pleiohomeotic

Targets of Activity

Mutations in genes of the Polycomb (Pc) group cause abnormal segmental development due to ectopic expression of the homeotic products of the Antennapedia and bithorax complexes. This paper describes the requirements for Pc group genes in controlling the abdA and AbdB products of the bithorax complex. Examined were embryos containing mutations in the genes Polycomb (Pc), extra sex combs (esc),Enhancer of zeste [E(z)], polyhomeotic (ph), Sex comb on midleg (Scm), Polycomb-like (Pcl), Sex comb extra (Sce), Additional sex combs (Asx), Posterior sex combs (Psc) and pleiohomeotic (pho). In every case, both abdA and AbdB are expressed outside of their normal domains along the anterior-posterior axis, consistent with these Pc group products acting in a single pathway or molecular complex. The earliest detectable ectopic expression is highest in the parasegments immediately adjacent to the normal expression boundary. Surprisingly, in the most severe Pc group mutants, the earliest ectopic AbdB is distributed in a pair-rule pattern. At all stages, ectopic abdA in the epidermis is highest along the anterior edges of the parasegments, in a pattern that mimics the normal abdA cell-specific pattern. These examples of highly patterned mis-expression show that Pc group mutations do not cause indiscriminate activation of homeotic products. It is suggested that the ectopic expression patterns result from factors that normally activate abdA and AbdB only in certain parasegments, but that in Pc group mutants these factors gain access to regulatory DNA in all parasegments (Simon, 1992).

A 176 bp fragment located at -576 to -400 bp of the engrailed locus has been shown to act as a pairing-sensitive silencer in transgenic Drosophila. An evolutionarily conserved 17 bp sequence from this fragment was used to search for DNA binding proteins that might be required for silencing. Pleohomeotic is shown to bind to this 17 bp sequence and mediate a pairing-sensitive silencer function (Brown, 1998)

Polycomb response elements (PREs) in several genes contain conserved sequence motifs. One of these motifs is the binding site for the protein coded for by the recently cloned gene polyhomeotic (pho), the Drosophila homolog of mammalian YY1. The conserved sequence extends beyond the YY1 core consensus sequence suggesting that parts of Pho may impose additional DNA sequence requirements. In this respect and unlike YY1, PHO has an additional 45 amino acids following the fourth zinc finger. It is also possible that Pho may bind to PREs together with another protein in order to fully exploit the conserved sequence. The conserved sequence motif CNGCCATNDNND, includes the YY1 core consensus CCATNWY. Eight consensus sites have been identified in 6 PREs of the bithorax complex (BX-C): bxd, iab-2, Mcp, iab-6, iab-7 and iab-8. The bxd PRE harbors all three characteristics used to define PREs (maintenance of expression of a lacZ reporter assay throughout development; pairing-sensitive repression of a mini-white reporter, and creation of an additional chromosomal binding site of the PcG-repressing complex in a salivary gland assay). The iab-2 PRE contains two homology boxes (a and b) and has been identifed in the maintenance and pairing-sensitive assays. The Mcp and iab-6 PREs have been characterized in the pairing-sensitive assay. The iab-7 PRE contains two homology motifs, a and b. This PRE has been characterized in all three assays. The iab-8 PRE has been identified in the maintenance assay. The conserved sequence motif is found in three PREs from Sexcombs reduced regulatory regions, and has been identified in the pairing-sensitive assay. The sequence motif found in two PREs from the engrailed regulatory region has been characterized in the pairing-sensitive assay. The sequence motif is also found in polyhomeotic, and has been identified in the pairing-sensitive and salivary gland assays (Mihaly, 1998)

In Drosophila, two classes of genes, the trithorax group and the Polycomb group, are required in concert to maintain gene expression by regulating chromatin structure. Trithorax protein (Trx) binding elements have been identified within the bithorax complex. Within the bxd/pbx regulatory region these elements are functionally relevant for normal expression patterns in embryos and they confer Trx binding in vivo. Trx binding elements have been localized to three closely situated sites within a 3-kb chromatin maintenance unit with a modular structure. Results of an in vivo analysis have shown that these DNA fragments (each ~400 bp) contain both Trx- and Polycomb-group response elements (TREs and PREs) and that in the context of the endogenous Ultrabithorax gene, all of these elements are essential for proper maintenance of expression in embryos. Dissection of one of these maintenance modules has shown that Trx- and Polycomb-group responsiveness is conferred by neighboring but separable DNA sequences, suggesting that independent protein complexes are formed at their respective response elements. The activity of this TRE requires a sequence (~90 bp) that maps to within several tens of base pairs from the closest neighboring PRE and the PRE activity in one of the elements may require a binding site for Pho, the protein product of the Polycomb-group gene pleiohomeotic. These results show that long-range maintenance of Ultrabithorax expression requires a complex element composed of cooperating modules, each capable of interacting with both positive and negative chromatin regulators (Tillib, 1999).

Polycomb group (PcG) proteins repress homeotic genes in cells where these genes must remain inactive during development. This repression requires cis-acting silencers, also called PcG response elements (PREs). The Drosophila PcG protein Pleiohomeotic has been shown to bind to specific sites in a silencer of the homeotic gene Ultrabithorax. In an Ultrabithorax reporter gene, point mutations in these Pleiohomeotic binding sites abolish PcG repression in vivo. Hence, DNA-bound Pleiohomeotic protein may function in the recruitment of other non-DNA-binding PcG proteins to homeotic gene silencers (Fritsch, 1999).

To dissect the 1.6 kb Ubx PRE, a Ubx-lacZ reporter gene was used to monitor silencing capacity of PRE subfragments. PBX is an embryonic enhancer and IDE is an imaginal disc enhancer: Both are located about 30 kb upstream of the Ubx transcription start site. PBX directs expression in early embryos in a pattern similar to Ubx with a sharp anterior boundary in parasegment 6 (ps 6). In contrast, if IDE is linked to a reporter gene it activates transcription not only in haltere discs where endogenous Ubx is expressed but also in wing discs where Ubx is not expressed. A PBX-IDE reporter gene is thus active within Ubx expression boundaries in early embryos but is later expressed also outside of the Ubx domain, i.e. in the wing disc. A test was therefore performed to see whether PRE or subfragments thereof would silence this misexpression if inserted into the PBX-IDE reporter gene. The 1.6 kb PRE was inserted between the PBX and IDE enhancers and this reporter gene (PRE_1.6) was introduced into flies. Whereas PBX-IDE transformants without the PRE fragment show nearly uniform beta-galactosidase (beta-gal) expression in wing and haltere discs, beta-gal expression in PRE_1.6 transformants is confined to the posterior compartment of haltere discs. The boundary between beta-gal-positive and beta-gal-negative cells runs through the middle of the haltere disc and apparently coincides with the ps 6 compartment boundary. Thus, IDE activity is completely suppressed anterior to ps 6 but is unaffected in ps 6 itself. This suggests that PRE_1.6 silences the reporter gene anterior to ps 6 and thereby preserves the anterior expression boundary delimited by PBX in the embryo. The expression pattern directed by PBX in the embryo is not silenced by PRE_1.6 (Fritsch, 1999).

Subfragments of the 1.6 kb PRE have been tested for silencing function. PRE silencer is contained in the central 567 bp PRE_D fragment. It was asked whether the silencing mediated by the PRE fragments depends on PcG gene function. A reduction in Pc gene dosage leads to a partial loss of silencing; the extent of the observed misexpression is comparable to the misexpression of the endogenous Ubx gene in Pc heterozygotes. The patterns of PRE_D lines were examined in larvae homozygous for a pho mutation. In each case pho mutant wing and haltere discs show an extensive loss of silencing. These results demonstrate that silencing by PRE_D requires PcG gene function. It was next examined whether Pho protein binds directly to PRE_D. Pho contains a DNA-binding domain with very high similarity to the DNA-binding domain of YY1, which is known to bind to the sequence G/t C/t/a CATN T/a T/g/c. The PRE_D fragment contains several motifs that match versions of this YY1 protein binding site. Oligos spanning each of these motifs were tested for Pho binding in gel-shift assays. Pho protein forms a specific complex with six of the ten tested oligos. These and additional binding tests with other oligos suggest GCCATTAC as an optimal binding site for Pho. To test whether Pho protein binds to the PRE_D construct in vivo, antibodies were generated against the Pho protein. On polytene chromosomes from salivary glands, Pho antibodies bind to approximately 35 different loci. The strongest signal was found at the location of the Bithorax-Complex (BXC), suggesting that Pho protein is bound to the BXC genes. On polytene chromosomes of a PRE_D transformant line, a strong additional signal was found at the transposon insertion site. These data suggest that Pho protein binds directly to PRE_D in vitro and in vivo (Fritsch, 1999).

Are Pho protein binding sites needed for silencing in imaginal discs? All six Pho binding sites in the PRE_D fragment were mutated by altering two or three nucleotides in each CCAT core motif. The introduced base changes abolish binding of Pho protein in vitro. The mutated PRE_D fragment was inserted into the PBX-IDE reporter gene to obtain PRE_Dphomut. These PRE_Dphomut transformants show uniform beta-gal staining in wing and haltere discs that is comparable to transformants carrying the reporter gene without PRE. Thus, mutations in the Pho binding sites abolish PRE function. Taken together, these experiments provide strong evidence that Pho protein binds directly to PRE and is required for silencing (Fritsch, 1999).

Expression of the endogenous Ubx gene was examined in imaginal discs of pho mutants. Animals that are homozygous for pho null mutations develop into pharate adults with only relatively mild homeotic transformations. Consistent with this, it was found that pho¹ and pho^b homozygotes show only slight misexpression of Ubx in wing and antennal discs. The observed misexpression is comparable to the misexpression of Ubx in Pc heterozygotes. In pho mutants, the PRE_D reporter gene shows substantially more misexpression than the endogenous Ubx gene. Thus, silencing of the reporter gene is more sensitive to the lack of pho product than the native Ubx gene. Animals that are mutant for two different PcG mutations often show more severe misexpression of homeotic genes and consequently enhanced homeotic transformations, when compared to the single mutants by themselves. pho homozygotes that are also heterozygous for Pc show very dramatic misexpression of Ubx in wing and other discs. Thus, in this genetically sensitized background due to only one rather than two copies of Pc, pho is required to repress Ubx in all imaginal disc cells (Fritsch, 1999).

What is the role of maternal Pho? Most pho mutant embryos, which lack maternal wild-type pho product, fail to develop altogether and the rare putatively paternally rescued embryos that do develop die with segmentation defects and homeotic transformations. In contrast, if maternal pho product is present, pho homozygotes survive to pharate adults. This suggests that pho function is particularly important in the very early embryo. Mutation of the Pho binding sites in the PRE_Dphomut reporter gene abolish silencing in all disc cells. Thus, it appears that if Pho protein is prevented from binding to PRE, i.e. in the PRE_Dphomut reporter gene, silencing is probably never established. Conversely, silencing of the PRE_D reporter gene is only partially lost in larvae homozygous for a pho null mutation. Thus, in pho homozygous embryos (which contain maternal Pho protein) silencing of the PRE_D reporter is probably established but is subsequently lost in imaginal discs. In summary, these observations strongly suggest that maternally deposited Pho protein is crucial for the establishment of silencing but that zygotic Pho protein is required for complete silencing (Fritsch, 1999).

The Mcp element from the Drosophila melanogaster bithorax complex (BX-C) was initially identified because deletions of the element cause a dominant gain-of-function transformation of PS9 into PS10. This transformation in parasegmental identity is due to the inappropriate activation of the iab-5 cis-regulatory domain (which specifies PS10 identity) of the Abd-B gene in PS9 (a parasegment in which Abd-B is normally turned off). Two models have been proposed to explain the gain-of-function phenotypes associated with Mcp deletions. In the first, the Mcp deletions remove a PS10 silencer that functions to keep the iab-5 cis-regulatory domain off in PS9. When this silencer is removed, iab-5 is activated in PS9, turning on Abd-B. In the second model, Mcp corresponds to a boundary element that functions to preserve the functional autonomy of the iab-4 and iab-5 cis-regulatory domains. While the question of whether Mcp corresponds to a silencer, a boundary, or both (as is the case for the element deleted by another BX-C gain-of-function mutation, Fab-71, a novel activity has been uncovered. Sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and they silence adjacent reporters by means of a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a 'pairing-sensitive' silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression (Muller, 1999).

The Mcp element in BX-C is defined by three overlapping deletions. Although these deletions differ in size and location, all three have indistinguishable, dominant gain-of-function phenotypes: they transform PS9 into PS10. This transformation in parasegmental identity is due to the ectopic activation of Abd-B in PS9, a parasegment in which Abd-B is normally off. The three deletions remove a common region of ~450 bp in length. This common region spans a major ~400-bp chromatin-specific, nuclease-hypersensitive site that is present throughout embryogenesis and in tissue culture cells. The smallest deletion, Mcp^B116, is slightly larger than this common region, and it removes an additional ~350 bp proximal to the major nuclease-hypersensitive region. DNA fragments extending to either side of the small Mcp deletion have silencing activity when linked to either a mini-white or y reporter. Like other silencers in the PRE class, silencing activity depends on Pc-G proteins. Included in the Pc-G group is pleiohomeotic, a gene that encodes a DNA-binding protein that appears to be closely related to the mammalian YY1 transcription factor. As has been found for several other PREs in BX-C, the major Mcp nuclease-hypersensitive region has a consensus Pho/YY1-binding sequence. The presence of this sequence, together with the fact that silencing activity depends on the pho gene, argue that this DNA-binding protein may play a key role in the assembly of Pc-G-silencing complexes by the Mcp element (Muller, 1999).

The most unusual feature of the Mcp element is its ability to promote long-distance interactions. Regulatory interactions are observed between Mcp transgenes inserted at different sites on the same chromosomal arm, on different chromosomal arms, and even between transgenes inserted on different chromosomes. The long-distance regulatory activity of the Mcp element is unusual. Among the previously characterized PREs from BX-C and other genetic loci, pairing-sensitive silencing is generally observed only between interacting partners inserted at the same site. Only in a few instances have interactions been observed between partners inserted at different sites, and these usually involved PRE transgenes located in quite close proximity. How does Mcp promote regulatory interactions over long distances? One model suggests that Mcp might function by dragging paired DNA into a heterchromatic nuclear compartment. Contrary to the expectations of the compartmentalization model, the Mcp element can mediate not only long-distance silencing, but also long distance activation. An alternative, more plausible model suggests that the Mcp element facilitates long-distance regulatory interactions because it is able to locate and then pair with Mcp elements at other sites. After this locating process the formation and spread of a function silencing complex around each element would occur. Specificity is likely to be generated by a combination of proteins, some that are found in most PREs and some that are unique to the 800-bp MCP element (Muller, 1999).

Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the gap gene products and the Polycomb group proteins. DNA elements and trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein have been investigated. Analysis of a 1.7-kb enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that both Hb and Kruppel (Kr) are required to set the Abd-A anterior boundary in parasegment 7. DNase I footprinting and site-directed mutagenesis show that Hb and Kr are direct regulators of this iab-2 enhancer. The single Kr site can be moved to a new location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results suggest that Kr repression occurs through a local quenching mechanism. The gap repressor Giant (Gt) initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. This iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (Pho). These iab-2 Pho sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the Pho sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer (Shimell, 2000).

After Hb and Kr decay during early gastrulation, the repressed state is propagated through later stages of development by the PcG proteins. How the transition from early gap repressors to long-term PcG repressors occurs at the molecular level is not known. Two basic models have been proposed: (1) direct recruitment, and (2) chromatin recognition. Model (1): The gap gene products, especially Hb, have been proposed to help recruit PcG proteins directly to specific DNA sites. Based upon its early time of action, a role for the PcG protein Extra sex combs (Esc) as a molecular bridge between the two sets of repressors has been suggested. However, direct interactions between Esc and gap repressors have not been reported. A better candidate for such a molecular link is dMi-2, which binds directly to Hb and behaves genetically as an enhancer of PcG repression. In its simplest form the direct recruitment model is unlikely because the iab-2, bx, and pbx enhancers all contain Hb sites but do not effectively recruit PcG proteins. These elements fail to maintain A-P boundaries of expression and are unable to attract PcG proteins to sites on chromosomes. Furthermore, the continuous requirement for PRE sequences during development shows that DNA site recognition by PcG proteins can occur long after Hb and Kr have decayed. Model (2):The second model proposes that PcG proteins recognize some feature of silenced chromatin, rather than particular gap repressors. This model is supported by patterns of PcG-dependent silencing that reflect patterns of early gene activity rather than the distributions of gap proteins. In this view, PcG proteins sense the transcriptional off state and then assemble locally to imprint this state through later stages. These two models are not mutually exclusive. Both the Hb-interacting protein dMi-2 and the Kr-interacting protein dCtBP have mammalian homologs that interact with histone deacetylases. Perhaps the gap repressors work by targeting these deacetylases, whose action alters the local acetylation state of the histone tails. This could provide a feature of silenced chromatin that is recognized by PcG proteins and that promotes their association at nearby PREs (Shimell, 2000 and references therein).

In addition to sites for the gap repressors, the role of iab-2 binding sites was characterized for the recently identified PcG protein, Plieohomeotic. Pho sites on the iab-2(1.7) fragment are required for pairing-sensitive repression (PSR) of a mini-white reporter. Thus, Pho can mediate this type of gene repression in the context of a homeotic regulatory fragment, analogous to its activity with an engrailed regulatory fragment. Similarly, Pho binding sites are required for function of a different PRE located in the bxd region. Are the iab-2(1.7) Pho sites sufficient for full PcG repression? The results suggest that they are not, since lacZ maintenance in the embryo, as opposed to PSR function during late stages, requires more distally located iab-2 sequences in combination with the iab-2(1.7) fragment. Thus, assays for PSR and for lacZ maintenance are not measuring precisely the same activity. In molecular terms, this could reflect association of distinct complexes at PSR sites as opposed to sites that supply full PRE function. Alternatively, a larger critical number of Pho sites might be needed for lacZ maintenance and fewer sites might suffice for PSR. The iab-2(534) fragment, which enables lacZ maintenance, contains two additional Pho consensus core sites. However, three lines of evidence indicate that Pho is not likely the sole factor that recruits PcG proteins either to PREs or to PSR sites. (1) A multimerized Pho site is insufficient to mediate PSR; (2) in vivo crosslinking studies show heterogeneity among PcG proteins assembled onto DNA from different regions of the engrailed locus, and (3) the DNA-binding GAGA protein has also been implicated in PRE function and has been found associated with PRE sequences in chromatin binding assays. These observations strongly suggest that multiple DNA-binding factors form the landing pad for association of distinct types of PcG complexes. What might be the in vivo role of PSR sites, such as the one on the iab-2(1.7) fragment, which by themselves cannot provide full PRE activity? One possibility is that, in their normal context, they act as secondary recruitment sites to extend and/or stabilize chromatin changes that are nucleated at strong PREs. In agreement with this, is has been found that PC protein first assembles onto core PREs at the blastoderm stage and that high levels of PC association with fragments outside of these core regions do not occur until later in embryogenesis. The scattering of PSR sites throughout large regulatory domains, such as those within the BX-C, might assist assembly and propagation of repressive chromatin complexes over large DNA distances (Shimell, 2000 and references therein).

A functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila bithorax complex (BX-C) has been undertaken. Previous studies mapped the iab-7 PRE to an 860-bp fragment located just distal to the Fab-7 boundary. Located within this fragment is an ~230-bp chromatin-specific nuclease-hypersensitive region called HS3. HS3 has been shown to be capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of a mini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. GAGA and Pho interact with these sequences in vitro, and the consensus binding sites for the two proteins are critical for the silencing activity of the iab-7 PRE in vivo (Mishra, 2001).

Like the GAGA factor, Pho appears to function by directly interacting with target sequences in HS3. Several lines of evidence support this conclusion: (1) the silencing activity of the iab-7 PRE in vivo depends on pho function and is eliminated by mutations in the pho gene; (2) the Pho protein binds to two conserved target sequences in the iab-7 PRE; (3) mutations in these two sites not only eliminate binding in vitro but also compromise silencing activity in vivo. Pho has also been directly implicated in the silencing activity of three other PREs, one from the en gene and two from BX-C. The Pho protein has been shown to bind to these PREs in vitro, while mutations in either the Pho binding sites or in the pho gene itself reduce or eliminate silencing (Mishra, 2001).

Unlike that of Trl, the phenotypes of pho mutants are similar to those seen for other Pc-G genes. Animals homozygous for loss-of-function alleles die at the pupal stage and exhibit homeotic transformations of legs and abdomen. The late lethal phase is due to a substantial maternal contribution, and mutant embryos lacking a maternal source of wild-type Pho die with severe homeotic transformations and other developmental defects. The homeotic transformations evident in mutant animals indicate that pho is likely to have a direct role in Pc-G silencing. For the iab-7 PRE, the results argue that silencing activity depends on the binding of the Pho protein to the two target sites in HS3. Both sites seem to be important, since silencing activity is compromised when one site is deleted. Whereas it is supposed that the major function of the GAGA factor is to ensure that sequences in HS3 are accessible to other proteins, the phenotypic effects of pho mutations suggest that it plays a more active role in silencing. A plausible hypothesis is that it functions (perhaps together with as yet unidentified factors) to recruit components of the silencing machinery to the PRE, such as Polycomb or Sex Combs Midleg, which do not appear to interact directly with DNA. Supporting the possibility that other factors besides Pho play a critical role in recruiting Polycomb group complexes, a PRE fragment from iab-2, which contains Pho binding sites and which is able to silence mini-white, has been shown to be insufficient to confer full Pc-G maintenance activity. Moreover, mutations in the two Pho binding sites have only a minor effect on the maintenance activity of the 860-bp iab-7 PRE fragment in an iab-7 Ubx-LacZ assay system. Clearly it will be of interest to identify these other factors (Mishra, 2001).

Silencing of homeotic gene expression requires the function of cis-regulatory elements known as Polycomb Response Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene assays, a 138 bp sequence has been defined within the MCP silencer that is sufficient for silencing of a reporter gene in the imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (Pho) and two binding sites for the GAGA factor, encoded by the Trithorax-like gene. PHO and the Trl proteins bind to these sites in vitro. Mutational analysis of Pho and Trl binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of Trl, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that, individually, either Pho or Trl binding sites retain only weak silencing activity. However, when both Pho and Trl binding sites are present, they achieve strong silencing. A model is presented in which robust silencing is achieved by sequential and facilitated binding of Pho and Trl (Busturia, 2001).

How does Trl or perhaps another GAGA binding protein contribute to the silencing by MCP, and what is its relationship to the Pho protein function? Two models to explain their relationship which leads to strong silencing are suggested. These models are based on the following observations. (1) Pho binding sites by themselves show little silencing activity (MCP1 and MCP7* constructs). (2)Trl or some other protein that binds to MCP can weakly recruit silencing complexes in the absence of Pho binding (5MPho construct). (3) When present together, Trl and Pho binding sites exhibit robust silencing activity (MCP7 construct). In the first model, Trl and Pho bind to the MCP silencer in a sequential order. One version would be that Trl binding is absolutely required for binding or activity of Pho. Trl may open up chromatin at MCP, allowing binding of Pho. Upon binding, Pho may recruit PcG silencing complexes, although there is still little evidence that this happens. Trl has been shown to induce DNase I hypersensitive sites, or nucleosome-free regions, and this may create a prerequisite condition for Pho to bind to its recognition sites. There is indeed a DNase hypersensitive region associated with MCP that includes the location of the Trl binding site (Busturia, 2001).

In a second version of the model, Pho acts as a facilitator of Trl binding by creating some pre-condition, perhaps by bending DNA as YY1 does. Since Pho binding sites are not absolutely required for MCP silencing activity, Trl presumably can bind weakly to MCP in the absence of Pho. Enhanced binding of Trl leads to increased recruitment of silencing complexes. Trl bound to MCP may recruit PcG silencing complexes by directly interacting with PC or other members of PcG complexes. Alternatively, Trl could first recruit SIN3 histone deacetylation complexes through its interaction with SAP18, which then might generate a chromatin state favorable for PcG complex binding. Whichever version of the model is correct, the important feature of the model is the sequential recruitment of DNA binding proteins, Trl and Pho, to MCP. Binding of one protein creates a condition favorable to the binding of a second protein, eventually leading to the recruitment of PcG complexes. Note that the requirement of Trl and Pho proteins applies to MCP silencing, but not necessarily to all PREs. Other PREs may use other combinations of proteins. This model is analogous to Swi5 protein binding to the yeast HO promoter and recruiting the chromatin remodeling complex Swi/Snf. Swi/Snf in turn recruits the histone acetylase complex SAGA, eventually leading to the binding of the transcription factor SBF to the HO promoter. In such a sequential recruitment model, compromising one step in the sequence may become rate limitating so that combining two mutations that disable two different steps may not necessarily lead to synergistic effects. This may explain why no synergistic effects are observed when Trl and PcG mutations are combined. In the second model, Trl and Pho bind to MCP independently of one another. Each protein may induce a unique chromatin modification that, together, can have a positive synergistic effect on the recruitment of PcG silencing complexes (Busturia, 2001).

Regulatory DNA from engrailed causes silencing of a linked reporter gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transgene and has been called 'pairing-sensitive silencing.' The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), the activities of each of these engrailed fragments were tested in a construct designed to detect PRE activity in embryos. Both fragments behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment shows additional silencing capabilities. Using the mini-white reporter gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggest that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function (Americo, 2002).

Transcription factor YY1 functions as a PcG protein 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).

Genome-wide prediction of Polycomb/Trithorax response elements

Polycomb/Trithorax response elements (PRE/TREs) maintain transcriptional decisions to ensure correct cell identity during development and differentiation. There are thought to be over 100 PRE/TREs in the Drosophila genome, but only very few have been identified due to the lack of a defining consensus sequence. The definition of sequence criteria that distinguish PRE/TREs from non-PRE/TREs is reported in this study. Using this approach for genome-wide PRE/TRE prediction, 167 candidate PRE/TREs are reported, that map to genes involved in development and cell proliferation. Candidate PRE/TREs are shown to be bound and regulated by Polycomb proteins in vivo, thus demonstrating the validity of PRE/TRE prediction. Using the larger data set thus generated, three sequence motifs that are conserved in PRE/TRE sequences have been identified (Ringrose, 2003).

The detection of PRE/TREs by prediction generates a large data set that can be used to search for further common sequence features. To this end, the 30 highest scoring PRE/TRE hits were scanned for motifs that occur significantly more often in PRE/TREs than in randomly generated sequence. Five significant motifs were found. Not surprisingly, but reassuringly, two known motifs, the GAF and PHO binding sites were found. The Zeste binding motif was not found by this analysis, although it occurs as frequently as GAGA factor in the 30 sequences analyzed. This is probably due to the shortness and degeneracy of the Zeste motif, and suggests that other such short motifs will also be missed by this approach (Ringrose, 2003).

Nevertheless, three additional motifs were found. The first, called GTGT, is found several times in 14 of the sequences. The second motif, poly T, is found several times in almost all 30 PRE/TRE sequences analyzed. Some variants of this site match the binding consensus for the Hunchback protein, which has been shown to be an early regulator at some PRE/TREs. The third motif, TGC triplets, occurs several times in 13 of the PRE/TRE sequences. No binding factor for this sequence has yet been identified (Ringrose, 2003).

To further examine these three motifs, motif occurrence was evaluated in all 167 predicted PRE/TREs and in the promoter peaks described above. In contrast to the known GAF, Z, and PHO motifs, the three motifs each occur in only a subset of predicted and known PRE/TREs, and do not occur significantly together. These motifs may thus each define a subclass of PRE/TREs. Consistent with this idea, some of the lowest scoring known PRE/TRE sequences indeed contain one or more of the three motifs (Ringrose, 2003).

Although no correlation between particular sites and high scores was found, a negative correlation was found between numbers of GAF/Z and PHO sites (a correlation coefficient of -0.78, indicating that when many GAF/Z sites are present, there are few PHO sites, and vice versa). This suggests that each PRE/TRE may have a preferred ground state, in which it is either predisposed to silencing (many PHO sites) or to activation (many GAF/Z sites) (Ringrose, 2003).

In summary, this analysis identifies three motifs that occur significantly in association with known PRE/TRE motifs. Further functional characterization of these motifs and the proteins that bind them may contribute to a more complete definition of the sequence requirement for PRE/TRE function, and of subclasses of PRE/TREs (Ringrose, 2003).

This study offers four main contributions to the understanding of PRE/TRE function. First, a larger set of sequences have been defined that will facilitate the more complete definition of PRE/TRE sequence requirements. Three motifs have been identified that may contribute to this goal. The definition of the minimal requirement for PRE/TRE function will not be a trivial task. Analysis of motif composition and order in the 167 predicted PRE/TREs reveals that there is a great diversity of patterns, with no preferred linear order. It is possible that each different pattern of motifs reflects a subtly different function. However, the concept of a linear order of motifs may well be irrelevant, because these elements operate in the three-dimensional context of chromatin. The fact that such a diversity of PRE/TRE designs exist indicates that the vast majority of them would defy detection by conventional pattern-finding algorithms, and underlines the advantages of the approach described in this study (Ringrose, 2003).

Although no linear constraints on motif order were found, the fact that only motif pairs, and not single motifs, are able to identify PRE/TREs strongly suggests that this close spacing of sites has functional significance. Multiple sites may work in concert, to promote cooperative binding of similar proteins (e.g., repeated PHO sites) or to provoke competition between dissimilar proteins (e.g., closely spaced GAGA factor and PHO sites). In addition, in chromatin, only a subset of sites will be exposed and optimally available for binding at any one time, while others will be occluded by nucleosomes. The trxG includes nucleosome remodeling machines, raising the intriguing possibility that remodeling of PRE/TREs in chromatin may contribute to epigenetic switching by exposing different sets of protein binding sites (Ringrose, 2003).

Second, a PRE/TRE peak is observed at the promoter of all the genes examined. This strongly suggests that promoter binding is a general principle of PRE/TRE function. It has been reported that PcG proteins can interact with general transcription factors. It has hitherto been unclear whether the observed PcG/trxG binding at promoters of the genes they regulate is mediated indirectly via such an interaction, or whether the PcG and trxG bind directly to PRE/TREs at the promoters. The high scores observed at promoters favor the latter interpretation (Ringrose, 2003).

Third, it has been shown that in most cases, PRE/TREs do not occur in isolation, but are accompanied by one or more other peaks nearby. These grouped PRE/TREs may create multiple attachment sites for PcG and trxG proteins, which come together to build a fully operational complex at the promoter. Alternatively, grouped PRE/TREs may be individually regulated by tissue-specific enhancers as in the BX-C. Thus, each of the many PRE/TREs of the homothorax gene may interact with the promoter PRE/TRE in different tissues. This idea is consistent with the fact that Homothorax has specific roles in diverse developmental processes (Ringrose, 2003).

Finally, the current list of about ten PcG/trxG target genes has been expanded to over 150 genes, identifying candidates for epigenetic regulation. The genes thus identified encompass every stage of development, suggesting that the PcG/trxG are global regulators of cellular memory. Experiments to further investigate and compare this regulation for individual genes are currently underway (Ringrose, 2003).

Hierarchical recruitment of polycomb group silencing complexes

Polycomb group (PcG) proteins maintain the transcriptional silence of target genes through many cycles of cell division. This study provides evidence for the sequential binding of PcG proteins at a Polycomb response element (PRE) in proliferating cells in which the sequence-specific DNA binding Pho and Phol proteins directly recruit E(z)-containing complexes, which in turn methylate histone H3 at lysine 27 (H3mK27). This provides a tag that facilitates binding by a Pc-containing complex. In wing imaginal discs, these PcG proteins also are present at discrete locations at or downstream of the promoter of a silenced target gene, Ubx. E(z)-dependent H3mK27 is also present near the Ubx promoter and is needed for Pc binding. The location of E(z)- and Pc-containing complexes downstream of the Ubx transcription start site suggests that they may inhibit transcription by interfering with assembly of the preinitiation complex or by blocking transcription initiation or elongation (L Wang, 2004; full text of article).

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).

Replacement of a Drosophila Polycomb response element core, and in situ analysis of its DNA motifs

Long-term repression of homeotic genes in the fruit fly is accomplished by proteins of the Polycomb Group, acting at Polycomb response elements (PREs). This study used gene conversion to mutate specific DNA motifs within a PRE to test their relevance, and PREs were exchanged to test their specificity. Previously it was shown that removal of a 185 bp core sequence from the bithoraxoid PRE of the bithorax complex results in posteriorly directed segmental transformations. Mutating multiple binding sites for either the Polyhomeotic (Pho) or the GAGA factor (Gaf) proteins separately in the core bithoraxoid PRE resulted in only rare and subtle transformations in adult flies. However, when both sets of sites were mutated, the transformations were similar in strength and penetrance to those caused by the deletion of the 185 bp core region. In contrast, mutating the singly occurring binding site of another DNA-binding protein, DSP1 (Dorsal switch protein 1; reportedly essential for PRE-activity), had no similar effect in combination with mutated Pho or Gaf sites. Two minimal PREs from other segment-specific regulatory domains of the bithorax complex could substitute for the bithoraxoid PRE core. In situ analysis suggests that core PREs are interchangeable, and the cooperation between Pho and Gaf binding sites is indispensable for silencing (Kozma, 2008).

This study used gene conversion to test variant forms and substitutes of a PRE core in its normal chromosomal context. The conversion strategy retained the wild type PRE sequence in the initial convertant, so that the convertant animals were normal in their segmental identities. The wild type PRE sequence could then be removed, at will, to test the remaining function from the mutated PRE. The ability to recreate the mutant multiple times proved essential to phenotypic assays, since the strength and penetrance of the phenotypes faded with successive generations of heterozygous or homozygous mutant flies (Kozma, 2008).

In case of larger deletions, such as the 665 bp Δ1-2 or the 280 bp Δ10, the penetrance of posteriorly directed transformations stays stably high in homozygous stocks. In stocks homozygous for the 280 bp deletion, only the strength of transformations gets weaker, while in the 185 bp deletion homozygous stock both the strength and the penetrance of transformations decline. Very likely, sequences neighboring the bxd PRE core are involved in the decline of the penetrance in the 185 bp deletion Δ17 homozygotes. The neighboring sequences appear to be somewhat redundant with the bxd PRE core, as suggested by the 3.5-fold higher penetrance observed in the 280 bp deletion heterozygotes. In addition, neighboring PREs on the mutated chromosome (in the bx and the iab-2 cis-regulatory regions) and PREs on the wild type homolog may also compensate for the loss of the bxd PRE core (Kozma, 2008).

The adult transformations must be due to misexpression of the Ubx gene, but no corresponding misexpression of Ubx was detected in embryonic or larval stages. Most cells in the embryo do not give rise to adult tissues, and the adult lineages may have higher sensitivity to Ubx levels. Cells of the adult tissues have gone through one (or two) more cell division(s) than their ancestral cells in the larval imaginal discs. Consequently, adult cells can accumulate more mistakes in the maintenance of the cellular memory. It is also possible that clones expressing Ubx in wing discs escaped detection because of the low penetrance (Kozma, 2008).

The Drosophila Pho and Pho-like proteins are the only PcG proteins known to bind to specific DNA-sequences, and they recognize the DNA motifs GCCAT, ACCAT and GCCAC. The first two CCAT motifs in the 185 bp core bxd PRE are separated from each other by only a single base, which may be important for the PRE-function. Similar pairs of CCAT-sites can also be found twice in the iab-7 PRE. The tested iab-7 PRE-fragment, with one such CCAT-pair, could fully replace the core bxd PRE. However, mutating all five CCAT motifs in the core bxd PRE results in only a low penetrance of posterior transformations (gain-of-function phenotypes, GOF), partly due to the compensating effect of neighboring sequences. Although the effect of point mutations in CCAT motifs is weak, it shows a GOF penetrance at least two times higher than in the case of mutated GAGA motifs. This difference is even more pronounced (6.5 times) when the neighboring distal 228 bp region is removed. These data correlate well with a prior study of a 567 bp fragment from the bxd PRE, which found no effect of mutating GAGA sites, using transgene reporter assays in imaginal discs. However, another transgene study of the 138 bp Mcp PRE fragment suggested that GAGA motifs were more important for silencing than CCAT motifs (Kozma, 2008).

Three different proteins, GAF, PIPSQUEAK and BATMAN, were suggested to act in concert, binding to the same GA-repeats, at least in the bxd PRE. As the N-terminal BTB/POZ domain of the GAF protein self-associates, the C terminal Zn-finger of GAF favors paired or clustered binding sites. However, GAF was also demonstrated to bind even GAG triplets with only a slightly reduced affinity as compared to GAGAG pentamers. Therefore, GAGA tetramers, not only the canonical GAGAG sequences, usually claimed as the minimal binding site of GAF, were mutated. Despite the suggested importance of GA-repeats in silencing, the effect of destroying only GAGA motifs is extremely weak, even when the distal 228 bp neighboring sequence is removed. It is possible that the proximal neighboring sequences can also compensate for the loss of the mutated GAGA motifs in the bxd core PRE. Indeed, this proximal ~200 bp region contains a 70 bp fragment (named MHS-70) with multiple, non-overlapping d(GA)₃ repeats, which were found to be important for silencing in embryos in transgenic assays. This prior study also showed that the ~20 bp GA-repeats in the core bxd PRE compete strongly with MHS-70 in gelshift assays, suggesting the binding of the same proteins (Kozma, 2008).

Although mutating either the CCAT or the GAGA motifs alone had only a modest phenotypic effect, simultaneous mutations in both types of motifs completely eliminated the core PRE function. The resulting penetrance of GOF phenotypes is equivalent to that of a deletion of the 185 bp core PRE. This suggests a highly cooperative effect between factors binding to the two different DNA motifs. In biochemical studies, GAF was found to facilitate Pho binding to chromatin, perhaps by interacting with NURF to create nucleosome-free regions or DNAse I hypersensitive sites. There are indeed such hypersensitive sites in the Mcp, iab-7 and bxd PRE regions. Both the Mcp and the iab-7 PREs required the presence of both Pho and GAF binding sites for efficient silencing in transgenic assays (Kozma, 2008).

Although the combination of mutated CCAT and GAGA motifs eliminates the core PRE function, it is still possible that other protein binding sites are also required for silencing. For example, the HMG-group protein DSP1, which was known previously to bind without sequence-specificity to the minor groove of DNA, was recently reported to bind to the GAAAA DNA-motif. Little or no effect was found of mutating the singly occuring putative DSP1 binding site in the core bxd PRE. Even when four other putative DSP1 binding sites were mutated by the removal of the neighboring 228 bp DNA region, distal to the reintroduced core PRE with mutated GAGA motifs, only a very weak GOF phenotype was observed. Thus, in contrast to the results of the transgenic assay studying the iab-7 PRE, it seems that the putative DSP1 sites have little or no role in silencing in the 413 bp region of the bxd PRE studied in situ (Kozma, 2008).

The 191 bp fragment of the iab-7 PRE was able to fully substitute for the 185 bp core bxd PRE, irrespective of its orientation. No posterior or anterior transformations were observed in flies with this PRE replacement, in either hetero- or homozygous animals. There is a pronounced similarity between the bxd core and iab-7 fragment in the pattern of CCAT and GAGA DNA-motifs, although there is no other apparent homology between these sequences. The perfect substitution of these PREs demonstrates that core PREs alone do not carry positional information; they act only as simple silencers. This finding is in agreement with previous studies, which used bigger PRE-fragments fused to different enhancers in transgenic assays (Kozma, 2008).

The 189 bp iab-5 PRE-fragment, which shows much less similarity in the pattern of DNA-motifs to the bxd PRE, is also a perfect substitute. This observation reinforces the notion that only the CCAT and GAGA sites matter for PRE function. In contrast, the 263 bp human H1 and the 222 bp human H2 fragments completely fail to substitute the core bxd PRE. H1 has only three CCAT motifs, perhaps one less than necessary (the bxd, iab-5 and iab-7 core PREs each have at least 4 CCAT motifs), or perhaps the CCAT and GAGA motifs in the human DNA-fragment are not sufficiently close-packed. Alternatively, the BX-C PREs might share some other protein binding sequence, yet unrecognized, that is also necessary for the core PRE function. In any case, the negative results with human sequences exclude the possibility that the 185 bp Drosophila bxd PRE-fragment has only a spacer function. Indeed, sequences without CCAT and GAGA motifs (such as H2) can clearly interfere with the impaired PRE-activity (Kozma, 2008).

These experiments demonstrated that three core PREs from different regulatory regions of the bithorax complex are functionally equivalent in situ, suggesting the interchangeability of PRE cores; CCAT and GAGA DNA-motifs act in concert and, unlike GAAAA/GATAA motifs, they are absolutely necessary for the function of the bxd PRE. The versatile gene conversion strategy and the sensitive phenotypic assay developed in this study can now be used to ask more detailed questions about the number and spacing of the GAGA and CCAT motifs, and about the function of other sequence regions in the core bxd PRE. Molecular definition and artificial assembly of a functional PRE core may become also possible with the help of these further studies. The marker gene, Gal4-VP16, used in these gene conversion events, can also be used to monitor subtle changes in the local chromatin structure, which may not result in any detectable phenotypic changes. This system should also be useful to test PREs from other Drosophila loci, like engrailed or polyhomeotic, and to assay potential PREs predicted by computational analyses of mammalian genomes (Kozma, 2008).

PcG-mediated higher-order chromatin structures modulate replication programs at the Drosophila BX-C

Polycomb group proteins (PcG) exert conserved epigenetic functions that convey maintenance of repressed transcriptional states, via post-translational histone modifications and high order structure formation. During S-phase, in order to preserve cell identity, in addition to DNA information, PcG-chromatin-mediated epigenetic signatures need to be duplicated requiring a tight coordination between PcG proteins and replication programs. However, the interconnection between replication timing control and PcG functions remains unknown. Using Drosophila embryonic cell lines, this study found that, while presence of specific PcG complexes and underlying transcription state are not the sole determinants of cellular replication timing, PcG-mediated higher-order structures appear to dictate the timing of replication and maintenance of the silenced state. Using published datasets it was shown that PRC1, PRC2, and PhoRC complexes differently correlate with replication timing of their targets. In the fully repressed BX-C, loss of function experiments revealed a synergistic role for PcG proteins in the maintenance of replication programs through the mediation of higher-order structures. Accordingly, replication timing analysis performed on two Drosophila cell lines differing for BX-C gene expression states, PcG distribution, and chromatin domain conformation revealed a cell-type-specific replication program that mirrors lineage-specific BX-C higher-order structures. This work suggests that PcG complexes, by regulating higher-order chromatin structure at their target sites, contribute to the definition and the maintenance of genomic structural domains where genes showing the same epigenetic state replicate at the same time (Lo Sardo, 2013).

The epigenome in its overall complexity, including covalent modifications of DNA and histones, higher-order chromatin structures and nuclear positioning, influences transcription and replication programs of the cell. It is well known that timing of DNA replication is correlated with relative transcription state, in particular transcriptionally active genes tend to replicate early and inactive genes tend to replicate late. However, in recent years, genome-wide analyses revealed several exceptions to this rule. These and other evidence suggested that the transcriptional potential of chromatin, expressed as histone modifications and transcription factors binding (rather than the process of transcription per se) is most closely related to replication timing. A recent work in Drosophila has shown that the selection and the timing of firing of replication origins are associated with distinct sets of chromatin marks and DNA binding proteins (Eaton, 2011). This reinforces previous works showing that mutation, overexpression, depletion or tethering of chromatin modifying proteins to specific loci in yeast, Drosophila and vertebrates determines changes in replication timing locally or/and at a global level. In mammals, it has been suggested that higher-order chromatin structures more than basal epigenome modifications better correlate with replication timing profiles. Although several proteins have been reported to control higher-order chromatin structure formation, their role in replicon structure and replication timing regulation remains to be elucidated. Among these, cohesins have been shown to co-localize with ORC binding sites and to influence replication origin choice and density through the regulation of specific chromatin loops. Previously, it has been reported that PcG proteins are key regulators of higher-order chromatin structures and that condensins complex components and Topoisomerase II take part in PRE and BX-C silencing functio. Moreover, depletion of the mammalian PC homologue M33 determines a switch of the INK4a/ARF locus replication timing, suggesting a role for PcG proteins in the regulation of replication programs at their targets (Lo Sardo, 2013).

However, the interplay between PcG-mediated silencing, higher-order structures and control of replication timing in Drosophila has not been elucidated. This issue has been addressed on a genome-wide level; H3K27me3 enriched and PRC2 bound sequences were found to replicate later than their unbound counterparts. Surprisingly, the same is not true for PRC1 or PhoRC target sites, where the binding of PcG proteins does not significantly correlate with genome-wide replication timing distributions, highlighting a difference between PRC1, PhoRC and PRC2 complexes at a genome-wide scale. Notably, replication timing is more correlated to PRC1 binding at transcribed TSSs than at silenced TSSs (Lo Sardo, 2013).

To investigate the possible contribution of PRC1 and other PcG complexes at repressed genes, the functional interplay between PcG-dependent epigenetic signatures and maintenance of replication programs were analyzed at one of the major PcG targets: the Drosophila BX-C. After depletion of single PcG proteins in S2 cells, reactivation was found of BX-C genes and their related PREs. Interestingly, depletion of PHO protein causes only a mild effect on homeotic genes transcription, although this protein has been reported to be required for recruitment of PRC1 and 2. This suggests that multiple additional mechanisms of recruitment, such as ncRNAs or other protein partners, may act simultaneously at PcG target loci, as described particularly in mammalian cells. Interestingly, also 3C analysis in single PcG depleted cells reveals a different response to E(z) depletion with respect to PC and PHO depletions. In particular, in PC and PHO depleted cells no change or the small reduction of some PRE-PRE and PRE-promoter BX-C interactions were seen, while in E(z) depleted cells even an increase in specific crosslinking frequencies for some interactions was observed. Moreover, both 3C and replication timing analysis in single PcG depleted cells show that transcription per se cannot dramatically perturb the BX-C higher-order structures neither change the timing of replication. This result is in agreement with recent findings in mammalian cells showing that spatial chromatin organization and replication timing are not a direct consequence of transcription (Lo Sardo, 2013).

Conversely, simultaneous depletion of components of the three major PcG complexes (PhoRC, PRC2 and PRC1) determines major changes in BX-C transcription as well as in higher-order structure and an anticipation in replication timing, suggesting that PcG proteins act synergistically on three-dimensional structures and on replication program maintenance. In line with these findings, in recovered cells, BX-C topological structure and PRE replication timing are indistinguishable from controls, suggesting that the observed variations are not sufficient to determine a stable epigenetic switch. In this context the more stable contacts might hamper an irreversible disruption of the three-dimensional BX-C structure (Lo Sardo, 2013).

These findings were further confirmed by the comparison of two different cell lines: S2 and S3 that differ for their embryonic origin. Previous studies have shown that in S3 cells, active transcription of AbdB is associated with different topological conformation of the locus, where AbdB gene and its regulative PREs are topologically separated from the other repressed and clustered epigenetic elements of the locus. This study found that distinct chromatin structures in S2 and S3 are associated with different replication timings, thus confirming that these epigenetic parameters vary in parallel (Lo Sardo, 2013).

This analysis, in line with recent observations, indicates that the genome may be organized into distinct structural and functional domains in which DNA regions that stay together replicate together as a stable unit for many cell generations irrespective of single gene transcription state. It was shown that major adjustments of chromatin higher-order structure and replication program are necessary for a correct differentiation and are required for reprogramming of cell identity. The high stability of higher-order chromatin structures and replication programs can explain one of the underlying molecular basis counteracting cellular reprogramming and representing an epigenetic barrier and PcG complexes may play an important role in the maintenance of this barrier. The data show that correct levels of PcG components can fully restore silencing, higher-order structures and late replication timing at derepressed BX-C gene loci. Of course, additional functions may be involved in the maintenance of these epigenetic parameters either at the BX-C and in the rest of the genome. For example, other factors involved in the regulation of higher-order chromatin structure, including the insulator CTCF protein, condensin complex subunits and Topoisomerase II, were shown to have a role in PcG-mediated gene silencing function. Interestingly, Topoisomerase II has been shown to be required for a global resetting of replicon organization in the context of somatic cell reprogramming. Hence, a deeper understanding of the functional interplay between epigenetic mechanisms modulating the stability of higher-order chromatin structure and replication program will be crucial to unravel the molecular basis (Lo Sardo, 2013).

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

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

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

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

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

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

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

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

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

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

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

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

Dynamic regulation by polycomb group protein complexes controls pattern formation and the cell cycle in Drosophila

Polycomb group (PcG) proteins form conserved regulatory complexes that modify chromatin to repress transcription. This study reports genome-wide binding profiles of PhoRC, the Drosophila PcG protein complex containing the DNA-binding factor Pho/dYY1 and Scm-related gene containing four mbt domains (dSfmbt). PhoRC constitutively occupies short Polycomb response elements (PREs) of a large set of developmental regulator genes in both embryos and larvae. The majority of these PREs are co-occupied by the PcG complexes PRC1 and PRC2. Analysis of PcG mutants shows that the PcG system represses genes required for anteroposterior, dorsoventral, and proximodistal patterning of imaginal discs and that it also represses cell cycle regulator genes. Many of these genes are regulated in a dynamic manner, and the results suggest that the PcG system restricts signaling-mediated activation of target genes to appropriate cells. Analysis of cell cycle regulators indicates that the PcG system also dynamically modulates the expression levels of certain genes, providing a possible explanation for the tumor phenotype of PcG mutants (Oktaba, 2008).

It was asked whether the chromosomal intervals identified by Pho or dSfmbt ChIP-chip are enriched for particular DNA sequence motifs. To this end, de novo sequence motif discovery was performed on the Pho^E-, Pho^L-, dSfmbt^L-bound regions (superscript E refers to embryos and superscript L to larvae) and on the 196 core-PhoRC regions. Several 8-mers, based on a GCCAT core were significantly overrepresented in the Pho datasets and were used as the basis to reconstruct a position sequence-specific matrix (PSSM). This sequence, G^C/_A^C/_GGCCAT^T/_CTT, closely matches the motif previously identified in vitro as an optimal Pho-binding site using electro mobility shift assays. However, in vivo binding data suggest a more extensive Pho-binding motif containing an additional Guanine nucleotide at the -3 position and a Thymine pair at positions +7 and +8. This extended Pho-binding motif is enriched in all data sets. However, it is noteworthy that no Pho-binding site was identified as an overrepresented motif in the subset of dSfmbt^L regions not bound by Pho (i.e., the 328 dSfmbt^L regions). The lack of Pho enrichment by ChIP and the absence of detectable Pho binding sites in this fraction of the dSfmbt^L dataset indicate that dSfmbt is targeted to these regions independently of Pho (Oktaba, 2008).

Global identification of genomic locations to which PcG protein complexes bind and unraveling how expression of target genes is regulated by these complexes is important to understand how the PcG system controls transcription of the genome. The following main conclusions can be drawn from the work presented in this study: (1) PhoRC is sharply localized at discrete PRE sequences, many of which are co-occupied by PRC1 and PRC2. (2) 196 PREs were identified in the Drosophila genome where PhoRC is constitutively bound in embryos and larvae. In general, these PREs are located within ±1 kb from the closest gene transcription start site and the majority of target genes contain only one PRE. (3) Sequence analyses of identified PREs allowed definition of an extended Pho-binding motif that is part of the signature of PhoRC-bound PREs. (4) Functional analyses in Drosophila reveal that PcG proteins repress transcription of several key developmental regulators. In particular, the PcG system is required for maintaining the subdivision of segment primordia into anteroposterior, dorsoventral and proximodistal compartments by repressing the genes en, ap, pnr, tsh, and Dll. (5) As discussed in detail below, these analyses suggest that extracellular signaling can selectively induce transcription of previously silent PcG target genes, even though PcG protein complexes are bound at their PREs. (6) Among the PcG target genes are also cell cycle regulators such as Rbf, E2F, Dp, and CycB, and evidence is providedthat the PcG system regulates expression levels of the CycB gene in Drosophila (Oktaba, 2008).

Genome-wide binding profiling and ChIP analyses at selected target genes revealed an extensive overlap between PhoRC-, PRC1-, and PRC2-bound regions. These results, together with previous reports that Pho interacts with and is required for targeting of PRC1 and/or PRC2 at HOX gene PREs, suggest that PhoRC is needed for PRC1 and PRC2 binding at many PcG target genes. It should also be recalled that in larvae, PhoRC, PRC1, and PRC2 are all constitutively bound to PREs of the HOX gene Ubx, both in cells where Ubx is repressed and in cells where it is active. The observation that the same PRE sites are occupied in both tissue culture cells and in developing Drosophila thus further implies that PhoRC, PRC1, and PRC2 may be constitutively bound to a large fraction if not most of their target genes (Oktaba, 2008).

What are the sequences that make up a PRE? Using an algorithm based on binding site motifs for Pho, GAF/Trl, and Zeste proteins, 167 PREs have been predicted across the Drosophila genome. This study detected PhoRC binding at 26 of these predicted PREs (15%). Intriguingly, this study found a significant overlap between PhoRC-bound regions and regions bound by GAF/Trl but only a limited overlap with Zeste-bound regions. GAF/Trl mutants do not show HOX misexpression phenotypes, making its role at HOX gene PREs somewhat enigmatic. However, it is possible that GAF/Trl is required for PRC1 and/or PRC2 targeting to PREs of non-HOX genes. Finally, it is important to keep in mind that PRC1 and PRC2 are also bound at genomic regions where PhoRC has not been detected, suggesting that they are targeted there by other factors (Oktaba, 2008).

The most obvious phenotype of PcG mutants in Drosophila are homeotic transformations, caused by the global misexpression of multiple HOX genes. In this study, loss of repression of several non-HOX target genes was monitored and it was shown that these genes are indeed also misexpressed in PcG mutants. In particular, it was found that the PcG system represses key regulator genes required for the subdivision of appendages into anteroposterior, dorsoventral, and proximodistal compartments. This suggests that the PcG system is responsible for maintaining many more cell fate decisions than may be immediately evident from the phenotype. In this context, it is important to note that at some target genes, control by PcG proteins is masked by other regulatory interactions and can only be revealed in the absence of those regulatory inputs. The downregulation of Dll in PcG mutant clones by HOX proteins represents a prime example for such a masking effect; regulation of Dll by the PcG system could only be revealed in cells lacking both PcG and HOX gene functions (Oktaba, 2008).

Finally, it was found that some target genes are only strongly misexpressed in certain PcG mutants but not in others, even though PhoRC, PRC1, and PRC2 are all cobound at these genes. This implies that in those cases not all PcG protein complexes or complex components are required for repression. In the most extreme scenario, recruitment of all three complexes reflects the default state that even occurs at genes that require i.e., only H2A ubiquitylation by PRC1 but not H3-K27 methylation by PRC2 for repression (Oktaba, 2008).

The finding that PcG protein complexes are constitutively bound at PREs of target genes in both embryonic and imaginal disc cells has implications for understanding regulation by the PcG system. In particular, target genes such as ap, Dll, or pnr are not active in the wing disc primordium in embryos, remain silent as the primordium grows during the early larval stages and become transcriptionally active only at later larval stages. The factors responsible for activating ap transcription during the second larval instar are not known, but induction of Dll expression in the wing blade primordium during the third larval instar occurs in response to wg signaling, and expression of pnr in the notum primordium during the second larval instar is activated by dpp signaling. The most straightforward explanation of these observations is that these signaling pathways are able to switch on expression of these target genes even though PcG complexes are bound at their PREs and even though their chromatin bears the Polycomb-repressive H3-K27me3 mark in embryos. In wild-type wing discs, Wg-signaling would thus be able to overcome PcG repression at the Dll gene in wing pouch cells but not in the notum and hinge region where Wg protein is also present. Similarly, in wild-type animals, Dpp-signaling activates pnr expression only in the notum and not along the whole length of the anteroposterior compartment boundary where Dpp protein is expressed. One possible explanation for this selective activation in discrete parts of the disc would be a requirement for signaling pathways to act in a combinatorial manner with other (unknown) factors to relieve PcG repression. Consistent with this, removal of PcG function often results in misexpression of target genes only in specific regions of the disc, and these regions receive the same signals that are also responsible for activation of these target genes in their normal wild-type expression domain. For example, misexpression of pnr mainly occurs in PcG mutant clones in the wing blade and thus in cells that receive Dpp, the signal that also activates pnr expression in its wild-type expression domain in the notum. This raises the intriguing possibility that an important function of the PcG system may be to spatially restrict activation of target genes in response to more widely distributed extracellular signals (Oktaba, 2008).

Previous ChIP-chip studies showed that PcG protein complexes are bound to a large number of developmental regulators in mammalian embryonic stem cells. The majority of the genes bound by PcG proteins in stem cells are orthologs of PcG target genes identified in flies, including the family orthologs of en, ap, pnr, Dll, eve, and Doc whose regulation was analyzed in this study. The finding that these genes are regulated by the PcG system during Drosophila development implies that the mammalian PcG system may also regulate the orthologous genes in differentiating cells and tissues, beyond the known regulation in stem cells (Oktaba, 2008).

This study identified cell cycle regulator genes as PcG targets, and evidence is provided that the PcG system directly regulates CycB expression. Control of cell cycle regulators by the PcG system may provide a molecular explanation for the tumor phenotype observed in proliferating imaginal disc cells lacking the PRC1 components Psc-Su(z)2 or ph. The observation that the PcG system controls transcription of genes whose expression is modulated during the cell cycle suggests that the PcG system is also used to regulate target genes more dynamically than previously thought. In mammalian cells, knockout of the Psc homolog bmi-1 results in cellular senescence via loss of p16/INK4A transcriptional repression, a cyclin D regulator without any obvious ortholog in the Drosophila genome. It therefore appears that the PcG system has a conserved role in regulating expression of genes involved in body patterning but that it evolved in different ways to control cell growth and proliferation in mammals and flies (Oktaba, 2008).

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

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

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

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

Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene

Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, chromatin immunoprecipitation studies were performed using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. It was found that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. These results are discussed in light of current models for recruitment of PcG complexes to chromatin targets (Wang, 2010).

How might SCM fit in molecularly with the other PcG components Although in vitro associations of SCM with PRC1 subunits have been described, the ChIP analyses here indicate that SCM can associate with the Ubx PRE despite the loss of PRC1. Similarly, although SCM can bind to the PHO-RC subunit SFMBT in a pairwise assay, SCM localization at the PRE does not appear to be dependent on PHO. Taken together, these ChIP results are consistent with biochemical studies that reveal SCM separability from PHO-RC, PRC1, and PRC2 in fly embryo extracts (Wang, 2010).

An intriguing finding from the matrix of molecular epistasis tests is that SCM exhibits recruitment properties very similar to those of PHO. Specifically, both SCM and PHO can localize to the Ubx PRE independent of all other PcG components tested, and loss of either SCM or PHO diminishes PRC2 and PRC1 association with the PRE. This similarity suggests that SCM may function, like PHO-RC, at an early step in PcG recruitment. In this context, it is worth emphasizing the striking overall similarities between SCM and the PHO-RC subunit SFMBT. Perhaps SCM partners with a yet-to-be identified PcG DNA-binding protein, akin to the functional partnership of SFMBT with PHO. Indeed, since PHO-binding sites are insufficient for PRE function in vivo and many other DNA-binding proteins have been implicated in Drosophila PcG silencing, there is abundant evidence that PRE recognition involves more than just PHO-RC. The common view is that many PREs contain a composite of PHO sites plus additional types of factor-binding motifs. At present, little is known about the nature of SCM-containing complexes beyond the detection of an approximately 500-kDa moiety in fly embryo extracts. It will be informative to characterize stably associated SCM partner proteins and evaluate their potential roles in binding to PRE DNA (Wang, 2010).

Although the ChIP assays presented in this study emphasize SCM separability from other PcG components, SCM must still integrate with its PcG cohorts to achieve gene silencing. This interdependence is highlighted by in vivo assays where robust silencing of a miniwhite reporter by a tethered form of SCM is disrupted if the PRC1 subunit PH is compromised by mutation. Despite advances in understanding biochemical activities of individual PcG complexes, it is not yet clear how their multiple functions are integrated to achieve gene silencing. Further studies will be needed to determine how SCM functions in concert with other PcG components at target chromatin (Wang, 2010).

Ultimately, a precise understanding of SCM function requires deciphering the mechanistic contributions of each of its three identified domains. SCM contains a C-terminal SPM domain, two mbt repeats, and two Cys₂-Cys₂ zinc fingers. Strikingly, each of these domains is also present in SFMBT, suggesting that the overall biochemical roles of these two PcG components may be very similar. Indeed, a recent study provides evidence of functional synergy between SCM and SFMBT (Grimm, 2009). In addition, the PH PcG protein possesses two of these three homology domains. This presents the curious situation of three different PcG proteins related by shared domains yet with none appearing to reside in a stable common complex in nuclear extracts (Wang, 2010).

There are currently in vitro and in vivo data on roles of the SPM domain and mbt repeats but little knowledge yet about the zinc fingers. The SPM domain is a subtype within the broader category of SAM domains that mediate protein interactions. The SCM version of this domain is capable of robust self-binding and cross-binding to the PH version in vitro. The importance of SPM domain interactions in vivo is emphasized by PcG phenotypes observed after overexpressing a dominant-negative isolated SPM domain in developing flies (Peterson, 2004). However, it remains unclear precisely what SPM interactions contribute to the PcG silencing mechanism. The simple idea that they constitutively glue PcG complex subunits together is at odds with the biochemical separabilities in embryo extracts. Perhaps SPM interactions function primarily directly at chromatin targets, where they could sponsor contacts among different PcG complexes rather than among subunits within the same complex. Such chromatin-specific interactions could contribute to intralocus loops, which have been hypothesized to exist at PcG silenced loci (Wang, 2010).

The functional significance of the SCM mbt repeats is reflected by partial loss-of-function alleles that alter the first repeat and by Hox gene silencing defects observed after disruption of the second repeat. Structural determinations and in vitro binding studies have revealed that mbt repeats are modules for binding to methylated lysines. Since trimethylated H3-K27 is a prominent feature of PcG-silenced chromatin, the mbt repeats could, at first glance, play a role akin to that of the PC chromodomain. However, there are important differences between the substrate-binding properties of these mbt repeats and the PC chromodomain. First, the mbt repeats prefer mono-and dimethylated lysines, whereas the chromodomain prefers the trimethylated form. An intriguing hypothesis is that this mono/di preference could reflect a 'grappling hook' function whereby hypomethylated nucleosomes are recognized and brought into proximity for trimethylation by PRC2. Another distinction is that the binding mode of mbt repeats is not much influenced by peptide sequence context, whereas chromodomain binding features extensive contact with residues flanking the methylated lysine. Consistent with this, the SCM mbt repeats lack binding preference for any particular histone tail lysines. Thus, mbt repeats provide a pocket for methyl-lysine binding, but it is not yet clear if the relevant substrate for SCM is a particular methylated histone residue or even a nonhistone protein. Certainly, the in vitro binding preferences could be modified by additional associated factors in vivo (Wang, 2010).

A sequence alignment of the Cys₂-Cys₂ fingers present in SCM, SFMBT and PH shows that this zinc finger is a distinct subtype that adheres to the consensus sequence CXXCG-X_n-K/R-X-F/Y-CSXXC. These fingers do not appear to function by binding DNA, since sequence-specific binding is not observed in vitro for any of them. Thus, their molecular role is unknown, but their common inclusion in these related fly PcG proteins suggests some key contribution to PcG chromatin function. Curiously, both the SCM and SFMBT human homologs appear to have lost their Cys₂-Cys₂ fingers, whereas all three human PH homologs have retained them. Thus, if these zinc fingers are critical in PcG silencing, then they apparently can be supplied from different combinations of PcG proteins in flies and in mammals. It will be important to test the genetic requirement for the SCM zinc fingers in Drosophila and to further define the mechanistic contributions of all three SCM functional domains to PcG chromatin silencing (Wang, 2010).

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

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

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

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

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

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

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

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

Mapping Polycomb Response Elements at the Drosophila melanogaster giant Locus

Polycomb-group (PcG) proteins are highly conserved epigenetic transcriptional regulators. They are capable of either maintaining the transcriptional silence of target genes through many cell cycles or enabling a dynamic regulation of gene expression in stem cells. In Drosophila melanogaster, recruitment of PcG proteins to targets requires the presence of at least one Polycomb Response Element (PRE). Although the sequence requirements for PREs are not well defined, the presence of Pho, a PRE-binding PcG protein, is a very good PRE indicator. This study identified two PRE-containing regions at the PcG target gene, giant, one at the promoter and another approximately 6 kb upstream. PRE-containing fragments, which coincide with localized presence of Pho in chromatin immunoprecipitations, were shown to maintain restricted expression of a lacZ reporter gene in embryos and to cause pairing-sensitive silencing of the mini-white gene in eyes. The results also reinforce previous observations that, although PRE maintenance and pairing-sensitive silencing activities are closely linked, the sequence requirements for these functions are not identical (Alhaj Abed, 2013).

Cooperativity, specificity, and evolutionary stability of polycomb targeting in Drosophila

Metazoan genomes are partitioned into modular chromosomal domains containing active or repressive chromatin. In flies, Polycomb group (PcG) response elements (PREs) recruit Pho and other DNA-binding factors and act as nucleation sites for the formation of Polycomb repressive domains. The sequence specificity of PREs is not well understood. This study used comparative epigenomics and transgenic assays to show that Drosophila domain organization and PRE specification are evolutionarily conserved despite significant cis-element divergence within Polycomb domains, whereas cis-element evolution is strongly correlated with transcription factor binding divergence outside of Polycomb domains. Cooperative interactions of PcG complexes and their recruiting factor Pho stabilize Pho recruitment to low-specificity sequences. Consistently, Pho recruitment to sites within Polycomb domains is stabilized by PRC1. These data suggest that cooperative rather than hierarchical interactions among low-affinity sequences, DNA-binding factors, and the Polycomb machinery are giving rise to specific and strongly conserved 3D structures in Drosophila (Schuettengruber, 2014).

Comparative epigenomics was used to demonstrate that Polycomb domains are an extremely well conserved feature of the genome during fly evolution. In fact, the evolutionary profile of epigenomic domain organization in embryos of five Drosophila species indicates a complete lack of divergence of H3K27me3- marked Polycomb domains in syntenic regions. A similar high conservation of the H3K27me3 pattern across Drosophila species was recently described. Polycomb domains typically harbor several PH-marked PREs, and a comparative analysis showed that these are also highly conserved and the few loci that show a divergence of PRC1 occupancy patterns are not correlated with overall domain divergence. Likewise, the binding of PHO and DSP1 is highly conserved (to a degree at least as strongly, and possibly more strongly, than binding of individual factors), but even cases of diverged factor occupancies are usually not correlated with overall PRE divergence. In marked contrast, the sequences underlying PREs and Polycomb domains are diverging extensively, and sequence-based prediction of PREs across Drosophila species suggested that divergence of PREs could occur frequently. However, neither ChIP-seq experiments nortransgenic reporter assays support this dynamic behavior. Instead, such sequence divergence is buffered by the epigenetic targeting mechanisms to maintain Polycomb domains. It is suggested that the multilayered organization uses redundancy and cooperativity to facilitate the remarkable Polycomb domain conservation. This is occurring both in cis, where several TFs collaborate to define a regulatory element even when the underlying sequence is imperfect, and at the domain level, where several PREs participate to define the PcG domain structure and possibly stabilize each other (Schuettengruber, 2014).

Although PREs are associated with several known sequence features (such as GAGA- and PHO-binding motifs) in a statistically significant way, these features are not sufficient to distinguish many PREs from the genomic background and from other PHO- or DSP1-bound active chromatin elements. There are many possible explanations for this lack of specificity, including the existence of additional, yet-to-be-characterized sequence-specific recruiting factors; the involvement of nucleosome positioning; transcription of non-coding RNAs; or imperfect modeling of the sequence specificity of the known factors. The data presented in this study, however, introduce a new perspective that can help resolve this conundrum. In contrast to previous hypotheses, the data show that even when strong binding sites are lacking, PHO and DSP1 may bind PREs directly through weak (but highly nonrandom) motifs. Remarkably, sequence affinities that are completely nonspecific on a genomic scale (possibly defining millions of spurious sites) are still highly informative for predicting the binding intensity within the context of a PRE. The strong correlation of PHO binding with weak but nonrandom motifs makes it unlikely that binding to these sites represents indirect binding via interaction/looping with strong binding sites. The data show that in order to understand PRE sequence specificity, multiple potential binding sites with variable affinities and fidelities must be taken into account, and their cooperative interaction must be considered in the context of the PRE chromosomal landscape. This idea is compatible with the evolutionary constraints on PRE sequences, which has been demonstrated in this study to affect a spectrum of binding affinities rather than to conserve classical binding sites alone (Schuettengruber, 2014).

What might be the molecular mechanism that allows the specific binding of weak sites in the context of Polycomb domains? One possibility is that cooperative binding of TFs at PREs supports their occupancy of weak motifs. Indeed, this study found that PHO and DSP1 are bound jointly at PREs (with weak underlying sequence motifs), whereas at other regions of the genome where the factors bind alone, they are usually associated with strong sequence motifs. This observation is in agreement with the recently proposed 'TF collective model,' according to which combinatorial TF binding occurs with little or no apparent sequence motifs for at least a subset of the bound factors (Schuettengruber, 2014).

In addition, it was shown that transient interactions of DNA-binding proteins with weak affinity sites are stabilized by the presence of the PcG proteins themselves. A similar observation of a positive feedback of PRC1 on PHO binding was recently reported (Kahn, 2014) and is further supported by the fact that cooperative binding of PHO and Polycomb to PREs can occur even in vitro. In vivo, long-range contacts involving remote PREs within the same (or even a different) Polycomb domain may contribute to this process. Clustering of multiple flanking PREs in the 3D space of the nucleus might generate Polycomb compartments characterized by high concentrations of PcG proteins as well as their recruiting DNA-binding proteins. In this scenario, loss of occupancy following the dissociation of any of these factors from DNA may be more easily replenished by the concentrated stock of factor within a Polycomb compartment compared with individual binding sites present elsewhere in the genome. This may push the equilibrium toward increased PHO and DSP1 binding to low-affinity sites and partially reduce the evolutionary pressure to maintain the nucleotidic sequence of recruiter motifs at PREs. Structural long-range effects may also inhibit PcG recruitment in cases where active enhancers and TSSs are in proximity to a candidate PRE sequence. This analysis suggests that H3K4me3-marked loci are also highly conserved, but the low-affinity PHO- or DSP1-binding sites in them are completely uncorrelated with occupancy of these factors, further supporting a model of highly organized and cooperative epigenomic organization (Schuettengruber, 2014).

In conclusion, the data indicate that sequence conservation collaborates with 3D chromatin architecture to maintain an exceptional evolutionary stability of Polycomb-regulated loci in fly genomes. This phenomenon highlights the contribution of chromosome domains and their particular looping structures to epigenomic specificity and genome evolution. Hi-C analysis in mammals has revealed that topological domains are a strikingly conserved feature between the mouse and human genomes. The current data raise the possibility that, beyond combinatorial contributions by TF-binding sites in close proximity, the confinement of regulatory elements within TADs and their frequent DNA contacts constitute significant driving forces that also affect DNA sequence evolution in these and possibly many other specie (Schuettengruber, 2014).

Protein Interactions

GAGA facilitates binding of Pleiohomeotic to a chromatinized Polycomb response element

Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Using DNaseI footprinting analysis, a high-resolution map is provided of sites for the sequence-specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. While GAGA can efficiently bind to a chromatinized PRE, PHO alone is incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, is strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins (Mahmoudi, 2003).

This study has determined the precise distribution within the Ubx PRE of the recognition elements for four sequence-specific DNA-binding proteins that have all been implicated in Ubx regulation in vivo: PcG protein PHO, gap protein HB and trxG proteins GAGA and Zeste. The results indicate that, rather than a random collection, the binding site distribution within the Ubx PRE reflects a functional arrangement, allowing cooperation between distinct PRE binding proteins. Of particular interest is the observation that chromatin binding by the PcG protein PHO is strongly facilitated by the trxG protein GAGA. This finding provides a molecular mechanism for the requirement for both factors during PRE-directed silencing in vivo, and suggests that PHO and GAGA elements together may form a functional module (Mahmoudi, 2003).

Several independent genetic studies have pointed to a concurrent requirement for GAGA and PHO during gene silencing directed by distinct PREs. The PcG-dependent silencing conferred by a 230 bp fragment of the iab-7 PRE is dependent on both GAGA and PHO binding. Similarly, a 138 bp fragment of the MCP silencer, which was found to be sufficient for maintenance of embryonic silencing, contains PHO and GAGA sites. Mutations in either PHO or GAGA sites compromised silencing and revealed cooperation between both proteins. Particularly relevant for the current study are results that support a critical role in PcG silencing for GAGA and PHO sites within the Ubx PRE (Mahmoudi, 2003).

Functional dissection of the Ubx PRE has revealed that a Pc-dependent PRE silencer is contained in the central 567 bp fragment from position 577 to 1143, which includes all PHO and the highest density of GAGA sites. Another study showed that an oligomerized subfragment, corresponding to positions 890-1079 within PRE C, harboring two PHO and five GAGA elements, is able to confer PcG silencing in vivo. Finally, deletion of a 160 bp region corresponding to positions 851-1011 within PRE C impairs maintenance of silencing. The large extent of overlap between the DNA fragments identified in these independent studies strongly suggests that the common region within PRE C represents the critical core of the Ubx PRE. The most noticeable feature of this region is the many alternating GAGA and PHO binding elements. Moreover, it is of interest to note that footprinting analysis revealed the presence of Zeste as well as HB sites within this region, which may also contribute to the in vivo maintenance of repression (Mahmoudi, 2003).

The identification of Zeste as a component of the PRC1 PcG complex, suggests that it may play a direct role in PcG complex recruitment to the Ubx PRE. Further evidence for the involvement of Zeste in the maintenance of Ubx repression as well as activation has been provided by transgene experiments. Finally, the presence of HB sites within the Ubx PRE suggests a potential role for HB, not only during the initiation of Ubx repression, but also during the transition from establishment to maintenance. One attractive possibility is that this transition involves dMi-2 recruitment by HB. It should be noted that in the absence of initiating activation and repression elements, HB-independent PcG repression of the Ubx promoter has been documented (Mahmoudi, 2003).

Although there is substantial evidence for the notion that the proteins discussed above are involved in PcG silencing of homeotic genes, it remains unclear whether they can be sufficient for targeting or whether additional factors are required. One way to determine a minimal set of protein recognition sequences that can mediate PcG silencing will be the generation of synthetic PREs, which should be tested in vivo. The results suggest that, within such a PRE, PHO sites will need to be flanked by GAGA sites in order to facilitate chromatin binding. The proteins GAGA and Zeste may be particularly well adapted for such a purpose. Both GAGA and Zeste form large homo-oligomers that bind cooperatively to the multiple sites present in their natural response elements, such as the Ubx PRE and promoter. This cooperative mode of DNA-binding may allow these proteins to first bind an accessible site within a nucleosomal array and then progressively displace histones during binding to flanking sites. In addition, GAGA and Zeste have both been shown to recruit selective ATP-dependent chromatin remodeling factors. The process of targeting of remodelers to specific DNA elements may enable GAGA and Zeste to create nucleosome-free or remodeled areas, thus facilitating binding of other regulators. It is considerede likely that the remodeling complexes present in the chromatin preparations used in assays, are involved in the observed synergistic binding between PHO and either GAGA or Zeste (Mahmoudi, 2003).

GAGA oligomerization may also promote the communication between the Ubx PRE and promoter. Both elements, which are separated by ~24 kb of intervening DNA, contain a preponderance of binding sites for GAGA. GAGA oligomerization through its POZ domain allows it to form a protein bridge that directs long-range enhancer-promoter association. In fact, GAGA could even mediate enhancer function in trans by simultaneous binding of two separate DNA fragments. Thus, it is tempting to speculate that GAGA may link the Ubx PRE to the Ubx promoter. It should be noted that both the chromatin remodeling and long-range bridging functions of GAGA might accommodate PRE-mediated activation as well as repression (Mahmoudi, 2003).

The interdependence between proteins belonging to antagonistic genetic groups for efficient chromatin binding described it this study will have to be taken into account when interpreting mutational analysis of PRE function. Thus, removal of recognition sequences for the trxG protein GAGA may block its activation function but could also affect binding of the PcG protein PHO. Moreover, recent results suggest additional opportunities for cross-talk during recruitment of non-DNA-binding PcG complexes. Although a clear consensus between different studies is still lacking, there is experimental evidence for PcG complex recruitment by PHO, GAGA and Zeste. Because binding sites for either one of these proteins alone do not confer PRE function, it appears likely that they work in a combinatorial fashion. Depending on their context, the multitude of distinct binding elements that constitute a PRE might be redundant, cooperative or antagonistic to each other. Furthermore, distinct PREs may require different sets of PRE-binding proteins, and additional recruiters may be involved in PcG-silencing. Attractive candidates are GAGA-related factors batman and the PHO-related factor PHO-like (Mahmoudi, 2003).

In conclusion, current evidence suggests that PRE-directed maintenance of gene activation or repression is not achieved by a simple binary switch set by competing trxG and PcG proteins. Although their relative ratios vary considerably and correlate with transcription levels, they coexist at PREs during gene activation as well as repression. Likewise, genetic suppressor studies indicated extensive cross-talk between PcG and trxG proteins. This study has shown that, already at the level of PRE binding, there is strong interdependence between trxG protein GAGA and PcG protein PHO. The results demonstrate a direct biochemical mechanism for the cooperation between PcG and trxG proteins during PRE binding (Mahmoudi, 2003).

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).

Requirement for Sex comb on midleg protein interactions in Drosophila polycomb group repression

The Drosophila Sex comb on midleg (Scm) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish Scm as a crucial PcG member, its molecular role is not known. To investigate how Scm might link to PcG complexes, the in vivo role of a conserved protein interaction module, the SPM domain was analyzed. This domain is found in Scm and in another PcG protein, Polyhomeotic (Ph), which is a core component of Polycomb repressive complex 1 (PRC1). Scm-Ph interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize greater than 30 missense mutations in the SPM domain of Scm. Genetic rescue assays show that Scm repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of Scm with a reconstituted PRC1 core complex shows that Scm can partner with PRC1. However, gel filtration chromatography showed that the bulk of Scm is biochemically separable from Ph in embryo nuclear extracts. These results suggest that Scm, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing (Peterson, 2004).

Purifications of nuclear complexes and in vitro studies have identified eight proteins that are core components of two distinct fly PcG complexes: Esc, E(z), Su(z)12, and NURF-55 in the ESC-E(Z) complex plus Pc, Ph, Psc, and Sex combs extra/dRING1 in PRC1. One function of the Esc-E(z) complex is histone H3 methylation on K27. Further studies are needed to address whether the Esc-E(z) complex has additional functions. The molecular mechanism of PRC1 is not yet known. Studies to date suggest that it represses transcription through a noncatalytic mechanism that restricts template access, but it is not yet clear how PRC1 molecularly affects nucleosome array organization and/or packaging of the chromatin fiber. Since genetic studies in Drosophila identify at least 15 genes involved in PcG repression, many additional components need to be fit into the framework of PcG complexes and functions. In addition to identifying the players, analyses of loss of function for individual PcG genes distinguishes those repressors with central PcG roles from those that are more peripheral. In good agreement with the biochemical studies, loss of function for core subunits of either PcG complex produces severe homeotic defects. These mutants show robust Hox misexpression and die as embryos with most segments transformed into copies of the eighth abdominal segment. By these criteria, Scm is clearly a central player in the PcG repression system. In contrast, other repressors such as Asx and Pcl appear more peripheral since their complete loss from embryos yields significantly weaker homeotic defects (Peterson, 2004).

In this work, a combination of in vivo and in vitro approaches are presented to address Scm molecular function. Mutational analysis shows that Scm function absolutely depends upon an intact SPM protein interaction domain. There is a strong correlation between disruption of protein interactions in vitro and failure of Scm function in vivo. These results agree with the finding that Scm repressor function in an in vivo tethering assay requires its SPM domain. The importance of SPM domain interactions is also revealed by PcG loss-of-function phenotypes produced by overexpression of an isolated SPM domain. It is suggested that this dominant negative reflects SPM domain interactions critical for PcG repression that are disrupted by this avidly binding but otherwise nonfunctional competitor. The embryonic lethality of SPM domain mutants, together with embryonic and imaginal defects seen with SPM overexpression, indicate that SPM interactions contribute to PcG repression at both embryonic and postembryonic times. Thus, these interactions appear required for long-term maintenance of PcG silencing in vivo (Peterson, 2004).

The biochemical properties of the SPM domain suggest three potential types of Scm interactions in vivo: (1) binding to PRC1, (2) binding to other fly SPM domain proteins, or (3) binding to itself. Although the data do not rule out contributions from any of these, several lines of evidence favor Scm interaction and function with PRC1. (1) in vivo evidence derives from studies showing that Scm can repress reporter genes when tethered by fusion to a DNA-binding domain. Since this repression depends upon Ph function, Scm cannot repress on its own but rather requires PRC1 to repress in this context. (2) Substoichiometric quantities of Scm consistently copurify with tagged PRC1 complexes from both fly and mammalian extracts. Although the majority of Scm appears to not be stably bound, the conserved association of some Scm with purified PRC1 likely reflects in vivo interactions. (3) No stably associated partner proteins have been detected that copurify when FLAG-Scm is affinity purified from embryo extracts. Thus, there is no evidence for a heteromeric Scm-containing complex that could repress independently of PRC1 (Peterson, 2004).

If Scm does work with PRC1, then what might explain its substoichiometric association with purified PRC1? One possibility is that Scm assembles only into a subset of PRC1 complexes, perhaps restricted to certain tissues or times of development. Such a model has been proposed to explain how Asx contributes to PcG repression in the embryonic epidermis but not in the central nervous system. This explanation for Scm, however, is not favored because its requirement in PcG repression is widespread in both embryonic and imaginal tissues. Another possibility is that Scm interaction with PRC1 is robust in chromatin but is not fully preserved during preparation of soluble nuclear extracts used in purification. In this view, nucleosome arrays might provide a platform that promotes Scm-PRC1 binding. Indeed, both PRC1 and Scm have affinity in vitro for nucleosome arrays. Additional in vitro studies will be needed to address the nature of Scm-PRC1 interactions in the context of chromatin templates. It is noted that the GAGA factor provides an example of a protein that is not stably associated with PRC1 in embryo extracts but can nevertheless help recruit PRC1 to nucleosomal templates in vitro (Peterson, 2004).

At present, the evidence favors a noncatalytic role for PRC1 in PcG repression. How might Scm, which also lacks recognizable catalytic domains, contribute to PRC1 mechanism? Recent in vitro studies show that mouse PRC1 bound to a single nucleosome array can recruit a second chromatin template that then also becomes repressed. These bridging interactions between repressed templates in vitro may reflect the PcG-dependent chromosome-pairing and chromosome-chromosome interactions frequently observed in vivo. Thus, one role of PRC1 may be to promote higher-order chromosome interactions that spread or stabilize repression. Intriguingly, among the core PRC1 components, the mouse Ph protein was found most critical for in vitro bridging activity. Since Ph is the key subunit that mediates Scm interaction with PRC1, the possibility is raised that Scm could facilitate PRC1-mediated long-distance chromatin interactions. In this view, Scm might work by helping to anchor PRE-promoter and/or PRE-PRE interactions needed for PcG repression in vivo (Peterson, 2004).

A second type of potential Scm-PRC1 partnership in chromatin has been proposed on the basis of structural properties of the SPM domain. The SPM domain of fly Ph, determined by X-ray crystallography, is a five-helix bundle that has the special property of forming helical self-polymers in vitro. The possibility of an extended protein polymer that could bind alongside nucleosome arrays has prompted speculation that SPM proteins might organize higher-order chromatin arrangements. In such a model, SPM domain-containing proteins or complexes form a core helical polymer around which the chromatin fiber could be wrapped. This model, although speculative, is appealing since it brings structural data to bear upon the long-standing hypothesis that PcG proteins create extended tracts of repressed chromatin. Intriguingly, when mixed together, the SPM domains of Ph and Scm can also form copolymers in vitro. Thus, PH and Scm could collaborate in forming the proposed higher-order chromatin structures. In this context, the dominant-negative properties of overexpressed SPM domain could reflect disruption of contacts needed to produce PH-Scm chromatin polymers. To evaluate this model, it will be necessary to test if full-length PcG proteins or their intact complexes can form polymers in vitro like those seen for their isolated SPM domains. If so, then further studies would need to address the existence and roles of such polymers in vivo (Peterson, 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).

The Drosophila RYBP gene functions as a Polycomb-dependent transcriptional repressor

The Polycomb and trithorax groups of genes control the maintenance of homeotic gene expression in a variety of organisms. A putative participant in the regulation of this process is the murine RYBP (Ring and YY1 Binding Protein). Sequence comparison between different species has identified the homologous gene in Drosophila, the dRYBP gene. Whether dRYBP participates in the mechanisms of silencing of homeotic genes expression was investigated. dRYBP expression, examined by RNA in situ hybridisation, was found ubiquitously throughout development. Moreover, a polyclonal anti-dRYBP antibody was generated that recognises the dRYBP protein. dRYBP protein is nuclear and expressed maternally and ubiquitously throughout development. To study the transcriptional activity of dRYBP, a fusion protein was generated containing the entire dRYBP protein and the GAL4 DNA binding domain. This fusion protein functions, in vivo, as a transcriptional repressor throughout development. Importantly, this repression is dependent on the function of the Polycomb group genes. Furthermore, using the GAL4/UAS system, dRYBP was over-expressed in the haltere and the wing imaginal discs. In the haltere discs, high levels of dRYBP repress the expression of the homeotic Ultrabithorax gene. This repression is Polycomb dependent. In the wing discs, dRYBP over-expression produces a variety of phenotypes suggesting the overall miss-regulation of the many putative genes affected by high levels of dRYBP. Taking together, these results indicate that dRYBP is able to interact with PcG proteins to repress transcription suggesting that the dRYBP gene might belong to the Polycomb group of genes in Drosophila (Bejarano, 2005).

The mouse homologous gene, RYBP, was identified in a two-hybrid screen for murine Ring1 interacting proteins. RYBP family members include the human YEAF1 homologous gene and the murine and human YAF2 gene coding for structurally related proteins. Although very similar in sequences, they seem to have different functions as transcriptional regulators of the hGABP gene, i.e. YAF2 positively regulates the transcriptional activity of hGABP but YEAF1 negatively regulates this activity (Sawa, 2002; Bejarano, 2005).

dRYBP is expressed maternally and throughout development in all the nuclei of the embryo and the imaginal discs cells. The murine RYBP gene is also expressed ubiquitiously in the mouse embryo (Garcia, 1999). The ubiquitous and nuclear pattern of dRYBP expression coincides with the pattern of expression of the Polycomb group proteins so far described (Bejarano, 2005).

When dRYBP is tethered to DNA sequences, it is able to repress the transcriptional state of minigene reporter constructs. Moreover, GALDB-dRYBP transcriptional repression function requires the products of at least the Pc, Sce and pho genes, suggesting that GALDB-dRYBP represses transcription by interacting with PcG protein complexes. The Pho protein (homologous to mouse YY1) is able to bind DNA in a sequence specific manner and it has been proposed to recruit the PcG complexes to DNA. However, the results show that the transcriptional repression function of GALDB-dRYBP cannot be achieved in the absence of Pho protein. Although silencing in these experimental conditions could formally result solely from the interaction of dRYBP with Pho, the need of Pho to execute the transcriptional repression may also suggest that in the process of maintenance of homeotic gene expression, the Pho protein serve other functions than the recruitment of PcG complexes to DNA (Bejarano, 2005).

Additional evidence for the transcriptional repressor function of dRYBP comes from the experiments of over-expression of dRYBP using the GAL4/UAS system. UbxGAL4/UASdRYBP halteres show partial transformation towards wing which is correlated with the repression of UBX expression in the haltere imaginal discs due to high levels of dRYBP. The partial transformation of the haltere towards wing is not fully understood. It is speculated that the over-expression of dRYBP may also affect genes involved in proliferation that act downstream the Ubx gene. The repressive effect is Polycomb dependent, suggesting that dRYBP transcriptional repression function needs the interaction with Polycomb proteins. Moreover, although no changes have been detected in the levels of engrailed expression, some of the phenotypes observed in enGAL4/UASdRYBP flies are indicative of engrailed repression, revealing again the repressor effect of dRYBP over expression (Bejarano, 2005).

A model has been proposed in which RYBP protein, through its interaction with DNA-binding proteins like YY1, function as a ‘bridge’ to ensure interactions of DNA and non-DNA binding proteins in multimeric protein complexes. It is not yet known if dRYBP serves a similar bridging function in Drosophila. The YY1 protein (homologous to Drosophila Pho) is able to bind DNA in a sequence specific manner and directly interacts with dRYBP. It is speculated that dRYBP, serves a similar bridging function, bridging between DNA binding proteins like Pho and the multimeric PcG complexes. Further work and mutations in the dRYBP gene will be necessary to define whether dRYBP serves this putative bridge function (Bejarano, 2005).

dRYBP over-expression in the wing produces homeotic and non-homeotic phenotypes indicative of miss regulation of a variety of genes. High levels of dRYBP in the wing (i.e. sdGAL4/UASdRYBP flies) produces, among others, transformation towards haltere with the corresponding expression of the Ubx protein in the wing cells, i.e. outside its normal domain of expression. This effect could seem opposite to the repressor effect observed when dRYBP is tethered to DNA (GALDBdRYBP) or when dRYBP is over expressed under the control of the UbxGAL4 line. However, interference with the assembling/recruting of the PcG and trxG complexes either because of sequestration of PcG/trxG proteins, perturbation of the PcG/trxG balance or disruption of the cross regulatory interactions between PcG proteins could perhaps explain the observed expression of UBX protein in the wing disc due to over-expression of dRYBP. Alternatively, over abundance of dRYBP or dRYBP containing complexes might lead to a unique target gene repretoire that lead to the effects observed. Finally, the cross regulatory interactions between the genes patterning the wing, that are perhaps being miss regulated by the high levels of dRYBP could also explain the range of phenotypes observed in the wing due to over expression of dRYBP (Bejarano, 2005).

In conclusion, these results show that dRYBP protein is nuclear, maternal and ubiquotiously expressed throughout development. The results also show that dRYBP functions, in a Polycomb dependent manner, as a transcriptional repressor, suggesting that dRYBP is able to interact with the PcG proteins to repress transcription and therefore might belong to the Polycomb group of genes of Drosophila. Finally, the study of the multiple phenotypes produced by high levels of dRYBP in the wing might be indicative of the involvement of dRYBP on the regulation of many genes as also described for the PcG genes in Drosophila (Bejarano, 2005).

Efficient and specific targeting of Polycomb group proteins requires cooperative interaction between Grainyhead and Pleiohomeotic

Specific targeting of the protein complexes formed by the Polycomb group of proteins is critically required to maintain the inactive state of a group of developmentally regulated genes. Although the role of DNA binding proteins in this process has been well established, it is still not understood how these proteins target the Polycomb complexes specifically to their response elements. The grainyhead gene, which encodes a DNA binding protein, interacts with one such Polycomb response element of the bithorax complex. Grainyhead binds to this element in vitro. Moreover, grainyhead interacts genetically with pleiohomeotic in a transgene-based, pairing-dependent silencing assay. Grainyhead also interacts with Pleiohomeotic in vitro, which facilitates the binding of both proteins to their respective target DNAs. Such interactions between two DNA binding proteins could provide the basis for the cooperative assembly of a nucleoprotein complex formed in vitro. Based on these results and the available data, it is proposed that the role of DNA binding proteins in Polycomb group-dependent silencing could be described by a model very similar to that of an enhanceosome, wherein the unique arrangement of protein-protein interaction modules exposed by the cooperatively interacting DNA binding proteins provides targeting specificity (Blastyak, 2006).

The iab-7 PRE lies next to the Fab-7 boundary, a chromatin domain insulator element between the neighboring iab-6 and iab-7 cis-regulatory domains of BX-C. Fab-7 ensures the functional autonomy of these cis-regulatory domains; iab-7 is inactive in the sixth abdominal segment (A6), where iab-6 is active, while iab-7 is activated in segment A7. A large set of internal BX-C deficiencies is available, making this region ideal for genetic studies (Blastyak, 2006).

Class II deletions, which remove only the boundary region, fuse the otherwise intact cis-regulatory elements iab-6 and iab-7. The consequence of this fusion is that in some A6 cells iab-6 is inactivated by iab-7, while in some other A6 cells iab-6 ectopically activates iab-7. As a result, A6 will become a mixture of cell clones with either A5 or A7 identity. Due to the fact that the Abd-B gene, the expression of which is controlled by these cis regulators, is haploinsufficient, such transformations are evident even under heterozygous conditions. Class I deletions, which remove both the Fab-7 boundary and the adjacent iab-7 PRE, transform A6 into a perfect copy of A7, suggesting that in the case of class II deletions it is the iab-7 PRE that mediates the inactivation of iab-6 in A6; thus, the inactivation may depend on Pc-G-mediated silencing. Indeed, if a class II deletion is combined with some, but not all, Pc-G mutations, the resulting phenotype is indistinguishable from that of class I deletions. Based on this result, it should be possible to identify mutations in factors that specifically interact with the iab-7 PRE as enhancers of the phenotype of class II deletions (Blastyak, 2006).

Accordingly, several X-ray mutagenesis screenings were performed with the class II allele Fab-7². Among the enhancer mutants, one complementation group, represented by five alleles in the collection, is described here. Two alleles are associated with a cytologically visible breakpoint in 54F, and deficiency mapping placed the locus between the proximal breakpoints of the Pcl11b and Pcl7b deletions. Previously, four complementation groups were isolated within this interval. Noncomplementation with alleles of one of the four complementation groups showed that new mutant alleles were isolated of the previously described gene grainyhead (grh). The previously isolated grh alleles, including the molecularly characterized amorphic allele B37, are also strong Fab-7² enhancers, indicating that loss-of-function grh mutations affect the function of the iab-7 PRE (Blastyak, 2006).

Genome-wide prediction has indicated that the occurrence of the same limited set of consensus motifs can fairly accurately predict the PRE function of a DNA sequence (Ringrose, 2003). This observation suggests that many, if not all, PREs use the same set of DNA binding proteins. One of the frequently occurring consensus sequences within PREs is a poly-T motif. Many, although not all, GRH binding sites are T rich, and the current studies indicate that at least in some cases the poly-T consensus sequence may be a binding site for this protein. However, like other DNA binding proteins involved in PRE function, GRH alone cannot explain the specificity of targeting, since its function is not limited to PREs. In other contexts, GRH acts as a transcriptional activator. The fact that an array of distinct sequence motifs is required to accurately predict PREs probably means that there is no single major targeting activity. Indeed, in the case of the engrailed PRE it was demonstrated that all binding sites of DNA binding proteins are equally important for silencing activity. Identification of GRH as a PRE-related DNA binding protein and, in particular, its cooperative interaction with another member of this group both in vivo and in vitro may help in understanding the targeting of PC-G to PREs during development (Blastyak, 2006).

A cooperative interaction between GAF (Trithorax-like) and PHO has been demonstrated (Mahmoudi, 2003). In contrast to the case of GRH and PHO, cooperation between GAF and PHO is independent of the physical interaction between the two proteins and requires a nucleosomal context. Although the physical basis of this cooperative interaction is not understood, it also suggests that cooperativity may be an important principle in the organization of nucleoprotein assembly at PREs (Blastyak, 2006).

What could be the impact of cooperativity on PC-G targeting? Theoretically, one of the most significant problems encountered by a DNA binding protein is the huge excess of potential binding sites in the genome, including both functional sites and pseudosites. It can be assumed that if any of the DNA binding proteins involved in targeting are present in limited amounts in the nucleus, then their binding occurs only at the highest-affinity sites, where a combination of certain binding sites facilitates their cooperative binding. Several observations contradict this simple model. First, if the amount of these DNA binding proteins were limited, their mutations would be expected to result in strong haploinsufficient phenotypes, which is not the case. Second, studies on the DNA binding proteins EVE, FTZ, and GAF demonstrated that in vivo they also bind to genes that are not controlled by them. These functionally irrelevant sequences may represent pseudosites, and the relatively low level of binding at these sites may indicate a low binding affinity. Thus, it appears that restricted binding site occupancy of DNA binding proteins is not necessary for specificity in gene regulation. Likewise, even though the DNA binding proteins present on PREs may bind to nonfunctional sites, it is likely that the functionally relevant high-affinity sites are distinguished from pseudosites in vivo by the unique arrangement of distinct, stably bound cooperative partners. However, although in this model of targeting of PRC1 to the iab-7 PRE, cooperativity at the level of the DNA binding proteins is critically required for binding stability, by itself it is insufficient to provide the required specificity of the targeting process (Blastyak, 2006).

In contrast to the DNA binding components, other constituents of the silencing complex appear to be limiting factors. This is suggested by the fact that most Pc-G genes were identified either on the basis of their characteristic haploinsufficient phenotypes or on the basis of their dominant genetic interaction with other known Pc-G members. The number of potential PRE sequences is also relatively small, as a genome-wide survey estimated it to be not more than a few hundred in Drosophila. This brings us to the question of how the abundant DNA binding proteins link the limited amount of PC-G complexes to the low-frequency target sites with high specificity (Blastyak, 2006).

The first clue comes from studies showing that all of the PRE DNA binding proteins have the ability to interact with various PC-G proteins that are all subunits of the same preformed protein complex, PRC1. These interactions appear to be weak by themselves, as illustrated by the fact that although the occurrence of these interactions can be demonstrated by using short protocols like immunoprecipitation, the resulting complexes do not survive nonequilibrium methods used for traditional biochemical purification of protein complexes. The consequence of the cooperativity at the level of DNA binding proteins is that the otherwise weak interaction surfaces are integrated into a stable composite surface that can serve as a high-affinity docking site for the limited amount of PRC1 complex. In the model, this second level of cooperativity would provide targeting specificity (Blastyak, 2006).

Notably, the same DNA binding proteins involved in PC-G targeting can separately participate in weak interactions with various other protein complexes involved in processes unrelated to, or the opposite of, Pc-G-dependent silencing, such as TFIID-dependent transcription or chromatin remodeling by SWI/SNF. Based on the available data, interaction surfaces of any such complex are not shared by these DNA binding proteins, and according to this model, their concerted recruitment to PREs is unlikely. Also, in agreement with the experimental data, this model predicts that in the absence of DNA none of the DNA binding proteins will be able to interact stably with the complex to be recruited. The integration of several weak protein-protein interaction modules into a single entity is a prerequisite for the complex to dock on chromatin (Blastyak, 2006).

It has been shown that transcription through the iab-7 PRE displaces PC-G proteins and results in concomitant recruitment of the TRX and BRM proteins. Thus, iab-7 PRE appears to be a switchable element and the potential, for example, of PHO to interact with protein partners having a function that is the opposite of PC-G silencing might be realized under certain circumstances. There is insufficient data to explain the mechanism underlying this switch. One possibility is that binding of some DNA binding proteins to DNA or to their interacting partners is modified by posttranslational modifications, as it was shown in the case of the human homologue of Grh. According to the model, even the modification of a single actor (e.g., GRH) can radically influence the overall assembly configuration of the targeting complex and might be responsible for the dynamic nature of the iab-7 PRE (Blastyak, 2006).

This model shows remarkable similarity to the functional and structural organization of enhanceosomes. For example, multimerization of the binding sites of any of the DNA binding proteins involved in beta interferon (IFN-ß) enhanceosome formation does not reproduce faithfully the virus inducibility of the intact enhancer. Instead, these synthetic enhancers respond promiscuously to inducers that are normally not involved in regulation of the IFN-ß gene. The molecular basis of the selective inducer response of the enhanceosome is established by the following cooperative interactions. First, in their original context, the mutually cooperative interactions at the level of DNA binding proteins promote binding stability. Second, on the resulting spatially arranged protein surface, each DNA binding protein contributes to the recruitment of a protein complex through interactions with one of its subunits. It is concluded that the integration of different, hierarchical levels of cooperativity could be a general principle in the targeting of protein complexes to chromatin (Blastyak, 2006).

The validity of the enhanceosome model has already been demonstrated by in vitro reassembly of the IFN-ß enhanceosome with well-defined recombinant components. In vitro studies with a nucleosomal template have provided valuable insights into the role of PRC1 in regulation of the chromatin structure. However, in this experimental system the excess of PRC1 and nonspecific DNA binding of PRC1 complex members overcomes the problem of targeting. An initial attempt to reconstitute cooperativity at the level of DNA binding proteins failed, possibly because the simultaneous presence of several other DNA binding proteins is required for cooperative assembly. Until these components of PREs are identified, it is likely that PC-G targeting cannot be faithfully reconstituted in vitro. Hopefully, the identification of as-yet-unknown DNA binding protein components of PREs, together with the conceptual framework presented here, will facilitate these studies (Blastyak, 2006).

Recent results showed that in vivo stable recruitment of PC to the Ubx PRE critically depends on the presence of the E(Z) protein. E(Z) is a member of a PC-G complex, which is distinct from PRC1, and possesses histone methyltransferase activity. These findings led to a model wherein, upon binding of the EZ complex, its enzymatic activity could provide the mark for the specific targeting of PRC1. Hence, recruiting of PRC1 would only indirectly depend on sequence-specific DNA binding proteins, as they primarily act as recruiters of the E(Z) complex, but not PRC1. Contrary to the predictions of this model, it was found that although mutations in PRC1 complex members are similarly strong dominant enhancers of the Fab-7² phenotype as grh and pho, amorphic E(z) alleles in heterozygous condition are not. Thus, the current results indicate a rather intimate link between these DNA binding proteins and PRC1 complex members. However, it is still possible that in a nucleosomal context the histone mark could provide an additional constituent for binding whose presence can be critical in vivo in certain tissues. Certain PC-G group members have a tissue-specific phenotype, and GRH is also not ubiquitously expressed, which supports this notion (Blastyak, 2006).

Architecture of a polycomb nucleoprotein complex; Chromatin immunoprecipitation and nuclease mapping demonstrate that PREs are nucleosome depleted

Polycomb group (PcG) epigenetic silencing proteins act through cis-acting DNA sequences, named Polycomb response elements (PREs). Within PREs, Pleiohomeotic (Pho) binding sites and juxtaposed Pc binding elements (PBEs) function as an integrated DNA platform for the synergistic binding of Pho and the multisubunit Polycomb core complex (PCC). This study analyzed the architecture of the Pho/PCC/PRE nucleoprotein complex. DNase I footprinting revealed extensive contacts between Pho/PCC and the PRE. Scanning force microscopy (SFM) in combination with DNA topological assays suggested that Pho/PCC wraps the PRE DNA around its surface in a constrained negative supercoil. These features are difficult to reconcile with the simultaneous presence of nucleosomes at the PRE. Indeed, chromatin immunoprecipitations (ChIPs) and nuclease mapping demonstrated that PREs are nucleosome depleted in vivo. The implications of these findings for models explaining PRE function are discussed (Mohd-Sarip, 2006).

How specialized DNA elements such as PREs can bring a linked gene under epigenetic control remains poorly understood. An important breakthrough was the identification of Pho as a sequence-specific PcG protein. Subsequent research firmly established that Pho forms a critical component of the 'PRE code.' Another building block of PREs, the PBE is located directly downstream of Pho binding sites. Pho and PCC interact only weakly in solution, but docking onto Pho/PBE modules drives the assemblage of a stable Pho/PCC/PRE silenceosome. The mechanistical properties of silenceosome and enhanceosome formation are strikingly similar. Both involve synergistic interactions between a stereo-specific arrangement of binding sites and a reciprocal network of protein-protein interactions. This study investigated the architecture of a PcG silenceosome (Mohd-Sarip, 2006).

The results revealed that Pho/PCC contacts the bxd PRE over 400 bp and wraps the PRE DNA around its surface in a constrained negative supercoil. It has been found that Pho/PCC binding to the PRE can overcome chromatinization. Moreover, the resulting DNase I digestion pattern of the Pho/PCC/PRE complex suggested the absence of nucleosomes. It is estimated that a Pho/PCC oligomer can wrap more DNA around its surface than a nucleosome. The extensive contacts between Pho/PCC and PRE DNA together with the left-handed wrapping are likely to affect histone-DNA interactions (Mohd-Sarip, 2006).

The 400 bp bxd PRE core is nucleosome poor in vivo, as revealed by nuclease mapping and quantitative ChIP assays. Likewise, the PREs from the Abd-B cis-regulatory domains were found to be nuclease hypersensitive in chromatin digests. Other researchers have also suggested that the core PRE region is largely devoid of nucleosomes. While this work was in progress, ChIP analysis by others independently established that PREs are depleted for histones. The iab-7 PRE sequences required for the pairing-sensitive silencing of mini-white in transgene assays closely coincide with the nuclease-hypersensitive region bound by Pho/PCC. Finally, there appears to be a good correlation between PRE activity and the extent of nuclease hypersensitivity (Mohd-Sarip, 2006).

These findings dovetail nicely with the results of recent genome-scale determination of nucleosome positioning in yeast. These studies suggested that RNA polymerase II promoters comprise a nucleosome-free region flanked by positioned nucleosomes, bearing a stereotyped pattern of histone modifications. It is proposed that, like promoters and enhancers, PREs are in a nucleosome-depleted conformation in vivo (Mohd-Sarip, 2006).

It is suggested that PcG-directed gene silencing is a multistep process, initiated by silenceosome formation on the PRE. The next step requires the establishment of a silenced state onto PRE-linked genes. PCC-histone interactions, modulated by covalent histone modifications, are likely to be the main driving force of sequence-independent spreading over a target gene. Thus, it is imagined that histone modifications would generally follow, rather than precede, Polycomb nucleocomplex formation on PREs. Collectively, the available evidence enforces the notion that a cooperative network of contextual protein-DNA and protein-protein interactions nucleates silenceosome formation. This work presents a view of the architecture of a Pho/PCC/PRE nucleoprotein complex and provides a framework for models explaining PRE function (Mohd-Sarip, 2006).

A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities

Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pleiohomeotic (Pho) is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, Pho protein complexes were purified from Drosophila embryos and it was found that Pho exists in two distinct protein assemblies: a Pho-dINO80 complex containing the Drosophila INO80 nucleosome-remodeling complex, and a Pho-repressive complex (PhoRC) containing the uncharacterized gene product Scm-related gene containing four mbt domains (dSfmbt). Analysis of PhoRC reveals that dSfmbt is a novel PcG protein that is essential for HOX gene repression in Drosophila. PhoRC is bound at HOX gene PREs in vivo, and this targeting strictly depends on Pho-binding sites. Characterization of dSfmbt protein shows that its MBT repeats have unique discriminatory binding activity for methylated lysine residues in histones H3 and H4; the MBT repeats bind mono- and di-methylated H3-K9 and H4-K20 but fail to interact with these residues if they are unmodified or tri-methylated. These results establish PhoRC as a novel Drosophila PcG protein complex that combines DNA-targeting activity (Pho) with a unique modified histone-binding activity (dSfmbt). It is proposed that PRE-tethered PhoRC selectively interacts with methylated histones in the chromatin flanking PREs to maintain a Polycomb-repressed chromatin state (Klymenko, 2006).

The regulation of gene expression by Polycomb group (PcG) and trithorax group (trxG) proteins represents a paradigm for understanding the establishment and maintenance of heritable transcriptional states during development. PcG and trxG genes were first genetically identified as regulators that are required for the long-term maintenance of HOX gene expression patterns in Drosophila. PcG proteins keep HOX genes silenced in cells in which they must stay inactive, whereas trxG proteins maintain the active state of these genes in appropriate cells. This regulatory relationship is conserved in vertebrates, where PcG and trxG proteins also regulate HOX gene expression. In addition, mammalian PcG and trxG proteins have also been implicated in X-chromosome inactivation, hematopoietic development, control of cell proliferation, and oncogenic processes (Klymenko, 2006).

Drosophila HOX genes are among the best-studied target genes of the PcG/trxG system. Different studies have led to the identification of specific cis-regulatory sequences in HOX genes that are called Polycomb response elements (PREs) and are required for silencing by PcG proteins. PREs are typically several hundred base pairs in length, and they function as potent transcriptional silencer elements in the context of HOX reporter genes as well as in a variety of other reporter gene assays. This operational definition of PREs is complemented by their classification as DNA sequences to which PcG proteins bind, directly or indirectly. Among the 14 cloned Drosophila PcG genes, only Pleiohomeotic (Pho) and Pho-like (Phol) encode sequence-specific DNA-binding proteins. Pho and Phol bind the same DNA sequence, and while the two proteins act to a large extent redundantly, double mutants show severe loss of HOX gene silencing. DNA-binding sites for Pho and Phol are present in all PREs that have been characterized to date, and mutational analyses of these binding sites have shown that they are essential for silencing by PREs. In contrast, none of the other 12 characterized PcG proteins bind DNA in a sequence-specific manner. However, formaldehyde cross-linking studies showed that several of these proteins specifically associate with the chromatin of PREs in tissue culture cells and in developing embryos and larvae. Biochemical studies revealed that most of these non-DNA-binding PcG proteins are components of either PRC1 or PRC2, two distinct PcG protein complexes that have recently been purified and characterized. Specifically, PRC1 contains the PcG proteins Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), Sex combs extra/Ring (Sce/Ring), and Sex combs on midleg (Scm), whereas PRC2 contains the three PcG proteins Extra sex combs (Esc), Enhancer of zeste [E(z)], and Suppressor of zeste 12 [Su(z)12] (Klymenko, 2006).

What is the role of Pho and Phol at PREs? Biochemically purified PRC1 and PRC2 do not contain Pho or Phol. Several recent studies investigated possible physical interactions between Pho and PRC1 or PRC2 complex components. Based on coimmunoprecipitation and GST pull-down assays, it was proposed that Pho directly interacts with several different PRC1, PRC2, and SWI/SNF complex components. However, on polytene chromosomes of phol; pho double mutants, the binding of PRC1 and PRC2 to HOX genes and at most other loci is largely unperturbed (Brown, 2003), suggesting that, at least in this tissue, Pho and Phol are not strictly required for keeping PRC1 and PRC2 anchored to HOX genes (Klymenko, 2006).

To gain insight into the biological function of Pho, Pho-containing protein complexes were biochemically purified from Drosophila. The data show that Pho exists in two distinct multiprotein complexes that, contrary to expectation, do not contain any of the previously characterized PcG proteins. The functional analysis of one of these Pho complexes that was named PhoRC provides evidence that its binding to PREs is required for maintaining repressive HOX gene chromatin (Klymenko, 2006).

A tandem affinity purification (TAP) strategy was used to purify Pho protein complexes from Drosophila embryonic nuclear extracts. A transgene that expresses a TAP-tagged Pho fusion protein (Pho-TAP) was expressed under the control of the Drosophila alpha-tubulin promoter, and transgenic flies were generated. To test whether the Pho-TAP protein is functional, the transgene was introduced into the genetic background of animals homozygous for pho¹, a protein-negative allele of pho. pho¹ homozygotes die as pharate adults, but they are rescued into viable and fertile adults that can be maintained as a healthy strain if they carry one copy of the transgene expressing Pho-TAP. The Pho-TAP protein can thus substitute for the endogenous Pho protein, and this shows that the fusion protein is functional (Klymenko, 2006).

Proteins that are associated with the Pho-TAP protein were purified from embryonic nuclear extracts, following the TAP procedure. Seven different polypeptides that consistently copurified with the Pho-TAP bait protein in several independent purifications were identified through sequencing of peptides from individual protein bands by nanoelectrospray tandem mass spectrometry. In addition to Pho, the isolated protein assembly contains the product of CG31212, a protein that is most closely related to yeast INO80, the SWI/SNF2-like nucleosome-remodeling subunit in the yeast INO80 complex. The CG31212 locus as will therefore be referred to as dINO80. Five other subunits of the Pho complex were identified as Reptin (Rept), Pontin (Pon), Actin (Act), and the two actin-related proteins dArp5 and dArp8, which are encoded by CG7940 and CG7846, respectively. These five proteins represent the Drosophila homologs of five core subunits that assemble together with INO80 to form the yeast INO80 complex. Specifically, Rept and Pont are homologs of the yeast Rvb1 and Rvb2 AAA-ATPases that constitute a DNA helicase in the INO80 complex. Act, dArp5, and dArp8 are homologs of the Actin, Arp5, and Arp8 proteins, respectively, that are present in the yeast INO80 complex. Thus, it appears that a Drosophila dINO80 complex copurifies with Pho. In addition, the purified material also contained the product of CG16975, a protein that is not conserved in yeast but is closely related to the product of the murine Scm-related gene containing four mbt domains (Sfmbt); the CG16975 gene is referred to as dSfmbt. The characteristic features of mammalian Sfmbt and the Drosophila dSfmbt protein are four malignant brain tumor (MBT) repeats and a sterile alpha motif (SAM) domain. The Drosophila genome encodes two other proteins that contain MBT repeats and show a similar domain architecture, l(3)mbt and the PcG repressor Scm. Taken together, these findings suggest that Pho exists in multiprotein assemblies that contain a dINO80 complex and dSfmbt but, unexpectedly, none of the previously characterized PcG proteins (Klymenko, 2006).

Since the yeast genome does not contain any dSfmbt-related protein, it was asked whether dSfmbt and dINO80 are part of distinct Pho protein complexes. To this end, crude embryonic nuclear extracts were fractionated by glycerol gradient sedimentation and individual fractions were probed by Western blotting with antibodies against Pho, Pho-like, dINO80, and dSfmbt. The results show that dINO80 and dSfmbt are present in separate fractions of the gradient but that Pho and Pho-like are present in both dINO80- and dSfmbt-containing fractions. dSfmbt and dINO80 thus exist in distinct protein complexes in embryonic nuclear extracts. It should be noted that Pho and Pho-like are also present in fractions that do not contain dINO80 or dSfmbt. This suggests that Pho and Pho-like also exists in soluble protein assemblies that are distinct from the complexes identified in this study, but that these assemblies are not stable enough to be isolated as complexes in the purification scheme (Klymenko, 2006).

It was asked whether components of the purified Pho complexes are associated with PREs in vivo. To this end, chromatin immunoprecipitation (X-ChIP) assays were performed. Drosophila embryos were treated with formaldehyde and DNA that was cross-linked to Pho, dSfmbt, dINO80, Reptin, Pontin, or Ph was immunoprecipitated with antibodies against these proteins. Real-time quantitative PCR was used to measure the abundance of the following endogenous and transgene PREs in the immunoprecipitates. The bxd and iab-7 PREs in the HOX genes Ultrabithorax (Ubx) and Abdominal-B (Abd-B), respectively, are well-characterized, and Pho binds to these PREs in vitro and in vivo. It has been reported that PRE_D, a 572-bp core fragment of the bxd PRE, silences a Ubx-LacZ reporter gene in imaginal discs and in embryos but that point mutations in all six Pho protein-binding sites in this fragment (PRE_{D pho mut}) completely abolish its silencing capacity (Fritsch, 1999). Therefore X-ChIP assays were performed in transformed embryos that carried either the wild-type PRE_D or the mutated PRE_{D pho mut} reporter gene; this allowed direct comparison of protein binding at the transgenic PRE with protein binding at the endogenous bxd and iab-7 PREs in the same preparation of chromatin. Specific PCR primer sets allowed X-ChIP signals at the reporter gene PRE to be distinguished from signals at the endogenous bxd PRE. It was found that Pho, Ph, and, importantly, also dSfmbt are specifically bound at the endogenous bxd and iab-7 PREs but not at sequences flanking those PREs. In contrast, binding of dINO80, Reptin, or Pontin at any of the sequences analyzed (data not shown). Pho, dSfmbt, and Ph are also bound at the PRE_D fragment in the transgene was not detected, but, strikingly, binding signals of Pho, dSfmbt, and Ph are severely reduced at the mutated PRE_{D pho mut} fragment. Taken together, these data show that Pho-dSfmbt complexes are bound at PREs in vivo and that binding of these complexes to PREs requires DNA-binding sites for Pho. Since association of dINO80 complex components with PREs was not detected in this assay, further analysis focused on the characterization of Pho-dSfmbt complexes (Klymenko, 2006).

Therefore, this study shows that the PcG protein Pho exists in two stable protein complexes, a Pho-dINO80 complex and PhoRC. Biochemical and genetic analyses identify PhoRC as a novel PcG protein complex that has a different subunit composition and molecular function than the previously described PcG complexes PRC1 and PRC2. The following conclusions can be drawn from these studies of PhoRC: (1) PhoRC contains Pho and dSfmbt, and these two proteins form a very stable complex that can be purified from embryos and reconstituted from recombinant proteins. (2) PhoRC is bound to PREs in vivo, and PRE-targeting of PhoRC requires intact Pho/Pho-like DNA-binding sites. (3) A dSfmbt knockout reveals that dSfmbt is a novel PcG protein that is critically needed for HOX gene silencing. (4) The MBT repeats of dSfmbt are a novel methyl-lysine-recognizing module that selectively binds to the N-terminal tails of histones H3 and H4 if they are mono- or di-methylated at H3-K9 or H4-K20, respectively. PhoRC thus contains sequence-specific DNA-binding activity via the Pho protein and methylated histone-binding activity via dSfmbt (Klymenko, 2006).

Pho and Pho-like are the only PcG proteins with sequence-specific DNA-binding activity. Therefore, it is likely that these factors might tether PRC1 or PRC2 to PREs. Unexpectedly, biochemical purification of Pho complexes revealed that Pho exists in stable assemblies with either the PcG protein dSfmbt or components of the Drosophila INO80 complex. However, native or recombinant Pho complexes that contain PRC1 or PRC2 components were not purified. Similarly, biochemically purified PRC1 and PRC2 also do not contain Pho. PhoRC, PRC1, and PRC2 thus seem to be separate biochemical entities (Klymenko, 2006).

Reconstitution of recombinant PhoRC shows that dSfmbt binds directly to Pho or to Pho-like to form stable dimeric complexes. Coimmunoprecipitation assays indicate that such interactions also take place in Drosophila, and it was found that dSfmbt is associated with Pho or Pho-like in vivo. Moreover, dSfmbt mutants and pho-like; pho double mutants show a comparable loss of HOX gene silencing with similar kinetics. These observations are consistent with dSfmbt being needed for repression by both Pho and Pho-like. Furthermore, the X-ChIP experiments show that Pho/Pho-like DNA-binding sites in PREs are critical for binding of both Pho and dSfmbt at PREs. These data thus suggest that PhoRC is tethered to PREs by Pho or Pho-like (Klymenko, 2006).

Binding of the PRC1 subunit Ph at the bxd PRE also depends on intact Pho protein-binding sites. Could dSfmbt in PRE-bound PhoRC interact with Scm or Ph, for example, through the C-terminal SAM domain and thereby tether PRC1 to PREs? In coimmunoprecipitation experiments, no association of dSfmbt with Ph or Scm was detected. These interactions, if they exist, might be either very weak or exist only transiently. Previous studies reported direct physical interactions between Pho and PRC1 or PRC2 subunits, respectively. A possible scenario could therefore be that multiple weak interactions between Pho and dSfmbt with PRC1 and/or with PRC2 subunits might help to stabilize the binding of these complexes to PREs. It is also possible that the lack of Ph binding to the PRE transgene with mutated Pho sites reflects an indirect role of PhoRC that does not involve direct physical interactions between PhoRC and PRC1. In this context, it is worth noting that, on polytene chromosomes, binding of Ph and other PRC1 components is largely unperturbed in animals that lack both Pho and Pho-like proteins (Klymenko, 2006).

Four consecutive MBT repeats are a key feature of the dSfmbt protein. Fluorescence polarization binding assays suggest that these MBT repeats selectively bind to the N-terminal tail of histones H3 and H4 if these are mono- or di-methylated, but not if the same sites are unmethylated or tri-methylated. This novel discriminatory methyl-lysine-binding activity of MBTs is in stark contrast to the well-documented preference of chromodomains for higher, i.e., tri-methylated, binding sites in histones and could constitute an important general function of chromatin-associated MBT-containing proteins. The dSfmbt methyl-lysine interaction seems to be specific for the H3K9 and H4K20 methylation sites since matched H3 peptides that are methylated at different lysine residues (i.e., H3-K4me instead of H3-K9me) or histone tail peptides in which the methylated lysine residue is embedded in the same amino acid sequence context (i.e., ARKmeS in H3-K27me instead of ARKmeS in H3-K9me) are bound with at least 20-fold lower affinity (Klymenko, 2006).

Since these results suggest that dSfmbt is targeted to HOX gene PREs primarily through interaction with Pho, it was reasoned that binding to methyl-lysine residues in histone tails is not a primary mechanism for targeting dSfmbt to HOX genes. Moreover, recent studies provide evidence that, in the PcG-repressed state, the silenced HOX gene Ubx is tri-methylated at H3-K9, H4-K20, and H3-K27 throughout the gene, whereas lower methylated states of these sites are largely absent. What, then, is the role of Sfmbt in binding histones that are mono- or di-methylated at H3-K9 and H4-K20 in silenced HOX genes? Mono- and di-methylation of H4-K20 are very abundant modifications in Drosophila chromatin, and mass spectroscopic analyses of histones in embryos imply that lower methylated forms of histone H4 (i.e., H4-K20me2) already exist prior to becoming incorporated into chromatin during S phase. It is therefore tempting to speculate that dSfmbt, tethered to PREs by Pho, scans the flanking HOX gene chromatin for nucleosomes that are only mono- or di-methylated at H3-K9 or H4-K20 and docks onto such nucleosomes through its MBT repeats. It is hypothesized that through this bridging interaction, nucleosomes of lower methylated states might be brought into proximity to PRE-bound PRC2 and other currently unknown HMTases that are responsible for local tri-methylation of H3-K9 and H4-K20 in silenced HOX genes. According to this model, PRE-bound PhoRC would act as a 'grappling hook' that tethers mono- and di-methylated histones in silenced HOX gene chromatin to PREs to ensure that they become hypermethylated to the tri-methylated state. Such a chromatin-scanning function might be particularly important during S phase, when newly incorporated histone octamers need to become fully tri-methylated in order to maintain silencing of HOX genes (Klymenko, 2006).

Recruitment of Drosophila Polycomb-group proteins by Polycomblike, a component of a novel protein complex in larvae

Polycomb-group (PcG) proteins are highly conserved epigenetic transcriptional repressors that play central roles in numerous examples of developmental gene regulation. Four PcG repressor complexes have been purified from Drosophila embryos: PRC1, PRC2, Pcl-PRC2 and PhoRC. Previous studies described a hierarchical recruitment pathway of PcG proteins at the bxd Polycomb Response Element (PRE) of the Ultrabithorax (Ubx) gene in larval wing imaginal discs. The DNA-binding proteins Pho and/or Phol are required for target site binding by PRC2, which in turn is required for chromosome binding by PRC1. This study identified a novel larval complex that contains the PcG protein Polycomblike (Pcl) that is distinct from PRC1 and PRC2 and which is also dependent on Pho and/or Phol for binding to the bxd PRE in wing imaginal discs. RNAi-mediated depletion of Pcl in larvae disrupts chromosome binding by E(z), a core component of PRC2, but Pcl does not require E(z) for chromosome binding. These results place the Pcl complex (PCLC) downstream of Pho and/or Phol and upstream of PRC2 and PRC1 in the recruitment hierarchy (Savla, 2008).

Drosophila Polycomb-group (PcG) genes were originally identified as negative regulators of Hox genes. PcG-mediated silencing in Drosophila occurs in essentially two broadly defined stages: assumption of transcriptional repression responsibilities from gene-specific transcription factors in early embryos, followed by maintenance of the silenced state through many cycles of cell division beginning in mid-late-stage embryos and continuing throughout the remainder of development (Savla, 2008).

Although much of the genetic analysis of PcG functions and studies of the mechanisms by which PcG proteins are targeted to specific genomic sites have focused on their activities in larval tissues, in vitro biochemical analyses have focused on PcG complexes isolated from embryos: PRC1, PRC2 and PhoRC. PRC1 possesses multiple chromatin modifying activities in vitro suggesting that it, among PcG complexes, might be most directly responsible for preventing transcription. The primary functions of PhoRC and PRC2 appear to be to recruit and/or stabilize target site binding by PRC1, and potentially other PcG proteins. PhoRC includes the DNA-binding PcG protein Pleiohomeotic (Pho), which binds to sites within Polycomb Response Elements (PREs) that serve as docking platforms for PcG proteins. Pho directly interacts with components of both PRC1 and PRC2, and is required for recruitment of both complexes. The E(z) subunit of PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), facilitating recruitment of PRC1 (Savla, 2008).

A variant of PRC2 has recently been described that includes the PcG protein Polycomblike (Pcl). On the basis of gel filtration analysis of native complexes in embryo nuclear extracts and the stoichiometry of the purified Pcl-PRC2 complex, it appears that the majority of embryonic Pcl is present in Pcl-PRC2, but that the other PRC2 core subunits, E(z), Su(z)12, Esc and NURF55 (also known as Caf1 - FlyBase), predominantly are in a complex(es) lacking Pcl. It has been proposed that inclusion of Pcl in PRC2 is required for high levels of H3K27me3 in vivo, although the in vitro histone methyltransferase activity of Pcl-PRC2 is indistinguishable from that of PRC2 lacking Pcl. In this study, a larval Pcl-containing complex is identified that is distinct from PRC2 and PRC1 and shown to be required for chromosome binding by these PcG complexes (Savla, 2008).

In order to examine potential differences between embryonic and larval stage PcG complexes, larval nuclear extracts were fractionated over a Superose 6 gel filtration column, and western blots of the fractions were probed with anti-E(z) and anti-Pcl antibodies. Larval E(z)-containing complexes have a relative mass of ~500 to 600 kDa, similar to that of embryonic PRC2 complexes that lack Pcl. However, Pcl was undetectable in E(z)-containing fractions and appeared to be in a complex with a relative mass of ~1500 kDa. This is different from the fractionation profile of Pcl from embryo extracts, in which it co-fractionates with E(z) in native complexes with relative mass estimates in the range of ~650 kDa to 1000 kDa, suggesting that, unlike its association with a subset of PRC2 complexes in embryos, Pcl functions as a component of a distinct complex in larvae, which will be referred to as the Pcl-Complex (PCLC) (Savla, 2008).

In order to further investigate the relationship of Pcl with other PcG proteins and its role in PcG-mediated silencing in larvae, chromatin immunoprecipitation (ChIP) assays were performed on wing imaginal discs. The PcG maintains the transcriptional silence of the Hox gene Ultrabithorax (Ubx) in the epithelial cells of wing discs. Other PcG proteins, including the DNA-binding proteins Pho and Phol and components of the PRC1 and PRC2 complexes, have previously been shown to be present at the major PRE in the Ubx cis-regulatory bxd region in this tissue. Consistent with a previous report, Pcl also was detected at the bxd PRE, and appears to largely colocalize with E(z) and Phol (Savla, 2008).

A hierarchical relationship among PcG proteins at the bxd PRE in which Pho and/or Phol are required, but are not necessarily sufficient, for recruitment of PRC2, which in turn facilitates recruitment of PRC1. In order to determine how Pcl might fit into this recruitment pathway, ChIP assays were performed on E(z) mutant wing imaginal discs. E(z)⁶¹ is a temperature-sensitive allele that displays nearly wild-type activity at 18°C, but strongly reduced activity at 29°C. Following shift from 18°C to 29°C, bxd PRE binding by E(z)⁶¹ protein is rapidly lost and along with it the detection of H3K27me3 and Pc in this region. ChIP assays of wing discs dissected from E(z)⁶¹ larvae 24 hours following shift from 18° to 29°C confirmed loss of E(z) from the PRE, but revealed no effect on Pcl and Phol binding to PRE fragments 3 and 4, but a slight decrease of both proteins at the PRE 2 fragment. It is speculated that Pcl and Phol signals at this proximal edge of the PRE are partly due to protein-protein cross-links, which might be reduced in the absence of PRC2. Retention of Pcl at the PRE in the absence of E(z) and by extension absence of PRC1, which requires PRC2 for binding to this region, confirms that Pcl is not a stable subunit of larval versions of either PRC1 or PRC2 and is consistent with its inclusion in a distinct complex (Savla, 2008).

Flies that are homozygous for null Pcl alleles die as embryos and no conditional Pcl alleles exist, precluding reciprocal experiments on Pcl mutant larvae. Therefore, transgenic fly lines were generated that contain inserts of a pWIZ-Pcl construct, which expresses Pcl shRNA under the control of Gal4, permitting inducible RNAi-mediated knockdown of Pcl in combination with Gal4 drivers. Individuals that contain both pWIZ-Pcl and P{GAL4-da.G32}, which constitutively expresses Gal4, died as early pupae and exhibited dramatically reduced levels of Pcl in wing imaginal discs. E(z) levels were not affected. ChIP assays of these Pcl-depleted wing discs confirmed reduced Pcl levels at the bxd PRE and revealed commensurate loss of E(z). Thus, although Pcl does not require PRC2 for PRE binding, Pcl, presumably functioning as a subunit of PCLC, is needed for stable binding of PRC2 to the bxd PRE. Phol remains at the PRE in the absence of Pcl (Savla, 2008).

In order to determine whether Pcl (like components of PRC1 and PRC2) requires Pho and/or Phol for PRE binding, ChIP assays were performed using wing imaginal discs from phol^81A; pho¹ larvae. Consistent with their role in recruiting other PcG proteins, Pcl was lost from the bxd PRE in the absence of Pho and Phol. These observations at the bxd PRE also appear to generally apply to PcG-binding sites throughout the genome (Savla, 2008).

These results demonstrate the existence of a distinct Pcl protein complex in larvae that is required for recruitment of PRC2 to chromosomal target sites and/or to stabilize its binding. As previously described, E(z), as a core subunit of PRC2, is required for target site binding by PRC1. Therefore, Pcl is indirectly required for chromosome binding by PRC1 as well, although direct interaction with PRC1 cannot be ruled out, similar to the way in which Pho may contribute to target site binding by PRC1 by interacting both with PRC2 subunits and with Pc, a core subunit of PRC1 (Savla, 2008 and references therein).

In vitro histone methyltransferase assays of Pcl-PRC2 show that its activity and specificity for methylation of H3K27 are essentially indistinguishable from that of PRC2 complexes lacking Pcl. ChIP analysis of Pcl mutant embryos has shown that Pcl does not seem to be required for target site binding by other PRC2 subunits, but that it may be needed for high levels of trimethylation of H3K27. One explanation for these observations is that the contribution of Pcl to Pcl-PRC2 in embryos might be to mediate interaction with other proteins that are yet to be identified. In larvae, Pcl exists as a subunit of a distinct complex. Given the ability of Pcl to directly interact with several PRC2 subunits, colocalization of Pcl and E(z) at the PRE, and dependence of E(z) on Pcl for binding to the bxd PRE and other genomic sites, it is likely that PCLC is closely associated with PRC2 at target sites in larvae. In both embryos and larvae, some of the activities attributed to Pcl might, upon further inspection, be due to the activities of other Pcl-associated proteins, the close apposition of which with PRC2 and other PcG complexes may be mediated by Pcl. The differential deployment of Pcl as a subunit of PRC2 and as a subunit of PCLC at distinct developmental stages is intriguing and might reflect the different molecular activities needed for establishment of silencing in embryos and maintenance of the silenced state in larval tissues. A more detailed understanding of the mechanisms by which Pcl contributes to PcG silencing will require identification of the other proteins contained within the larval PCLC complex and the potential biochemical activities of the complex (Savla, 2008).

Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners

The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function, an inducible version of the biotinylation tagging approach was established to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. Many TrxG related proteins were found, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, this analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function (Strübbe, 2011).

Combinations of one-step capture with streptavidin, low stringency washes, specific elution, and detection of peptides using a highly sensitive LTQ-FT-ICR mass spectrometer enabled the identification of even labile and transient interactions. It has been well recognized that PcG and TrxG proteins exert their counteracting activities at the level of chromatin by employing various biochemical activities directed against histones, like methylation, acetylation, and chromatin remodeling. Indeed, this study reveals a substantial number of Pc-interacting proteins implicated in TrxG action. The genes encoding for Rdx, Ebi, CG1845, Rad21, and Fs(1)h have been shown genetically to belong to the TrxG suppressing PcG mutant phenotypes and activating HOX gene expression, for example. Additionally, Pp1-87B has been found to interact with Trx or its homologue MLL. These data indicate that Pc and specific members of the TrxG may physically cooperate to maintain the on/off state of genes (Strübbe, 2011).

So far, the DNA-binding proteins Zeste, Gaf, Pho, Dsp1, Sp1/Klf family members, Psq, and Grh have been connected to PcG function on the basis of genetic interactions, biochemical copurification, functional assays, and/or colocalization on PREs. This study found direct biochemical interactions of Grh and Pho with Pc. Moreover, a Pc-interacting protein called Fs(1)h was identified that might, as well, contribute to recruitment of PRCs to chromatin. Fs(1)h interacts strongly with Ubx, trx, and ash1 mutations and leads to homeotic phenotypes when overexpressed. Fs(1)h is essential for development and conserved in mammals. Whether Pc is recruited by Fs(1)h or opposes its function in gene activation needs to be established. Beside the aforementioned DNA-binding proteins, Enok is a Pc interactor with a putative DNA-binding domain. Enok forms part of the MYST domain family of histone acetyl transferases (HATs), and mutants with defects in the HAT domain show retarded development and pupal lethality (Strübbe, 2011).

Enok's HAT domain is conserved in the vertebrate Moz/Morf proteins. They typically form complexes comprising one protein per BRPF-, ING-, and EAF family member. In Drosophila, a Moz/Morf like complex may consist of Enok, CG1845 (homologue of Brpf1-3), and Eaf6 as all these proteins copurified with Pc and were detected with high confidence. Moz and Brpf1 are TrxG proteins required for HOX gene expression in vertebrates. Although MYST-domain-containing HATs have generally been associated with transcriptional activation, there are also examples with a link to HOX gene repression in Drosophila (Strübbe, 2011).

This work uncovered a connection between Pc and the cohesin complex. Cohesin has been described in detail for its roles in mitosis and meiosis, embracing sister chromatids in mitotic cells. Interestingly, mutations in Ph-p, Psc, and Pc have been reported to result in chromosome missegregation phenotypes in embryos. Besides its traditional role in sister chromatid cohesion, cohesin has also been implicated in both activation and repression of transcription. Furthermore, mutations in the Rad21 subunit of the cohesin complex strongly enhance TrxG and suppress PcG loss of function phenotypes. Pc and cohesins are not colocalized on salivary gland chromatin, and removal of cohesin does not affect Pc binding. It cannot be ruled out that Pc is needed for recruitment of cohesin, however. For example, chromatin binding of cohesin in S. pombe depends on formation of heterochromatin, requiring another chromo domain protein, HP1 (Strübbe, 2011).

A hallmark of PcG repression in flies is pairing-sensitive silencing (PSS), depending on pairing of homologous chromosomes in interphase chromatin. It is known that multiple copies of a transgenic PRE interact with each other if inserted on the same or even on different chromosomes. Because cohesin plays a role in pairing of homologous chromosomes in meiosis and has been suggested to facilitate long-range DNA interactions, it may also facilitate PRE pairing. The transgenic reporter for PSS used in this study only showed PRE-dependent silencing upon PRE pairing. The observation that cohesin mutant alleles reduce PSS supports a model in which cohesins contribute to PRE pairing in interphase chromatin. The identification of Pc-interacting proteins was made possible by employing the in vivo biotinylation system combined with highly sensitive mass spectrometric analysis, thereby preserving near physiological conditions for protein purification. The identification of substoichiometric levels of interacting proteins shows that in vivo biotinylation was effective in capturing even weakly or underrepresented associated proteins. Inducible biotinylation tagging is currently limited to the use of Gal4 drivers that trigger biotinylation well above the background levels. Generation of libraries of different UAS-BirA transgenic lines with less leaky expression and flies carrying BirA under direct control of tissue-specific promoters will further improve and expand this tool, making it a versatile system for proteomic and genomic studies in specialized cell types. As a major advantage over tissue-specific expression of tagged bait proteins, biotin tagging allows to express the bait protein under control of endogenous promoter sequences, whereas the induction of the BirA ligase can be independently induced via the Gal4/UAS system avoiding bait protein misexpression artifacts. This work opens the perspective for tissue-specific applications, potentially enabling a systems analysis on how protein networks can control subsets of genes in specialized cells (Strübbe, 2011).

Structural basis for targeting the chromatin repressor Sfmbt to Polycomb response elements

Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. This study reports the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Scm-related gene containing four mbt domains (Sfmbt). The spacer region of Pho, separated from the DNA-binding domain by a long flexible linker, forms a tight complex with the four malignant brain tumor (4MBT) domain of Sfmbt. The highly conserved spacer region of the human Pho ortholog YY1 binds three of the four human 4MBT domain proteins in an analogous manner but with lower affinity. Structure-guided mutations that disrupt the interaction between Pho and Sfmbt abolish formation of a ternary Sfmbt:Pho:DNA complex in vitro and repression of developmental regulator genes in Drosophila. PRE tethering of Sfmbt by Pho is therefore essential for Polycomb repression in Drosophila. The results support a model where DNA tethering of Sfmbt by Pho and multivalent interactions of Sfmbt with histone modifications and other PcG proteins create a hub for PcG protein complex assembly at PREs (Alfieri, 2013).

What is the function of Pho-tethered Sfmbt at PREs? A straightforward scenario is that PhoRC functions as a platform for the recruitment of other PcG complexes and the interaction with chromatin. Unlike in the case of the Pho:Sfmbt interaction, it has not been possible to reconstitute stable Pho:PRC1 or Pho:PRC2 complexes with recombinant proteins. Previous studies nevertheless found both Pho and Sfmbt in purifications of the Polycomb protein from Drosophila embryos. Pho itself has been also reported to interact physically with subunits of both PRC1 and PRC2 . In addition, recombinant Sfmbt and the PRC1-associated Scm protein can be reconstituted into a stable complex. This interaction thus represents a direct molecular link between PhoRC and PRC1. The C-terminal SAM domain of Sfmbt is not required for Scm binding and may thus engage in interactions with other ligands. In addition, Sfmbt and Scm both bind to lower-methylated lysine residues in various histone tails, and Sfmbt can also bind methylated histone tails while bound to Ph. Taken together, this supports the view that PhoRC functions as a platform for the recruitment of various interactors. While Pho and Sfmbt bind each other strongly, their affinities for other binding partners such as individual PcG proteins or nucleosomes might be weaker and more transient. However, the modular architecture and the multivalency of PhoRC interactions create the hub that is necessary for the stable assembly of different PcG complexes at PREs (Alfieri, 2013).

YY1 DNA binding and interaction with YAF2 is essential for Polycomb recruitment

Polycomb Group (PcG) proteins are crucial for epigenetic inheritance of cell identity and are functionally conserved from Drosophila to humans. PcG proteins regulate expression of homeotic genes and are essential for axial body patterning during development. Previous studies have shown that transcription factor YY1 functions as a PcG protein. YY1 also physically interacts with YAF2, a homolog of Ring and YY1 Binding Protein (RYBP). This study has characterize the mechanism and physiologic relevance of this interaction. Drosophila RYBP mutant flies were phenotypically and biochemically corrected by mouse YAF2 demonstrating functional conservation across species. Further biochemical analysis revealed that YAF2 bridges interaction between YY1 and the PRC1 complex. ChIP assays in HeLa cells showed that YAF2 is responsible for PcG recruitment to DNA, which is mediated by YY1 DNA binding. Knock-down of YY1 abrogated PcG recruitment, which was not compensated by exogenous YAF2 demonstrating that YY1 DNA binding is a priori necessary for Polycomb assembly on chromatin. Finally, it was found that although YAF2 and RYBP regulate a similar number of Polycomb target genes, there are very few genes that are regulated by both implying functional distinction between the two proteins. A model is presented of YAF2-dependent and independent PcG DNA recruitment by YY1 (Basu, 2013). Ph to form functional, ordered assemblies via its SAM domain (Gambetta, 2014).