respectively), indicating the possibility that function of these complexes is regulated separately from binding. The challenges now are to understand how PcG regulated domains are established, differently in different parasegments, and to describe the molecular mechanisms, including changes in chromosome structure, that block gene activity in H3K27 trimethylated domains (Bowman, 2014).

Ecdysone-induced 3D chromatin reorganization involves active enhancers bound by Pipsqueak and Polycomb

Evidence suggests that Polycomb (Pc) is present at chromatin loop anchors in Drosophila. Pc is recruited to DNA through interactions with the GAGA binding factors GAF and Pipsqueak (Psq). Using HiChIP in Drosophila cells, this study found that the psq gene, which has diverse roles in development and tumorigenesis, encodes distinct isoforms with unanticipated roles in genome 3D architecture. The BR-C, ttk, and bab domain (BTB)-containing Psq isoform (Psq(L)) colocalizes genome-wide with known architectural proteins. Conversely, Psq lacking the BTB domain (Psq(S)) is consistently found at Pc loop anchors and at active enhancers, including those that respond to the hormone ecdysone. After stimulation by this hormone, chromatin 3D organization is altered to connect promoters and ecdysone-responsive enhancers bound by Psq(S). These findings link Psq variants lacking the BTB domain to Pc-bound active enhancers, thus shedding light into their molecular function in chromatin changes underlying the response to hormone stimulus (Gutierrez-Perez, 2019).

The BTB domain has been shown to contribute to the oncogenic roles of these proteins. Most BTB-containing transcription factors also encode isoforms that lack the BTB domain and the role of these short isoforms is uncertain. This study shows that different isoforms of Psq appear to play different roles in nuclear function, which may explain their opposing roles in tumorigenesis ascribed to the gene. The BTB-containing PsqL isoform colocalizes with a specific class of architectural proteins that includes Su(Hw), CP190, and Mod(mdg4)2.2. In contrast, the PsqS isoform, which lacks the BTB domain, colocalizes with GAF and Pc at developmental enhancers (dCP) and is mainly associated with active chromatin states. Therefore, PsqS appears to contribute to enhancer function, whereas PsqL is an architectural protein that binds to sequences that have insulator function. How these two isoforms display different genomic distributions while sharing the same DNA binding domain is unclear. However, based on previous findings, it is speculated that the conformation adopted by the protein in the presence of the BTB-interaction domain might inhibit its direct binding to DNA. In addition, the two isoforms coincide in regions in which both Pc and architectural proteins are found. This may explain the reported involvement of PsqL in the recruitment of PcG proteins to chromatin, where it might act with the help of other architectural proteins. In addition to its canonical role, Pc is found, together with PsqS, ISWI, GAF, and CBP, in regions containing H3K27ac and previously characterized experimentally as housekeeping enhancers (hkCP) or dCP enhancers. These findings, suggesting an association of Pc with active enhancers, agree with previous observations showing that PRC1 can be recruited to active genes by the cohesin complex, where it affects phosphorylation of Pol II and Spt5 occupancy (Gutierrez-Perez, 2019).

H3K27me3 is present in the genome of Kc167 cells at very high levels in Pc-repressed domains such as Hox genes. The rest of the genome containing silenced genes in Kc167 cells has low but significant levels of H3K27me3 that represent B compartment sequences. Pc HiChIP analysis provides insights into the dual role of Pc in regulating chromatin organization. Classical Pc-repressed domains interact with each other and with other B compartments with a frequency that correlates with the amount of H3K27me3 present in these compartments. Distinct from these interactions, Pc also forms punctate point-to-point contacts. Two types of loops, defined as puncta of an intense signal in Hi-C heatmaps, have been identified when analyzing changes in 3D organization during Drosophila embryonic development. These loops were classified as active loops containing H3K27ac, Zelda, and Pol II at their anchors or as Pc loops bound by GAF. Zelda loops are absent from Kc167 cells. Like Pc loop anchors observed in embryos, loops represented by puncta in Hi-C heatmaps of Kc167 cells are located within regions enriched in H3K27me3. However, this study found that the center of these sites in Kc167 cells is depleted of H3K27me3 and enriched in H3K27ac. The exact roles of H3K27ac, Pc, PsqS, and GAF found at these loop anchors are unknown, but it is speculated that maintaining a localized active chromatin state may be important for the binding of these proteins and the establishment of these loops. These results suggest a dual and context-dependent function of regulatory elements and agree with previous studies showing that dCP enhancers can act as PREs, and vice versa, during Drosophila embryogenesis (Gutierrez-Perez, 2019).

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