Posterior sexcombs and Suppressor two of zeste


Targets of Activity

Hunchback regulates knirps and giant repression in the anterior portion of the embryo. Enhancer of zeste (E[z])maintains the expression domains of knirps and giant inititated by the maternal HB protein gradient. A small region of the knirps promoter mediates the regulation by E(z) and HB. Other Pc-G genes are involved in silencing as well. Single mutants of the Su(z)2 complex have a small effect, but deletions of the whole complex, including Posterior sex combs, Su(z)2 and Su(z)2-D has a stronger effect (Pelegri, 1994).

The requirements for the multi sex combs (mxc) gene during development have been examined to gain further insight into the mechanisms and developmental processes that depend on the important trans-regulators forming the Polycomb group (PcG) in Drosophila. Although mxc has not yet been cloned, it is known to be allelic with the tumor suppressor locus lethal (1) malignant blood neoplasm [l(1)mbn]. The mxc product is dramatically needed in most tissues because its loss leads to cell death after a few divisions. mxc also has a strong maternal effect. Hypomorphic mxc mutations are found to enhance other PcG gene mutant phenotypes and cause ectopic expression of homeotic genes, confirming that PcG products are cooperatively involved in repression of selector genes outside their normal expression domains. The mxc product is needed for imaginal head specification, through regulation of the ANT-C gene Deformed. This analysis reveals that mxc is involved in the maternal control of early zygotic gap gene expression known to involve some other PcG genes and suggests that the mechanism of this early PcG function could be different from the PcG-mediated regulation of homeotic selector genes later in development (Saget, 1998).

Induction of uncontrolled growth and deregulation of Hox genes are linked in mammals, where Hox products can induce leukemia. In Drosophila, modification of homeotic gene expression causes homeosis, sometimes associated with increased proliferation but not with uncontrolled tumorous growth, possibly because the identity of each segment is specified by a combination of HOM products. Loss or gain of one HOM gene will likely lead to a new combination that is found elsewhere in wild type, and cells expressing this combination could be expected to follow the corresponding developmental pathway and give rise to homeotic transformations. However, because each cellular identity apparently corresponds to a given proliferation rate, loss or ambiguity of identity due to deregulation of several selector genes in a single cell, such as mxc mutations apparently induce, could lead to loss of proliferation control. Identification of mxc partners and targets, as well as of the molecular nature of the mxc product, may help throw light on the genes and mechanisms involved in this process (Saget, 1998).

It has been proposed that certain PcG genes are required for the maintenance of the expression domains of knirps and giant, through a mechanism similar to the regulation of homeotic genes. The regionalization of the Drosophila embryo depends on the maternally supplied products of bicoid (bcd), hunchback (hb), and nanos (nos). Nos represses the translation of the maternal HB mRNA in the posterior embryonic region. This permits the expression of the zygotic gap genes knirps (kni) and giant (gt), which specify posterior identities. These genes would otherwise be repressed by Hb. Embryos from nos/nos mothers form no abdominal segments, but this phenotype can be rescued by a total lack of hb in the maternal germline. It can also be dominantly rescued by the mutation of maternally supplied regulator molecules that normally repress kni and gt in the zygote. Pelegri and Lehmann (1994) have shown that certain mutant products of the PcG genes E(z), Psc, and pleiohomeotic can partially rescue nos by such a maternal effect. To determine if mutation of mxc also affects this regulation, the cuticles of embryos were examined from mxc/+;hb nos/nos mothers that were heterozygous for different mxc mutations. This genetic background was used because a decrease in the amount of maternal hb product can partially rescue the nos phenotype in F1 embryos. Such embryos can differentiate a few abdominal denticle belts and form an adequate background to evaluate increased rescue of nos. Thus loss-of-function PcG mutations should have a strong effect on rescue, and the embryos from hb nos/nos mothers that have two PcG mutations in their genetic background should permit increased rescue of the nos phenotype (Saget, 1998).

Any of three E(z)son (suppressor of nanos) alleles or a hypomorphic pleiohomeotic allele partially rescue the phenotypes of hb nos/nos progeny by a maternal effect; deficiencies covering E(z) or the Psc/Su(z)2 complex also allow some maternal rescue of hb nos/nos progeny, yet the strongest effect is observed with the gain-of-function E(z)son alleles. The EMS-induced allele mxcG48 rescues the hb nos/nos progeny phenotype, whereas a deficiency of mxc does not. Some rescue with the Psc/Su(z)2 complex deletion Df(2)vgB is also observed and strong rescue (consistently >50%) is observed with an EMS-induced pleiohomeotic allele phob, described as amorphic. This suggests that phob and mxcG48 are probably not amorphic alleles, and that maternal rescue of hb nos/nos progeny by a PcG gene is most efficient with a non-null mutation (Saget, 1998).

Segmentation of embryos from transheterozygous mothers was also examined. Because neither a reduction of wild-type PcG product nor two PcG mutations in trans in the hb nos/nos mothers increases nos rescue, these data strongly suggest that, whatever the mechanism of gap gene regulation by these PcG mutations may be, it does not function like the PcG-mediated maintenance of homeotic gene expression in embryos and in imaginal discs. The strong rescue provided by several non-null EMS-induced mutations, which may produce mutant proteins, leads to a proposal that modified PcG proteins are poisoning a normal process. How this process depends on wild-type regulation by PcG products has yet to be established (Saget, 1998).

Mutations in several Polycomb group genes cause maternal-effect or zygotic segmentation defects, suggesting that Pc-G genes may regulate the segmentation genes of Drosophila. Individuals doubly heterozygous for mutations in polyhomeotic and six other Pc-G genes show gap, pair rule, and segment polarity segmentation defects. Posterior sex combs and polyhomeotic interact with Krüppel and enhance embryonic phenotypes of hunchback and knirps (McKeon, 1994).

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. Using human cells lines, a study was carried out on the ability of insulators to block the action of chromatin-associated repressors, such as HP1, MeCP2 and PcG proteins. The Drosophila special chromatin structure insulator (scs, found to flank the hsp70 heat shock locus in Drosophila) completely blocks transcriptional repression mediated by all of the repressors tested. The Drosophila gypsy insulator is able to block the repression mediated by the PcG proteins Su(z)2 and Sex combs extra/Ring1, as well as mHP1, but not the repression mediated by methyl-CpG-binding protein (MeCP2) and the PcG protein HPC2. The 5'-located DNase I-hypersensitive site in the chicken beta-globin locus displays a limited ability to block repression, and a matrix or scaffold attachment region element is entirely unable to block repression mediated by any repressor tested. These results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high degree of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators (van der Vlag, 2000).

These results show that several insulators are able to block repression mediated by chromatin-associated repressors. Of these, the scs insulator is most efficient in blocking the repressors tested. This result demonstrates a striking evolutionary conservation, since in this study the scs insulator was used outside its natural Drosophila environment. This implies that there are human proteins that bind to the scs element in such a manner that the insulator becomes functional. Also, the function of the gypsy insulator is functionally conserved, since gypsy is able to block most of the repressors used. However, unlike scs, gypsy is not able to block repression mediated by HPC2 or MeCP2. There are several explanations for this observation: (1) it is an intrinsic characteristic of the gypsy insulator; (2) the DNA-protein interaction within the gypsy nucleoprotein complex is not sufficiently conserved to allow the insulator to function properly within the context of the human cell line; (3) the gypsy insulator does not obtain a proper chromatin structure that allows the insulator to become fully functional. At present, the data do not favor the last two options for the following reasons: (1) if the evolutionary conservation is insufficient, it is hard to explain why gypsy is very efficient in blocking repression mediated by RING1, Su(z)2, and mHP1. With insufficient conservation, one might expect that the gypsy insulator would block no vertebrate repressor at all. (2) To exclude an improper chromatin environment, stably transfected cell lines were made with the reporter construct that contains gypsy. Also, in this case, gypsy is unable to block repression mediated by HPC2 and MeCP2. (3) No significant differences in the nucleosomal chromatin structure of the reporter construct containing gypsy were detected. (4) If an improper chromatin structure plays a role, this would not explain why gypsy, under similar conditions, is very efficient in blocking repression mediated by RING1, Su(z)2, and mHP1. When taking these arguments together, the possibility is favored that these results indicate that it is an intrinsic characteristic of the gypsy insulator to block repression by RING1, Su(z)2, and mHP1 but not by HPC2 and MeCP2. Apparently, gypsy is able to block chromatin-associated repressors with a high level of selectivity. This establishes an important point: whereas both HPC2 and MeCP2 are able to very efficiently repress gene activity, they are different from the other repressors in the sense that their action cannot be blocked by the gypsy insulator. The assay used thus uncovers both differences in the ability of insulators to block repression and differences between chromatin-associated repressors. The differences between repressors do not become apparent when only their abilities to repress gene activity are being monitored (van der Vlag, 2000).

These results show an orientation dependence of the 5'-located DNase I-hypersensitive site (5'-HS) in the chicken beta-globin locus. Whereas a single copy of the entire element does not have much effect on the repression mediated by any of the repressors tested, a distinct effect has been found when a tandem of six core elements within the 5'-HS element is tested. Previously, it has been shown that the enhancer blocking ability of the 5'-HS element resides precisely in this core element. The finding that the tandem of 5'-HS core elements is very efficient in blocking repression, but only when cloned in the 5' to 3' orientation, comes as a surprise. The fact that this is true for all repressors tested gives weight to the idea that this orientation dependence is an intrinsic property of the 5'-HS element. No indication has been found that a Drosophila MAR/SAR element is able to block chromatin-associated repressors. This was observed in a stably transfected cell line and in a cell line 72 h after transfection. In either case, a bona fide nucleosomal chromatin structure was observed. It should also be pointed out that the ability of MAR/SARs to shield reporter genes against enhancers and repressing chromatin is controversial. It is concluded that the portion of the Drosophila histone MAR/SAR element tested does not possess an ability to block repression (van der Vlag, 2000).

The result that is most easy to interpret is the efficient blocking of repression by the scs insulator, since scs blocks each repressor tested. How do these findings relate to previous studies? The scs and gypsy insulators have been tested for their ability to protect a reporter gene against position effects in transformed flies. The white maxigene construct is able to confer high expression levels of white and is prone to repression due to position effects. The white maxigene has been artifically flanked with the scs and scs' elements. The elements have been shown to confer a consistently high level of white expression in the majority of transformants, independent of the integration position within euchromatic regions of the genome. These results strongly suggest that scs and scs' are able to efficiently block repression. Thus, the ability of the scs element to block repression, are in agreement with earlier studies (van der Vlag, 2000).

The gypsy insulator has not been tested in the context of the white maxigene. Instead, the gypsy insulator has been used to flank the white minigene. These constructs are considered to be easily affected by position effect variegation, a phenomenon that involves repression in a heterochromatin environment. The extent of position effect variegation increases when these constructs are tested in fly lines that lack functional Su(Hw) protein. Su(Hw) is the protein that binds to gypsy and is necessary for gypsy to function properly. These results have been interpreted as indicating that in these fly lines the gypsy insulator does not function properly and that, consequently, repression is blocked less efficiently (van der Vlag, 2000).

The 5'-HS element has been tested in Drosophila embryos within the context of the white minigene. This assay tests the ability of the 5'-HS element to protect against activation emerging from the surrounding chromatin, not the ability to protect against repression. However, recently the 5'-HS element has been found to convey position-independent expression levels to a reporter gene that was stably integrated in a chicken cell line. This favors a model in which the 5'-HS element protects a reporter gene against both activating and repressing influences emerging from the surrounding chromatin (van der Vlag, 2000).

What is the evidence from other studies that link PcG proteins to the function of insulators? The most convincing genetic evidence indicates that the function of gypsy depends on PcG-mediated repression. Mutations in PcG genes suppress the insulator properties of gypsy, as monitored by its ability to prevent enhancer-promoter interactions. It has further been found that when either gypsy or scs is placed between a polycomb response element and a promoter, the repression initiated from the polycomb response element is blocked. The data of the current study are in agreement with these earlier studies. Whether this also implies that insulators such as scs and gypsy function as stop signals to terminate spreading of the PcG complex remains speculative. Taken together, however, all data point toward an important role of PcG proteins in the function of the gypsy and scs insulators (van der Vlag, 2000).

These data indicate that insulators are conserved nucleoprotein structures that are able to efficiently block repression mediated by a variety of chromatin-associated repressors in evolutionarily nonrelated species. Whereas this statement is true in general, the data also show an unexpected level of selectivity toward specific repressors (HPC2 and MeCP2) as well as an orientation dependence of boundary function (the 5'-HS core elements). This suggests that there are distinct classes of insulators that may be well defined by their ability to block the action of specific chromatin-associated repressors. The repression assay that was developed may be a powerful tool in characterizing both putative insulators as well as novel repressors (van der Vlag, 2000).

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Ruscha, 2000).

During the late phase, two PcG genes, Psc and ph, have been identified that are involved in maintaining repression of pb outside its normal domain of expression. This result supersedes a previous report that the PcG genes do not regulate pb. No trxG genes have been identified that are required for the maintenance of pb expression. To function, the PcG genes are thought to assemble on DNA in large multimeric complexes. Unlike the genes of the BX-C, which are regulated, to a greater and lesser extent, by all the PcG genes that have been tested, pb is not regulated by the majority of known PcG genes. Assuming that the PcG genes function similarly at the pb locus, the implication is that not all multimeric complexes can be equal. However, it is not clear how these differences are established. One possibility is that complexes composed of different combinations of PcG genes are formed at different times during development, thereby regulating different loci. Interestingly, a vertebrate homolog of Psc has been shown to bind a specific DNA sequence. This exact sequence is also found in the regulatory elements of the pb reporter construct, indicating that Psc may bind directly, though this remains to be shown (Rusch, 2000).

Genome-wide analysis of Polycomb targets in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Regulation of Polycomb group genes Psc and Su(z)2 in Drosophila melanogaster

Certain Polycomb group (PcG) genes are themselves targets of PcG complexes. Two of these constitute the Drosophila Psc-Su(z)2 locus, a region whose chromatin is enriched for H3K27me3 and contains several putative Polycomb response elements (PREs) that bind PcG proteins. To understand how PcG mechanisms regulate this region, the repressive function of the PcG protein binding sites was analyzed using reporter gene constructs. It was found that at least two of these are functional PREs that can silence a reporter gene in a PcG-dependent manner. One of these two can also display anti-silencing activity, dependent on the context. A PcG protein binding site near the Psc promoter behaves not as a silencer but as a down-regulation module that is actually stimulated by the Pc gene product but not by other PcG products. Deletion of one of the PREs increases the expression level of Psc and Su(z)2 by twofold at late embryonic stages. Evidence is presented suggesting that the Psc-Su(z)2 locus is flanked by insulator elements that may protect neighboring genes from inappropriate silencing. Deletion of one of these regions results in extension of the domain of H3K27me3 into a region containing other genes, whose expression becomes silenced in the early embryo (Park, 2012).


The products of PSC and Su(z)2 were immunohistochemically detected at 80-90 sites on polytene chromosomes. The chromosomal binding sites of these two proteins were compared with those of Zeste protein and two other Polycomb group proteins, Polycomb and Polyhomeotic. The five proteins co-localize at a large number of sites, suggesting that they frequently act together on target genes. In larvae carrying a temperature sensitive mutation in E(z), the Su(z)2 and PSC products become dissociated from chromatin at non-permissive temperatures from most but not all sites, while the binding of the Zeste protein is unaffected. The polytene chromosomes in these mutant larvae acquire a decondensed appearance, frequently losing characteristic constrictions (Rastelli, 1993).

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

The proximal Polyhomeotic gene product has 193 amino-terminal amino acids that are absent from distal Ph; in addition, it makes use of internal initiation to give an alternate product that is shorter by 244 amino acids. A notable feature of this unique proximal domain is the presence of a PxxPxxPxxP motif (amino acids 156 to 165) with proline spacing the same as that of the polyproline type II helix recognized by the SH3 domain. Ph also has many glutamine repeats and a serine/threonine-rich region. Near the carboxyl terminus are two blocks of sequence (amino acids 1297 to 1388 and 1511 to 1576) that are shared with the mammalian Ph homologs. The first sequence, named H1, consists of 28 highly conserved amino acids followed by an unusual C4 zinc finger with intercysteine spacing Cx2C...Cx3C. The second sequence has been variously referred to as H2 or SEP (for the mouse homolog); as SPM (for the PcG protein Sex combs on midleg as well as for the human Ph homologs HPH1 and HPH2), and as SAM (for a variety of yeast signal transduction proteins). This domain can mediate homotypic and heterotypic self-association between Ph and Scm proteins in vitro. In view of this result, the domain is referred to as a self-association motif (SAM), but the specific subset of SAMs with greatest similarity to Ph and Scm is called SPM. The only internal region of sequence dissimilarity between the proximal and distal Ph are the 52 amino acids immediately preceding the SPM domain (Kyba, 1998).

Polyhomeotic and Posterior sexcombs are shown to coprecipitate with Polycomb from nuclear extracts. The domains required for the association of Psc with Ph and Pc were analyzed by using the yeast two-hybrid system and an in vitro protein-binding assay. Psc and Ph interact through regions of sequence conservation with mammalian homologs, i.e., the H1 domain of Ph (amino acids 1297 to 1418) and the helix-turn-helix-containing region of Psc (amino acids 336 to 473). Psc contacts Pc primarily at this region of Psc, and secondarily at the ring finger (amino acids 250 to 335). The Pc chromobox is not required for this interaction. There is also a discussion of the implication of these results for the nature of the complexes formed by Polycomb group proteins (Kyba, 1998).

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

Recruitment of components of Polycomb Group chromatin complexes in Drosophila

Polycomb Group complexes assemble at polycomb response elements (PREs) in vivo and silence genes in the surrounding chromatin. To study the recruitment of silencing complexes, various Polycomb Group (PcG) proteins have been targeted by fusing them to the LexA DNA binding domain. When LexA-Pc, -Psc, -Ph or -Su(z)2 are targeted to a reporter gene, they recruit functional PcG-silencing complexes that recapitulate the silencing behavior of a PRE: silencing is sensitive to the state of activity of the target chromatin. When the target is transcriptionally active, silencing is not established but when the target is not active at syncytial blastoderm, it becomes silenced. The repressed state persists through embryonic development but cannot be maintained in larval imaginal discs even when the LexA-PcG fusion is constitutively expressed, suggesting a discontinuity in the mechanism of repression. These proteins also interact with other PC-containing complexes in embryonic nuclear extracts. In contrast LexA-Pho is neither able to silence nor to interact with Pc-containing complexes. Analysis of pho mutant embryos and of PRE constructs whose Pho-binding sites are mutated suggests that, while Pho is important for silencing in imaginal discs, it is not necessary for embryonic PcG silencing (Poux, 2001).

These results show that several PcG proteins, targeted by fusion to a DNA-binding domain, can recruit a repressive PcG complex. Several new conclusions can be drawn from these experiments. (1) The recruitment of the silencing complex cannot occur before blastoderm. The alpha1-tubulin-LexA-Pc construct reveals that even when the protein is deposited in the egg during oogenesis, as well as being zygotically expressed, it does not block the initiation of transcription from the Ubx promoter. These results suggest that the LexA-PcG protein cannot establish repression at this early stage, just as the endogenous PcG proteins known to be present in the normal pre-blastoderm embryo do not prevent the initiation of Ubx transcription. Thus, PcG silencing directed by a PRE or by LexA-Pc appears only to set in after blastoderm. One possible explanation is that the assembly of a functional PcG complex at the PRE or at the LexA-binding sites is a multistep process that requires time and is not accomplished until after blastoderm. Another interesting possibility is that the state of the chromatin in nuclei whose very rapid nuclear divisions are just beginning to slow down, cannot yet support the establishment of PcG silencing, for example, because the nucleosomes still bear the deposition-associated histone acetylation pattern. A similar argument might explain why centric heterochromatin is not detectable until blastoderm (Poux, 2001).

(2) The repression established by the LexA-PcG protein is not unconditional but it is sensitive to the state of activity of the target. Like a genuine PRE complex, the LexA-PcG protein establishes silencing only in cells in which the reporter gene is inactive, thus discriminating between active and inactive chromatin targets. The later-acting Ubx H1 enhancer can still function but only in the progeny of cells that were active at early times. The fact that, like the endogenous PRE, the action of the LexA-PcG protein distinguishes between active and silent chromatin, indicates that the discrimination occurs after the binding of the first PcG protein. The sensitive step could be the assembly of a sufficient nucleus of PcG proteins or still later, the involvement of other factors that effect the silencing. It has been shown that Pc-containing complex purified from embryonic nuclear extracts can prevent chromatin remodelling in vitro if it is bound to chromatin before the addition of purified SWI/SNF complex but not if it is added simultaneously or afterwards. If the activation of the Ubx-lacZ reporter gene by the enhancers involves the recruitment of the Drosophila SWI/SNF complex, this observation could help to understand how the LexA binding sites function as a genuine synthetic PRE possessing at least one aspect of the cellular memory displayed by endogenous PREs (Poux, 2001).

(3) Nevertheless, the LexA-binding sites do not constitute a fully functional PRE. Silencing by LexA-PcG proteins is much less effective during larval development. It is possible that the activity of the Ubx H1 enhancer in imaginal discs is more difficult to repress, e.g. because of very high activator concentration. More likely, once the H1 enhancer is active, it is much more difficult to repress by LexA-Pc induced at later times. It cannot be explained, however, why neither daily heat shocks, nor constitutive expression of LexA-Pc can maintain a continuity between embryonic silencing and larval silencing. Repeated heat shocks do eventually reduce the level of expression of the reporter in imaginal discs but the memory of the early domains of repression is lost. The apparent discontinuity in PcG silencing between the embryo and the larva, suggests that there might be a real mechanistic difference between the two states. There are also differences in the maintenance properties of embryos and larvae. One possible explanation is that additional proteins, recruited at a true PRE but not by the LexA fusion proteins, might be necessary for continuous repression at postembryonic developmental stages (Poux, 2001).

LexA proteins unable to repress PcG proteins Psc, Su(z)2 and Ph are as effective as Pc in recruiting a silencing complex, implying that any one of the 'core' PcG proteins can reconstitute a silencing complex. In contrast, LexA-Gaga and -Pho do not silence the reporter gene. That LexA-Gaga does not silence is hardly surprising. Many promoters, including the hsp70 promoter, the alpha1-tubulin promoter or the Ubx promoter itself, contain Gaga-binding sites but are not thereby targets for PcG silencing in vivo. The possibility that the LexA-Gaga fusion protein is defective in some respect cannot be excluded, although it is able to bind to the normal endogenous sites on polytene chromosomes and participate in PcG complexes. Most likely, however, Gaga/Trithorax-like factor by itself cannot recruit PcG complexes. It is supposed that, like many other nuclear factors, Gaga can have either a stimulating or a repressing activity, depending on the binding of other proteins. In the chromatin context of the PRE, however, Gaga interacts with PcG proteins to form a stable complex (Poux, 2001).

Polycomb Group complexes assemble at polycomb response elements (PREs) in vivo and silence genes in the surrounding chromatin. To study the recruitment of silencing complexes, various Polycomb Group (PcG) proteins have been targeted by fusing them to the LexA DNA binding domain. When LexA-Pc, -Psc, -Ph or -Su(z)2 are targeted to a reporter gene, they recruit functional PcG-silencing complexes that recapitulate the silencing behavior of a PRE: silencing is sensitive to the state of activity of the target chromatin. When the target is transcriptionally active, silencing is not established but when the target is not active at syncytial blastoderm, it becomes silenced. The repressed state persists through embryonic development but cannot be maintained in larval imaginal discs even when the LexA-PcG fusion is constitutively expressed, suggesting a discontinuity in the mechanism of repression. These proteins also interact with other PC-containing complexes in embryonic nuclear extracts. In contrast LexA-Pho is neither able to silence nor to interact with Pc-containing complexes. Analysis of pho mutant embryos and of PRE constructs whose Pho-binding sites are mutated suggests that, while Pho is important for silencing in imaginal discs, it is not necessary for embryonic PcG silencing (Poux, 2001).

These results show that several PcG proteins, targeted by fusion to a DNA-binding domain, can recruit a repressive PcG complex. Several new conclusions can be drawn from these experiments. (1) The recruitment of the silencing complex cannot occur before blastoderm. The alpha1-tubulin-LexA-Pc construct reveals that even when the protein is deposited in the egg during oogenesis, as well as being zygotically expressed, it does not block the initiation of transcription from the Ubx promoter. These results suggest that the LexA-PcG protein cannot establish repression at this early stage, just as the endogenous PcG proteins known to be present in the normal pre-blastoderm embryo do not prevent the initiation of Ubx transcription. Thus, PcG silencing directed by a PRE or by LexA-Pc appears only to set in after blastoderm. One possible explanation is that the assembly of a functional PcG complex at the PRE or at the LexA-binding sites is a multistep process that requires time and is not accomplished until after blastoderm. Another interesting possibility is that the state of the chromatin in nuclei whose very rapid nuclear divisions are just beginning to slow down, cannot yet support the establishment of PcG silencing, for example, because the nucleosomes still bear the deposition-associated histone acetylation pattern. A similar argument might explain why centric heterochromatin is not detectable until blastoderm (Poux, 2001).

(2) The repression established by the LexA-PcG protein is not unconditional but it is sensitive to the state of activity of the target. Like a genuine PRE complex, the LexA-PcG protein establishes silencing only in cells in which the reporter gene is inactive, thus discriminating between active and inactive chromatin targets. The later-acting Ubx H1 enhancer can still function but only in the progeny of cells that were active at early times. The fact that, like the endogenous PRE, the action of the LexA-PcG protein distinguishes between active and silent chromatin, indicates that the discrimination occurs after the binding of the first PcG protein. The sensitive step could be the assembly of a sufficient nucleus of PcG proteins or still later, the involvement of other factors that effect the silencing. It has been shown that Pc-containing complex purified from embryonic nuclear extracts can prevent chromatin remodelling in vitro if it is bound to chromatin before the addition of purified SWI/SNF complex but not if it is added simultaneously or afterwards. If the activation of the Ubx-lacZ reporter gene by the enhancers involves the recruitment of the Drosophila SWI/SNF complex, this observation could help to understand how the LexA binding sites function as a genuine synthetic PRE possessing at least one aspect of the cellular memory displayed by endogenous PREs (Poux, 2001).

(3) Nevertheless, the LexA-binding sites do not constitute a fully functional PRE. Silencing by LexA-PcG proteins is much less effective during larval development. It is possible that the activity of the Ubx H1 enhancer in imaginal discs is more difficult to repress, e.g. because of very high activator concentration. More likely, once the H1 enhancer is active, it is much more difficult to repress by LexA-Pc induced at later times. It cannot be explained, however, why neither daily heat shocks, nor constitutive expression of LexA-Pc can maintain a continuity between embryonic silencing and larval silencing. Repeated heat shocks do eventually reduce the level of expression of the reporter in imaginal discs but the memory of the early domains of repression is lost. The apparent discontinuity in PcG silencing between the embryo and the larva, suggests that there might be a real mechanistic difference between the two states. There are also differences in the maintenance properties of embryos and larvae. One possible explanation is that additional proteins, recruited at a true PRE but not by the LexA fusion proteins, might be necessary for continuous repression at postembryonic developmental stages (Poux, 2001).

LexA proteins unable to repress PcG proteins Psc, Su(z)2 and Ph are as effective as Pc in recruiting a silencing complex, implying that any one of the 'core' PcG proteins can reconstitute a silencing complex. In contrast, LexA-Gaga and -Pho do not silence the reporter gene. That LexA-Gaga does not silence is hardly surprising. Many promoters, including the hsp70 promoter, the alpha1-tubulin promoter or the Ubx promoter itself, contain Gaga-binding sites but are not thereby targets for PcG silencing in vivo. The possibility that the LexA-Gaga fusion protein is defective in some respect cannot be excluded, although it is able to bind to the normal endogenous sites on polytene chromosomes and participate in PcG complexes. Most likely, however, Gaga/Trithorax-like factor by itself cannot recruit PcG complexes. It is supposed that, like many other nuclear factors, Gaga can have either a stimulating or a repressing activity, depending on the binding of other proteins. In the chromatin context of the PRE, however, Gaga interacts with PcG proteins to form a stable complex (Poux, 2001).

A Drosophila Polycomb group complex includes Zeste and dTAFII proteins

A goal of modern biology is to identify the physical interactions that define 'functional modules' of proteins that govern biological processes. One essential regulatory process is the maintenance of master regulatory genes, such as homeotic genes, in an appropriate 'on' or 'off' state for the lifetime of an organism. The Polycomb group (PcG) of genes maintain a repressed transcriptional state, and PcG proteins form large multiprotein complexes, but these complexes have not been described owing to inherent difficulties in purification. A major PcG complex, PRC1, has been purified to 20%-50% homogeneity from Drosophila embryos. Thirty proteins have been identified in these preparations, then the preparation has been further fractionated and Western analyses have been used to validate unanticipated connections. The known PcG proteins Polycomb, Posterior sex combs, Polyhomeotic and Sex combs extra/dRING1 exist in robust association with the sequence-specific DNA-binding factor Zeste and with numerous TBP (TATA-binding-protein)-associated factors that are components of general transcription factor TFIID (dTAFIIs). Thus, in fly embryos, there is a direct physical connection between proteins that bind to specific regulatory sequences, PcG proteins, and proteins of the general transcription machinery (Saurin, 2001).

The inheritance of established expression patterns of certain genes during multiple cell divisions is essential for the correct development of an animal. In Drosophila, the expression patterns of the homeotic genes that govern body segment identity are established early in embryogenesis by the products of the gap and pair-rule genes, but are maintained throughout the rest of development by proteins of the PcG and trithorax group (trxG). The trxG maintains the transcriptionally active state of the homeotic genes, whereas the PcG prevents ectopic expression by maintaining a repressive state. The PcG genes encode components of multiple complexes. One of these complexes, Polycomb repressive complex 1 (PRC1), contains the Polycomb (PC), Polyhomeotic (PH) and Posterior sex combs (PSC) proteins (Saurin, 2001).

To better understand the mechanisms of this cellular memory system, an epitope-tag strategy was used to purify PRC1 over 3,000-fold from Drosophila embryos. This complex has been extensively washed in 1 M salt and has a high specific activity in functional analyses; however, contaminating proteins remain associated. Extensive efforts to fractionate this complex to homogeneity in reasonable quantity were blocked by unacceptably low yields on a wide variety of subsequent purification steps. The advent of genome-wide sequence analysis provided an alternative route to identify the components of PRC1. Using mass spectrometry and the recently completed Drosophila genome, almost all of the proteins were identified in the highly fractionated material derived from the M2-affinity column. The sensitivity of Western analysis was used to validate of the association of proteins during subsequent chromatography steps (Saurin, 2001).

The presence of the previously identified PcG proteins PH, PC, and PSC was confirmed by mass spectrometry. In addition, a Drosophila homolog of the mammalian RING1 protein (dRING1) was identified; dRING1 has been found to colocalize with PC on polytene chromosome preparations and its mammalian counterparts have previously been shown to associate with mammalian PcG proteins. Thus, the PcG complement of PRC1 is made up from PH, PSC, PC, dRING1 and sub-stoichiometric amounts of Sex Combs on Midleg (SCM) (Saurin, 2001).

Of the remaining proteins identified by mass spectrometry, the presence of several dTAFII proteins and Zeste is particularly striking. Zeste is a sequence-specific DNA-binding factor, with binding sites in the promoter and regulatory regions of some homeotic genes. The dTAFII proteins were initially identified in the general transcription factor TFIID, a central component for transcriptional initiation, but are also found in histone acetyltransferase complexes. The dTAFII proteins identified by mass spectrometry and Zeste all appear approximately stoichiometric with the PcG complement in the M2 fraction, and maintain a quantitative association with PcG proteins on gel filtration and heparin agarose (Saurin, 2001).

PRC1 fractionates as an extremely large complex, and contains several other proteins in addition to the PcG, dTAFII and Zeste proteins described above. The sequence information provides speculative information on the identity of these proteins, but further work is needed to validate each of these associations. The constitutively expressed Heat shock cognate 3 and 4 (HSC3 and HSC4) proteins were found. The requirement for HSCs in PcG action during development has been demonstrated genetically in flies, where a mutant allele of HSC4 enhances the homeotic phenotype of PC-heterozygous flies. Proteins were found that have been linked to histone deacetylase complexes, including HDAC (RPD3), dMi-2, dSin3A, p55 and SMRTER, a functional homolog of the human SMRT/N-CoR corepressors. While dMi-2 has been linked genetically to PcG repression, and HDAC and p55 have been found present in the Esc/E(z) PcG complex, further studies are clearly needed to examine their association with PRC1. These proteins are present in low stoichiometry: cofractionation of these proteins with PcG on subsequent steps could not be accurately assessed owing to lack of signal, and PRC1 has low deacetylase activity when acetylated core histones and histone peptides are used as substrate (Saurin, 2001).

The most surprising connection revealed in this study is that between PcG proteins and several dTAFIIs. TAFII proteins have previously been found in TFIID and in histone acetyltransferase complexes, and in both contexts have been linked to transcriptional activation. This study suggests that they may also function in PRC1-mediated PcG repression. PcG complexes are targeted to specific genes by sequences called Polycomb response elements (PREs), but are also known to associate at promoters. The presence of dTAFII proteins in PRC1 provides a direct physical connection between PcG proteins and components of the general transcription machinery that bind at promoters. In addition, several of these dTAFIIs have similarities with core histone proteins (dTAFIIs 62, 42 and 30beta) and have been biochemically and structurally demonstrated to associate with each other in a histone octamer-like substructure. Although quite speculative, the structural similarities between the dTAFII42/62 heterotetramer with the histone H3/H4 heterotetramer might indicate a direct role in interacting with nucleosomes and/or DNA to help maintain a stable association of PcG proteins across the numerous rapid cell divisions of the embryo (Saurin, 2001).

The presence of Zeste in PRC1 may serve to assist in the targeting of PcG proteins to repressed loci. Indeed, Zeste can be found localized with PcG proteins at some PcG-repressed loci and recent data demonstrate that Zeste is directly involved in the maintenance of the repressed state of some of these loci. Zeste binds to both PRE and promoter sequences, and thus may serve to bridge the connection of the PcG proteins to these elements. Zeste has also been shown to interact directly with the BRM complex of the trxG. Zeste thus appears be involved in both PcG function and trxG function, consistent with previous genetic studies implying a role in activation and repression (Saurin, 2001).

Essential role of Drosophila Hdac1 in homeotic gene silencing

Deacetylation of the N-terminal tails of core histones plays a crucial role in gene silencing. Rpd3 and Hda1 represent two major types of genes encoding trichostatin A-sensitive histone deacetylases. Drosophila Rpd3, referred to here by its alternative name HDAC1, interacts cooperatively with Polycomb group repressors in silencing the homeotic genes that are essential for axial patterning of body segments. The biochemical copurification and cytological colocalization of HDAC1 and Polycomb group repressors strongly suggest that HDAC1 is a component of the silencing complex for chromatin modification on specific regulatory regions of homeotic genes (Chang, 2001).

Df(3R)10H deletes not only Hdac1, but several other genes as well. To show that deletion of the Hdac1 gene is responsible for the genetic interactions with Pc, genetic interactions between Pc-G mutants and Hdac1P-UTR, a semilethal mutant with a P element inserted at +47 of Hdac1 were examined. Hdac1P-UTR also shows dosage-sensitive genetic interactions with Pc and Psc mutations, indicating that Hdac1 is important in regulating the function of homeotic genes. In contrast to the results with Pc and Psc, no genetic interactions were observed between Hdac1P-UTR and extra sex combs (esc) or Enhancer of zeste [E(z)] mutations. The differences in the genetic interactions with the Pc-G mutations might reflect the presence of two physically distinct complexes formed by these proteins, because the PC and PSC proteins copurify in one Pc-G complex and the ESC and E(Z) proteins copurify in a different Pc-G complex. It is interesting to note that Df(3R)10H and Hdac1P-UTR by themselves did not cause leg transformation. Thus, the homeotic effects of Hdac1 appear to manifest themselves only in combination with Pc-G mutations as noted for several other Pc-G, including Enhancer of Polycomb, Suppressor 2 of zeste, and Mi-2 (Chang, 2001).

Experiments also were performed to explore the role of Hdac1 in regulating the embryonic expressions of two homeotic genes, Abd-B and Ubx. Abd-B proteins normally are expressed in a graded fashion in the posterior part of ventral nerve cord, starting from parasegment 10 (PS10). Although this pattern is not altered in homozygous Hdac1303 mutants, significant levels of Abd-B proteins are observed in more anterior parasegments of homozygous Psce24 mutants. Much higher levels of ectopic Abd-B proteins are found in Psce24 Hdac1303 double mutants, indicating a synergistic effect of Hdac1 and Psc on Abd-B repression. Consistent (but less striking) effects also are observed on Ubx protein levels. The anterior boundary of the Ubx expression domain is PS5, with the exception of a small cluster of cells in the middle of PS4 that also express Ubx proteins. Although homozygous Psce24 mutants only show sporadic low levels of Ubx expression in more anterior parasegments, Psce24 Hdac1303 double mutants show significantly higher levels of ectopic Ubx expression in more cells. In PS5, more cells with higher levels of Ubx proteins are observed in the double mutants than in either of the single mutants. In contrast, Ubx expression is reduced substantially in the abdominal parasegments of the double mutants compared with that in the single mutants, presumably reflecting Ubx repression by more extensive ectopic expression of Abd-B and possibly ABD-A. These data indicate that Hdac1 is essential for homeotic gene silencing in embryos (Chang, 2001).

The genetic interactions between Hdac1 and Pc-G mutations suggest that the encoded proteins might be physically associated. This idea was tested by examining whether HDAC1 and PC proteins can be copurified from cultured Drosophila cells. A permanent S2 cell line was established that expresses a PC protein with a FLAG-epitope tag at the C-terminal end under the control of a metallothionein promoter. Nuclear extracts prepared from induced cells were passed over a FLAG antibody column. After the addition of FLAG peptide, tagged PC and its associated proteins were eluted. Using 3H-labeled, acetylated core histones as substrates to assay these fractions, it was found that HDAC activity elutes with the same profile as PC. In addition, this activity is sensitive to the HDAC-specific inhibitor TSA (Chang, 2001).

To determine the identity of the HDAC associated with PC, immunoblotting was performed with an affinity-purified antibody against the C-terminal part of HDAC1. HDAC1 was detected in the eluted fraction. In addition, substantial amounts of PSC and PH also were copurified, consistent with previous findings that they are components of large PC protein complexes. Much lower amounts of another Pc-G protein, Sex combs on mid-leg (Scm), were detected in these preparations. Thus, these results indicate that HDAC1 is associated with the PC protein complexes in cultured cells (Chang, 2001).

To examine whether HDAC1 is associated with PC complexes in embryos, HDAC1 proteins were immunoprecipitated from embryonic nuclear extracts. PC was detected when an HDAC1 antibody was used for the immunoprecipitation. These results further support the idea that the associations between PC and HDAC1 proteins are physiologically relevant (Chang, 2001).

Given the genetic and biochemical interactions between Hdac1 and Pc, it might be anticipated that a fraction of HDAC1 would colocalize with Pc-G complexes on polytene chromosomes. Approximately 100 common binding sites have been identified for several Pc-G proteins. At least 70% of these sites (identified by staining with PSC mAbs) also stain with the HDAC1 antibody, including the Antennapedia complex at 84AB and the bithorax complex at 89E. These results suggest that HDAC1 proteins act together with a substantial fraction of the Pc-G complex. However, the relative intensities of the signals for PSC and HDAC1 at these sites do not always correlate, suggesting a regulatory, rather than a constitutive function. Furthermore, HDAC1 is much more widely distributed along the chromosomes than is PSC, consistent with its role in global gene regulation and/or chromatin structure (Chang, 2001).

The colocalizations of HDAC1 and PSC were examined further on polytene chromosomes from a transgenic line that carries a Ubx upstream cis-regulatory region (i.e., bxd-14) inserted at 62A. This insert contains a functional PRE and creates a new Pc-G-binding site. Staining with both PSC and HDAC1 antibodies reveals that a new PSC site coincides with a new HDAC1-binding site. This new binding site is beside an HDAC1 site present in the wild-type chromosome, creating a broader signal of HDAC1 at this site. These results strongly suggest that HDAC1 and Pc-G proteins are recruited to this ectopic PRE (Chang, 2001).

These results do not imply that there is a direct physical interaction between HDAC1 and any Pc-G proteins that have been characterized to date. It is possible that an adapter-like molecule might be involved. In several organisms, direct interactions or cytological colocalization between HDAC1 and SIN3A proteins have been demonstrated. However, there is currently no evidence that SIN3A is required for homeotic gene repression. The observations that HDAC1 is not detected at about 30% of PSC sites and that HDAC1 intensity does not always correlate with that of remaining PSC sites suggests that HDAC1 is not constantly associated with the PC complexes. This is consistent with a catalytic rather than a structural function. Different levels of HDAC1 staining might reflect varying degrees of repression at these sites. Because more acetyl groups need to be removed when a gene becomes repressed from an active state, it is also possible that higher levels of HDAC1 are required for the initiation of a repressed state than for the maintenance of a repressed state. Thus, the significance of the relative levels of HDAC1 should be interpreted with caution (Chang, 2001).

Reconstitution of a functional core Polycomb repressive complex

The opposing actions of polycomb (PcG) and trithorax group (trxG) gene products maintain essential gene expression patterns during Drosophila development. PcG proteins are thought to establish repressive chromatin structures, but the mechanisms by which this occurs are not known. Polycomb repressive complex 1 (PRC1) contains PcG proteins Polyhomeotic (Ph), Polycomb (Pc), Posterior sexcombs (Psc) and Ring and inhibits chromatin remodeling by trxG-related SWI/SNF complexes. A functional core of PRC1 has been defined by reconstituting a stable complex using four recombinant PcG proteins. One subunit, Psc, can also inhibit chromatin remodeling on its own. These PcG proteins create a chromatin structure that has normal nucleosome organization and is accessible to nucleases but excludes hSWI/SNF (Francis, 2001).

To assemble an active recombinant complex of PcG proteins that could be purified in large amounts and used for detailed functional analyses, Sf9 cells were coinfected with Flag-Ph, Pc, Psc, and Ring. Flag-Ph and associated proteins were purified by affinity chromatography. Pc, Psc, and Ring were purified quantitatively with Flag-Ph. If Sf9 cells are coinfected with Psc, Ph, and Pc, with either Psc or Ph carrying the Flag epitope, the three proteins copurify but the eluted fractions contain a significant excess of the epitope-tagged subunit; this suggests Ring is important for stable complex formation. Purification of Flag-Ph from cells expressing Flag-Ph, Ring, and Pc did not result in purification of a complex, but mainly of Flag-Ph. Thus, Pc, Ph, Psc, and Ring form the most stable complex, which will be referred to as PCC (PRC1 core complex) for simplicity. PCC fractions invariably contain a fifth, 70 kDa protein; this protein may be an HSC70 homolog from Sf9 cells since it is immunoreactive with antibodies to HSP70. HSC70 may interact specifically with PcG proteins since HSC proteins copurify with PcG proteins in PRC1 and mutations in HSC4 enhance Pc phenotypes; alternatively, it may associate with the complex simply because the PcG proteins are overexpressed (Francis, 2001).

To determine whether PCC shares the ability of PRC1 to block chromatin remodeling by hSWI/SNF, PCC was tested in the plasmid supercoiling assay originally used to define PRC1 activity. When incubated with a nucleosomal plasmid array and topoisomerase I, hSWI/SNF induces an ATP-dependent decrease in negative supercoiling, which can be identified on agarose gels as a change in the distribution of plasmid topoisomers. PRC1 blocks the ability of hSWI/SNF to alter plasmid topology when preincubated with the template, but not when added simultaneously with hSWI/SNF. Preparations of PCC inhibit remodeling in this assay in a preincubation-dependent manner that mimics the activity of PRC1 (Francis, 2001).

Each component of PCC was purified to determine whether any individual protein shared functional characteristics with PCC or PRC1. One subunit, Psc, also inhibits chromatin remodeling in the plasmid supercoiling assay in a manner that is strictly dependent on preincubation. Preparations of Pc, Ph, and Ring were less active than Psc in this assay. It is concluded that PCC and at least one of its subunits share the ability of PRC1 to inhibit chromatin remodeling (Francis, 2001).

To compare the relative efficiency of PRC1, PCC, and Psc, a quantifiable assay for inhibition was developed based on restriction enzyme accessibility. Assembly of DNA into chromatin blocks the ability of restriction enzymes to digest DNA at nucleosomal sites, but this decrease in accessibility can be counteracted by ATP-dependent chromatin remodeling factors such as hSWI/SNF. Nucleosomal arrays were assembled by the use of templates consisting of two sets of five 5S nucleosome positioning sequences that flank DNA sufficient to assemble two nucleosomes, one of which is predicted to overlap a unique HhaI site. The extent of digestion of the HhaI site in a chromatin template exposed to hSWI/SNF was used to quantify inhibition of chromatin remodeling by PcG proteins (Francis, 2001).

PRC1 blocks the ATP-dependent stimulation of restriction enzyme digestion by hSWI/SNF when preincubated with the template at a concentration of approximately 1 nM. Under the same conditions, approximately 2-4 nM PCC also inhibits chromatin remodeling, suggesting the core complex is up to 50% as efficient as PRC1. Psc inhibits chromatin remodeling at higher concentrations than PCC. Thus, both Psc and PCC inhibit chromatin remodeling and are nearly as efficient as PRC1 (Francis, 2001).

PcG preparations could inhibit remodeling through interactions with the nucleosomal array, by directly inhibiting hSWI/SNF function, or by a combination of these mechanisms. PRC1 does not efficiently inhibit remodeling of mononucleosomes. This suggestes that the complex might require a nucleosomal array substrate. Because large amounts of concentrated PCC and Psc could be purified, tests were performed to see whether these proteins inhibit remodeling of mononucleosomes over a wide range of protein concentrations in excess of nucleosomes. PCC and Psc were titrated into a restriction enzyme accessibility assay for hSWI/SNF remodeling of mononucleosomes assembled on a 155 bp DNA fragment. PCC and Psc have no effect on remodeling of mononucleosomes at concentrations greater than those required to completely inhibit remodeling of arrays, and Psc inhibits remodeling only when present at greater than 20-fold excess over template. These results suggest that PCC or Psc inhibit chromatin remodeling by interacting with the substrate and that these proteins have specific substrate requirements not present in mononucleosomes, such as linker DNA or multiple contiguous nucleosomes. These experiments also confirm that concentrations of PCC and Psc that inhibit remodeling of nucleosomal arrays do not directly inhibit hSWI/SNF activity (Francis, 2001).

These experiments demonstrate that the stable PcG complex PCC and the single subunit Psc share the ability of PRC1 to inhibit remodeling. The definition of this minimal system and the ability to obtain large quantities of highly purified PCC and Psc allows a detailed characterization of the ability of PcG proteins to inhibit remodeling. The ability of PCC and Psc to bind DNA and to interact with nucleosomal arrays to prevent remodeling was examined (Francis, 2001).

Although one mammalian Psc homolog, MEL-18, has been demonstrated to bind DNA, this has not been shown to be the case with the PcG proteins present in PCC. The ability of PCC and Psc to inhibit remodeling on arrays but not on mononucleosomes has suggested that they might recognize free DNA. PCC and Psc were tested for DNA binding activity using filter binding with a 155 bp probe; both PCC and Psc bind DNA with high affinity. The measured KDs of PCC and Psc for DNA were similar. Filter binding assays were used to determine what fraction of molecules in each of the protein preparations were active for DNA binding. A high fraction of the molecules in PCC and Psc preparations were active for DNA binding (PCC 30%-60%; Psc 20%-50%), assuming binding as monomers (Francis, 2001).

It was of interest to determine whether PcG proteins generally block access of all enzymes to chromatin or, more specifically, prevent chromatin remodeling. Considerable attention has been given to the ability of remodeling factors to increase accessibility of DNA sites, such as is measured in the restriction enzyme accessibility assay. However, nucleosome movement stimulated by remodelers can also cause previously exposed sites to become occluded. A SacI site in the 5S array template is predicted to reside between nucleosomes; consistent with this prediction, the SacI site is accessible in 40%-60% of the nucleosomal arrays in the absence of hSWI/SNF. If the template is remodeled with hSWI/SNF, the accessibility of this site is dramatically reduced. The SacI site therefore allows the dissociation of effects of PCC and Psc on chromatin remodeling from direct effects on the restriction enzyme or on template accessibility, since in this case inhibition of remodeling would be expected to increase SacI digestion after incubation with hSWI/SNF. When the template was remodeled by SWI/SNF for 30 min and subsequently digested with SacI for 5 min, a clear decrease in restriction enzyme accessibility was observed, consistent with repositioning of nucleosomes over this site on some templates. This hSWI/SNF-dependent decrease in accessibility is blocked by preincubation of the template with PCC or Psc so that digestion levels were similar to those in the absence of hSWI/SNF (40%-60% digestion). It is concluded that Psc and PCC block ATP-dependent remodeling without blocking access of restriction enzymes to the template (Francis, 2001).

To examine whether inhibition of remodeling by hSWI/SNF is accompanied by changes in nucleosome organization, the effect of PRC1, PCC, and Psc on MNase digestion of nucleosomal arrays was examined. MNase preferentially digests the linker DNA between nucleosomes. On arrays of nucleosomes that are positioned by 5S sequences, digestion with MNase produces a distinct ladder, which can be visualized by probing Southern blots of the digested array with the 5S sequence. Incubation of the array with hSWI/SNF and ATP causes a smearing of the banding pattern, reflecting randomization of nucleosome positioning. PRC1, PCC, and Psc block disruption of the MNase ladder by hSWI/SNF. Significantly, none of these PcG proteins or complexes alter the MNase digestion pattern of the array, implying that MNase is still able to access the array and that nucleosome organization is not grossly altered by the presence of these PcG proteins. These results suggest that complexing these PcG proteins with the template prevents hSWI/SNF-mediated nucleosome movement but not nuclease access to the template (Francis, 2001).

In these MNase experiments, it was possible that PCC or Psc altered nucleosome positions but nonetheless maintained a regular organization of nucleosomes on the template. To determine whether nucleosome positions were altered, indirect end labeling was carried out after digestion with MNase or DNaseI and nucleosome position was examined in the presence or absence of PCC or Psc. Neither Psc nor PCC altered nucleosome position as assayed by either MNase or DNaseI digestion, both of which yield nucleosomal ladders due to the tight positioning of the nucleosomes on the 5S array. Similar results were obtained in MNase experiments with PRC1. Interestingly, however, arrays incubated with PCC or Psc were approximately 5- to 10-fold less sensitive to digestion by MNase, but about 10-fold more sensitive to digestion by DNase I. Although as yet there is no explanation for this observation, it is consistent with PCC or Psc altering the structure of the chromatin template and perhaps the orientation of the linker DNA (Francis, 2001).

Psc and PCC could prevent remodeling by preventing hSWI/SNF from binding to the template, by interfering with the remodeling of chromatin carried out by bound hSWI/SNF, or by a combination of both mechanisms. To determine whether binding of Psc or PCC to the template interferes with the binding of hSWI/SNF, in vitro chromatin immunoprecipitations (ChIPs) were carried out using antisera to BRG1, the major ATPase subunit and remodeling protein of hSWI/SNF. The amount of DNA precipitated by anti-BRG1 was compared on templates incubated with or without Psc or PCC and these results were correlated with inhibition of remodeling in the same reactions. When arrays were preincubated with concentrations of Psc or PCC that inhibit chromatin remodeling, the amount of BRG1 bound to the template was decreased by at least 10-fold. Titrations of Psc and PCC indicate that exclusion of BRG1 from the template correlates with inhibition of remodeling. Thus, these experiments suggest that Psc or PCC can exclude hSWI/SNF from a nucleosomal array, and are consistent with exclusion accounting, at least in part, for inhibition of remodeling (Francis, 2001).

Psc is active as an isolated polypeptide, raising the possibility that this protein is an important component of PCC activity and could function in the absence of the other PCC subunits. Indeed, recent evidence suggests Psc is an essential component of the silencing mechanism and may function independently of the other PCC subunits in certain circumstances. (1) Loss of Psc and its homolog suppressor of zeste 2 [Su(Z)2] or Ph, but not of other PcG proteins, has been shown to result in rapid upregulation of homeotic genes. Repression can be restored by resupply of Psc [and Su(Z)2] provided only a few cell generations has passed. These results are consistent with an essential role for Psc in the actual silencing mechanism. (2) Immunocytochemistry in Drosophila embryos demonstrates that the majority of Ph, Pc, and Psc dissociate from chromosomes during mitosis; Psc reassociates with the chromosomes before Pc and Ph, suggesting Psc may function independently of these subunits in vivo during reestablishment of repression (Francis, 2001).

In vivo, PcG proteins are targeted to appropriate genes by PREs, but PCC does not require PRE sequences to prevent chromatin remodeling. Preliminary results with templates that include PRE sequences from the Ubx gene suggest that PREs may not target PCC and Psc in vitro. This raises the possibility that the basic mechanisms by which PcG proteins influence chromatin can be mechanistically separated from those which target repression to appropriate genes. It seems likely that PcG proteins interact, perhaps directly, with DNA binding factors that target them to PREs. The characterization of PCC activity provides a system in which targeting of PcG proteins can be analyzed in vitro (Francis, 2001).

Native and recombinant polycomb group complexes establish a selective block to template accessibility to repress transcription in vitro

Polycomb group (PcG) proteins are responsible for stable repression of homeotic gene expression during Drosophila melanogaster development. They are thought to stabilize chromatin structure to prevent transcription, though how they do this is unknown. An in vitro system has been established in which the PcG complex PRC1 and a recombinant PRC1 core complex (PCC; a stable complex of the core PcG subunits of the PRC1 complex—Polycomb, Polyhomeotic, Posterior Sex Combs, and dRING1) are able to repress transcription by both RNA polymerase II and by T7 RNA polymerase. Assembly of the template into nucleosomes enhances repression by PRC1 and PCC. The subunit Psc is able to inhibit transcription on its own. PRC1- and PCC-repressed templates remain accessible to Gal4-VP16 binding, and incubation of the template with HeLa nuclear extract before the addition of PCC eliminates PCC repression. These results suggest that PcG proteins do not merely prohibit all transcription machinery from binding the template but instead likely inhibit specific steps in the transcription reaction (King, 2002).

These results are consistent with the hypothesis that an interaction with the template is primarily responsible for PcG repression, rather than an interaction with the general transcription machinery. PRC1 represses transcription of both RNA Pol II and T7 RNA polymerase, and repression of both of these polymerases is quantitatively similar, suggesting that repression requires neither a specific interaction with either polymerase nor one with eukaryotic transcription factors. In addition, the enhancement of repression observed for Pol II transcription on a chromatin template in comparison to naked DNA is also quite similar to the enhancement observed with T7 transcription. Since T7 transcription does not involve activators or any of the general transcription factors required for Pol II transcription, it seems unlikely that PRC1 represses transcription predominantly by interacting with or sequestering these factors. The simplest way to explain how PRC1 and PCC behave in this system is that they interact with the template to block steps required for transcription (King, 2002).

Though this study indicates that PcG complexes are capable of repressing transcription in vitro through an interaction between the PcG complexes and the template, it is still unknown whether interactions with the general machinery also play a role in PcG repression. Indeed, the finding that PRC1 contains a number of TAF proteins, which are also a part of the TFIID complex, raises the possibility of interactions between PRC1 and general factors at the promoter. However, PCC, which contains only PcG proteins, is sufficient for repression in this system, implying that the TAF subunits of PRC1 are not essential for the repression observed here. The TAF subunits may be involved in targeting PcG activity, since TAF proteins are capable of interacting with promoter sequences and with transcription factors (King, 2002).

The Psc subunit of PCC has substantial activity as a single protein, suggesting that Psc may be central to the mechanism of transcriptional repression by PcG complexes. This is in accord with previous results that show that Psc on its own is sufficient to inhibit chromatin remodeling by Swi/Snf in vitro and raises the possibility that a template interaction mediated mainly by Psc is responsible for both of these in vitro activities. The Ph subunit also has a small amount of activity on its own, which is consistent with in vivo studies that have also indicated that Psc and Ph may be central to the mechanism of repression. When Psc and its homolog Su(z)2, or Ph, are removed from imaginal disc clones by recombination, homeotic gene expression is disrupted more quickly and to a greater extent than it is when other PcG genes including Pc are removed. The other subunits of PCC that do not appear to inhibit transcription might function primarily in other aspects of repression that are not limiting in this assay, such as the targeting of complexes to specific genes (King, 2002).

Polycomb complexes and the propagation of the methylation mark at the Drosophila Ubx gene

Polycomb group proteins are transcriptional repressors that control many developmental genes. The Polycomb group protein Enhancer of Zeste has been shown in vitro to methylate specifically lysine 27 and lysine 9 of histone H3 but the role of this modification in Polycomb silencing is unknown. This study shows that H3 trimethylated at lysine 27 is found on the entire Ubx gene silenced by Polycomb. However, Enhancer of Zeste and other Polycomb group proteins stay primarily localized at their response elements, which appear to be the least methylated parts of the silenced gene. These results suggest that, contrary to the prevailing view, the Polycomb group proteins and methyltransferase complexes are recruited to the Polycomb response elements independently of histone methylation and then loop over to scan the entire region, methylating all accessible nucleosomes. It is proposed that the Polycomb chromodomain is required for the looping mechanism that spreads methylation over a broad domain, which in turn is required for the stability of the Polycomb group protein complex. Both the spread of methylation from the Polycomb response elements, and the silencing effect can be blocked by the gypsy insulator (Kahn, 2006).

The experiments described in this study show clearly that all three PcG proteins tested, Pc, Psc, and E(z), are preferentially located at the PREs. This specificity is clearest and most sharply delineated in the case of Psc and E(z). In the case of PC, the peaks centered at the PREs are much broader, including secondary peaks, and although the binding detected at other Ubx regions decreases to low values, it never reaches the level seen at control sites such as the white gene that possess no PRE. The second basic conclusion from these experiments is that, in contrast to the localization of PcG proteins, the H3 me3K27 profile forms a broad domain that includes the entire Ubx transcription unit and upstream regulatory region. The third important observation is that, contrary to previously published accounts, the PREs themselves appear to contain the lowest levels of me3K27 of the entire domain. This surprising result will be considered first. The lack of apparent methylation at the PREs does not depend on the antibody used or on the level of cross-linking. Comparable results were obtained with two different anti-me3K27 antibodies and with anti-me3K9. Furthermore, the fact that a similar result was obtained with antibody against total histone H3 or histone H2B suggests that nucleosomes are underrepresented at the bxd PRE core. The result is not because of lack of accessibility to the histone or to the epitope: GAGA factor bound to the PRE appears as easily accessible as GAGA factor bound to the Ubx promoter. Salt extraction of the PcG complexes before cross-linking does not qualitatively change the me3K27 binding profile (Kahn, 2006).

In sum, careful quantitative analysis of ChIP indicates that while PcG proteins are principally localized at the PRE, the histone H3 methylation they produce is distributed over the entire Ubx gene. It is evident from this and from the undermethylation of the PRE core that that K27 methylation does not, by itself, recruit PcG complexes. This does not preclude an important role for methylation in PcG binding and silencing but suggests that the relationship between the two requires a more dynamic model (Kahn, 2006).

PREs have been shown to recruit PcG complexes and to produce new binding foci detectable in polytene chromosomes. It is not surprising therefore to find the three PcG proteins tested are associated with the two Ubx PREs. A much smaller peak in microarray profiles for all three proteins can be discerned in the vicinity of the Ubx 3'-exon but its significance is unknown. More surprising was the striking difference between the distributions of Psc and E(z) and that of Pc. E(z) and Psc belong to two different complexes that do not co-precipitate (except in the very early embryo) but are both recruited to the PRE. Pc and Psc are core components of the PRC1-type of PcG complex yet, while Psc is detected almost exclusively at the PREs, Pc has a much broader distribution peaking at the PREs but tailing over considerable distances along the Ubx gene and regulatory regions. The simplest interpretation of this is that a second type of complex containing Pc but not Psc is recruited by a different mechanism to the rest of the Ubx sequences. Alternatively, the same complex, containing both proteins is involved in both cases but the nature of the chromatin contact is different, such that in one case both proteins are well cross-linked to the chromatin but in the second case only Pc is efficiently cross-linked (Kahn, 2006).

Just as striking is the fact that, although the E(z) complex is responsible for the H3 K27 methylation spread over the entire Ubx gene, the E(z) protein is found localized at the PREs. It is concluded that the E(z) complex methylates the Ubx domain by a hit-and-run type of mechanism. Because the methylation is stable, the E(z) complex needs only visit each nucleosome once on the average every cell cycle. It is noted that E(z)-dependent histone H3 K27 dimethylation is highly abundant and widely distributed in the genome but E(z) complexes are not associated with it. Where then does the E(z) that methylates PcG target genes come from? While more complicated scenarios may be imagined, the simplest one involves the E(z) complex bound at the PRE (Kahn, 2006).

It is supposed that the PcG complexes are recruited to PREs by DNA-binding proteins independently of histone methylation. To methylate the entire Ubx domain, the E(z)/ESC histone MTase complex might then detach from the PRE and slide along the chromatin from one nucleosome to the next to survey the entire domain. However, it more likely that both the Pc and the E(z) complexes assembled at the PRE remain associated with the PRE sequences, where they are detected, but that the whole PRE assembly loops over to scan the entire region, methylating all accessible nucleosomes. Such looping models were originally proposed to be mediated by sites of weak PcG complex formation. In a modern version of this type of model, the looping activity would be mediated by the distinct affinity of the Pc protein for histone H3, which is greatly increased by K27 methylation. These affinities would mediate transient interactions of the complexes bound at the PRE with the surrounding chromatin and allow continuous scanning and methylation of unmethylated or hemimethylated nucleosomes (Kahn, 2006).

In such a model, ChIP experiments would always detect a strong PcG presence at the PRE but PcG interactions with the rest of the repressed gene would be distributed over a region, which is very large in the case of the Ubx gene, smaller in the case of the YGPhsW transposon, hence the signal detected at any one site would be weaker in proportion to the extent of the methylated domain. In addition, the contacts between the PcG complex and the rest of the silenced gene would be much more transient than contacts with the PRE. Together, these considerations would explain why ChIP assay gives such low values for PcG proteins over the rest of the methylated domain (Kahn, 2006).

The looping mechanism proposed for the PRE-bound complex strongly resembles that suggested for the interaction between the Locus Control Region and β-globin genes or for enhancer-promoter interactions. Like these interactions, the silencing of a promoter by the PRE is blocked by insulator elements. In transposon constructs, the insertion of a gypsy Su(Hw) insulator between PRE and promoter blocks the spread of methylation. At present, the mechanism of insulator action is not clear and how the block to methylation is achieved is unknown. It is possible that the insulator element produces topological constraints that prevent the PRE-bound complexes from looping beyond the insulator. This would be consistent with the observation that a significant level of Pc presence becomes detectable over the yellow gene when the insulator block is lifted (Kahn, 2006).

Although the data argue against a principal role of histone methylation in the recruitment of Polycomb proteins to their response elements, it seems to be important for both transcriptional repression and stable association of PcG proteins with chromatin. Loss of catalytic E(z) function eventually results in derepression of HOX genes and dissociation of PcG proteins from polytene chromosomes. It is speculated that once the me3K27 domain is established, modified nucleosomes will pave the way for looping interactions of the PRE-bound PcG proteins with the parts of the silenced gene including promoter or enhancer regions. Silencing might then result from hit-and-run interactions with either or both, possibly even resulting in methylation of the associated factors. Alternatively or in addition, trimethylation of K27 and possibly K9 may directly interfere with the signaling cascade of consecutive histone modifications that guide the multistep process of transcription initiation and elongation. Since histone methylation is thought to persist through cell division its immediate presence at the very beginning of the subsequent interphase might win the time necessary for the full assembly of PcG complexes on the PREs before competing transcription has taken over (Kahn, 2006).

dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing

Transcription regulation involves enzyme-mediated changes in chromatin structure. This study describes a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase Lysine (K)-specific demethylase 2 (dKDM2). In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression (Lagarou, 2008).

This study investigated the molecular mechanisms involved in PcG-mediated gene silencing. The major findings of this work are the following. First, a novel PcG silencing complex was idebtufued tat was named dRAF, harboring core subunits dKDM2, dRING, and PSC. Whereas dRING and PSC are also part of PRC1, the other two PRC1 core subunits, PC and PH, are absent from dRAF. In addition, it was found that significant amounts of PSC and PH are not associated with either PRC1 or dRAF, suggesting they might form part of other assemblages. In short, this work suggests a greater diversity among PcG complexes than previously anticipated. Second, genome-wide expression analysis revealed that dKDM2 and PRC1 share a significant number of target genes. Third, it was found that Pc and dkdm2 interact genetically and cooperate in repression of homeotic genes in vivo. Fourth, dKDM2 counteracts homeotic gene activation by the trxG histone methyltransferases TRX and ASH1. Fifth, a novel trans-histone pathway acting during PcG silencing was uncovered. dKDM2 plays a central role by removal of the active H3K36me2 mark and promoting the establishment of the repressive H2Aub mark by dRING/PSC. Finally, the observation that dKDM2 is required for bulk histone H2A ubiquitylation by dRING/PSC, suggests that dRAF rather than PRC1 is the major histone H2A ubiquitylating complex in cells (Lagarou, 2008).

The term trans-histone pathway was first coined to describe that H2B ubiquitylation is required for H3K4 and H3K79 methylation, whereas the reverse is not the case. Recently, it was found that H2Bub determines the binding of Cps35, a key component of the yeast H3K4 methylase COMPASS complex, providing insight in the molecular mechanism by which two positive marks are coupled. This study describes a different type of trans-histone regulation where the removal of the active H3K36me2 mark is directly linked to repressive monoubiquitylation of H2A. A recent study strongly argued that ASH1 mediates H3K36me2. Significantly, the current genetic and biochemical analysis revealed an in vivo antagonism between dKDM2 and ASH1. Thus, dKDM2 appears to reverse the enzymatic activity of trxG protein ASH1 through H3K36 demethylation, whereas it does not affect H3K4 methylation. The observation that chromatin binding of TRX is ASH1 dependent is likely to be part of the explanation of the strong genetic interaction between dkdm2 and trx. The association of the H3K27me2/3 demethylase UTX with the MLL2/3 H3K4 methylase complexes is an example of coupling removal of a repressive mark to the establishment of an active mark (Lagarou, 2008).

This work revealed that the key H2A E3 ubiquitin ligase dRING is part of two distinct complexes, PRC1 and dRAF. A previous study identified the mammalian BCOR corepressor complex, which harbors RING1, NSPC1, and FBXL10 and other proteins, absent from dRAF. These findings suggest that BCOR and dRAF represent a variety of related but distinct silencing complexes. Reduction of dKDM2 caused a dramatic loss of H2Aub levels, which was comparable with that observed after depletion of dRING or PSC. However, knockdown of PRC1 subunits PC or PH had no effect on H2Aub. These observations suggest that dRAF rather than PRC1 is responsible for the majority of H2A ubiquitylation in cells. This notion was reinforced by in vitro reconstitution experiments, suggesting that dRAF is a more potent H2A ubiquitin ligase than PRC1. An unresolved issue remains the molecular mechanisms that underpin the opposing consequences of either H2A or H2B ubiquitylation. It is intriguing that H2Aub appears to be absent in yeast, present but less prominent than H2Bub in Drosophila, and abundant in mammalian cells. An attractive speculation is that H2Aub becomes more important when genome size increases and noncoding regions and transposons need to be silenced (Lagarou, 2008).

In summary, this study identified the PcG complex dRAF, which employs a novel trans-histone pathway to mediate gene silencing. dKDM2 plays a pivotal role by coupling two distinct enzymatic activities, H3K36me2 demethylation and stimulation of H2A ubiquitylation by dRING/PSC. The results indicate that dRAF is required for the majority of H2Aub in the cell. dKDM2 cooperates with PRC1 but counteracts trxG histone methylase ASH1. These findings uncovered a repressive trans-histone mechanism operating during PcG gene silencing (Lagarou, 2008).

Polycomb proteins remain bound to chromatin and DNA during DNA replication in vitro

The transcriptional status of a gene can be maintained through multiple rounds of cell division during development. This epigenetic effect is believed to reflect heritable changes in chromatin folding and histone modifications or variants at target genes, but little is known about how these chromatin features are inherited through cell division. A particular challenge for maintaining transcription states is DNA replication, which disrupts or dilutes chromatin-associated proteins and histone modifications. PRC1-class Polycomb group protein complexes, consisting of four core PcG subunits, polyhomeotic (Ph), posterior sex combs (PSC), dRING, and Polycomb (Pc), are essential for development and are thought to heritably silence transcription by altering chromatin folding and histone modifications. It is not known whether these complexes and their effects are maintained during DNA replication or subsequently re-established. When PRC1-class Polycomb complex-bound chromatin or DNA is replicated in vitro, Polycomb complexes remain bound to replicated templates. Retention of Polycomb proteins through DNA replication may contribute to maintenance of transcriptional silencing through cell division (Francis, 2009).

The data suggest that PCC is not released into solution during passage of the DNA replication fork. Furthermore, nucleosomes facilitate PCC binding to and retention on templates, but are not essential for either. The finding that PCC can be maintained on either chromatin or naked DNA is interesting in light of the finding that PREs are sites of rapid histone turnover and can be depleted of nucleosomes (Francis, 2009).

One model for the transfer of PCC during DNA replication is that the complex remains in direct contact with DNA during passage of the DNA replication fork. Contacts between PcG proteins and nucleosomes or DNA could be disrupted in front of the replication fork, but replaced by contacts with nucleosomes or DNA behind the replication fork. This mechanism has been proposed for transfers of histone-DNA contacts during replication and transcription in vitro. PCC can likely contact multiple nucleosomes or a long stretch of DNA, which may allow the complex to remain on chromatin when some template contacts are disrupted. A second model is that PCC interacts with the replication machinery, either directly or through intermediary factors. These interactions could retain PCC near DNA during replication, even if direct DNA contacts are disrupted, allowing rapid rebinding of PCC to newly replicated chromatin. Consistent with this idea, several chromatin-modifying proteins can interact with components of the DNA replication machinery (Francis, 2009).

The inhibition of DNA and chromatin replication by PCC in vitro raises the question of how PcG-bound regions are replicated if PRC1-class complexes are indeed continuously bound. If PCC inhibits replication initiation but not elongation, as the results suggest, then PRC1-class complexes would limit replication only if they were bound near replication origins (Francis, 2009).

Intriguingly, targeting of Pc to a replication origin in Drosophila that mediates developmental chorion gene amplification in follicle cells decreased gene amplification (Aggarwal, 2004) and PcG-silenced regions of polytene chromosomes (such as Hox gene clusters) are underreplicated, although this involves additional genes such as Suppressor of DNA Underreplication (Marchetti, 2003; Moshkin, 2001; Francis, 2009 and references therein).

Reduction of PcG protein levels leads to reactivation of their target genes, suggesting that these genes are continuously susceptible to transcriptional activation. It may therefore be important that PRC1-class complexes, which can directly repress transcription, maintain constant association with genes marked for silencing (Francis, 2009).

It was surprising to find that H3K27me3 is not essential for maintaining PRC1-class complexes through DNA replication in vitro. It is possible that retention of parental PRC1-class complexes and recruitment of new complexes are mechanistically distinct because no evidence was found for recruitment of new PCC during replication, and in vivo data suggest that PSC is present on newly replicated chromatin but that additional PSC is recruited after replication. This may be similar to histone proteins in that it is thought that parental histones are transferred randomly to the two daughter strands, followed by deposition of new histones by replication-coupled assembly complexes. In vivo data raise the possibility that recruitment of new PRC1 is not directly coupled to DNA replication; perhaps it involves H3K27me3 (Francis, 2009).

In these experiments, PCC interacts with chromatin through mass action, but in vivo, PRC1-class complexes are specifically targeted to PREs. It is hypothesized that the stable association of PCC with chromatin observed in this study reflects how the complex could behave once it is recruited to a PRE, but it will be important to test this mechanism in a system where PCC is targeted (Francis, 2009).

In conclusion, the ability of parental PCC to be transferred to daughter chromatin may help explain how PcG-mediated repression established by transiently acting factors can be propagated through cell generations. These data also suggest that maintenance of chromatin regulatory proteins through DNA replication might be an important mechanism of epigenetic inheritance (Francis, 2009).

Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge

Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. This study shows that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. Experimental support is provided for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. It is inferred that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function (Grau, 2011).

This study shows that the predicted protein charge of a mouse PcG protein correlates with in vitro activity. This observation was extended by making a computational prediction of PcG activity in a variety of species, and it was demonstrated that activity can be predicted based on charge characteristics. These results support the hypothesis that one key function for PRC1 proteins is the ability to compact nucleosomal arrays and repress chromatin remodeling. The conservation of this basic, charged domain suggests that it may be important to silencing by PRC1 family proteins (Grau, 2011).

Natively unfolded or intrinsically disordered proteins were first described in the late 1980s. These early descriptions were focused on the proteins that are involved in transcriptional activation. Notably, it was observed that the negatively charged amino acids of proteins required for optimal transcriptional activation did not need to be precisely ordered. The critical parameter appeared to be amino acid composition. This study found that canonical transcription repressors, the PcG proteins, also appear to have regions of disorder, yet, in contrast to transcriptional activators, contain high concentrations of basic amino acids. It is tempting to speculate that these oppositely charged disordered regions play a 'yin-yang' role in transcriptional regulation. It is possible that, in addition to the roles in nucleosome interaction, these positively charged transcription repressors could directly interact with and inhibit the negatively charged activation domains of the transcriptional machinery (Grau, 2011).

There are several proposed reasons why proteins would contain regions of disorder. Disordered regions could potentially adopt different conformations that allow interactions with multiple binding partners. This 'hub' function is expected to be beneficial for regulatory proteins; a single protein could potentially regulate many different proteins in a context-specific manner. There is also the 'fly casting' model, where an extended conformation could allow a protein to 'sample' a larger amount of space, forming and breaking low-affinity contacts until conformational change induces tighter binding. This is expected to promote interactions of low affinity and high specificity. One computational predictor of protein disorder -- charge -- was found to also be predictive of PcG functional activity, suggesting that charged disordered regions could possibly play a general role in PcG-mediated repression (Grau, 2011).

What might be the biological role for PcG charged domains in the repression of transcription? They appear to be predictive for both inhibition of remodeling and compaction of chromatin in vitro. This paper proposes a model for how the charged domains of PRC1 function: (1) PRC1 is recruited to target loci and presents the charged domain to linker and/or nucleosomal DNA. (2) The charged domain initially interacts with a nucleosome and creates more interactions with other nucleosomes. (3) Finally, oligomerization occurs through Ph or other protein-protein interactions to promote spreading or formation of higher-order chromatin fibers (Grau, 2011).

The CBOX domain of M33 is not required for in vitro repression activities, yet this motif is conserved and required for the repression of template DNA in cell-based assays. This domain is required for interactions with Ring1A/B and Bmi1, which in turn interact with Ph proteins. Thus, it is imagined that an initially transient nucleosome-nucleosome interaction mediated by charged domains facilitates the further stabilization of a repressed chromatin structure that is mediated by other PRC1 proteins. Studies analyzing the dynamics of Cbx/chromatin interactions in culture cell models observe both transiently and stably associated Cbx proteins (Polycomb in Drosophila), consistent with an initial unstable interaction followed by step(s) that promote stable associations (Grau, 2011).

The precise molecular mechanisms behind PcG protein interactions with chromatin are not understood. The flexible charged domains might interact with linker DNA, nucleosomal DNA, the histones themselves, or a combination of these chromatin components. This study found that two non-PcG proteins with predicted charges similar to M33 do not inhibit remodeling as well as M33. This suggests a mechanism that does not rely solely on the amount of positive charge. It is possible that function involves a specific spacing of the charged residues and/or juxtaposition of the charged surface with other functional domains. For example, the majority of the active proteins that this study characterized also contain a CHD, a known histone-binding domain, opening up the possibility that both DNA and histone contacts are required for optimal PcG-repressive activities (Grau, 2011).

Transcription-independent function of Polycomb group protein PSC in cell cycle control

Polycomb group (PcG) proteins control development and cell proliferation through chromatin-mediated transcriptional repression. A transcription-independent function is described for PcG protein Posterior sex combs (PSC) in regulating the destruction of cyclin B (CYC-B). A substantial portion of PSC was found outside canonical PcG complexes, instead associated with CYC-B and the anaphase-promoting complex (APC). Cell-based experiments and reconstituted reactions have established that PSC and Lemming (LMG, also called APC11) associate and ubiquitylate CYC-B cooperatively, marking it for proteosomal degradation. Thus, PSC appears to mediate both developmental gene silencing and posttranslational control of mitosis. Direct regulation of cell cycle progression might be a crucial part of the PcG system's function in development and cancer (Mohd-Sarip, 2012).

Polycomb group (PcG) proteins are transcriptional repressors that maintain cell-fate decisions and control cell proliferation. They function as part of distinct multiprotein complexes that modulate chromatin structure. The RING domain protein Posterior sex combs (PSC) is a subunit of Polycomb repressive complex 1 (PRC1) and dRING-associated factors (dRAF), which mediate monoubiquitylation of histone H2A. A substantial portion of PSC is part of neither PRC1 nor dRAF, suggesting that PSC might have additional functions. The effects of depleting either Polycomb (PC), Polyhomeotic (PH), PSC, or dRING were compared by treating S2 cells with the appropriate double-stranded RNAs (dsRNAs). PC, PH, PSC, and dRING form the core of PRC1, whereas dRING, PSC, and dKDM2 are the central subunits of dRAF. Knock-down (KD) of PH or PSC decreased cell accumulation, whereas depletion of PC or dRING had no appreciable effects. Fluorescence-activated cell sorter (FACS) analysis indicated that cells lacking PSC primarily accumulated at the G2-M phase of the cell cycle. Loss of other PcG proteins did not give a clear cell cycle arrest. Therefore, PSC might function in cell cycle regulation, independent of PRC1 or dRAF (Mohd-Sarip, 2012).

Consistent with the G2-M arrest caused by loss of PSC, maternal effect mutations of Psc cause mitotic segregation defects in early Drosophila embryos. This is illustrated by the mitotic chromosome bridges, frequently detected in the progeny of Psch27 mutant mothers. Because early embryos have nonconventional checkpoint mechanisms, problems at either S phase or mitosis can lead to segregation defects. In S2 cells, which have a conventional cell cycle, depletion of PSC caused severe mitotic defects. After depletion of PSC, ~68% of mitotic cells displayed an abnormal phenotype, whereas loss of the other PcG proteins did not affect mitosis. The PcG system has been implicated in the regulation of cell cycle genes. Yet, because the integrity of PcG complexes is required for silencing, it was suspected that PSC's role in mitosis extends beyond transcription repression. Indeed, a portion of cellular PSC does not appear to be part of PRC1 nor dRAF (Mohd-Sarip, 2012).

To identify interaction partners, three distinct affinity-purified antibodies were used to isolate PSC from whole-cell extracts of 0- to 12-hour-old Drosophila embryos. Mass spectrometric analysis revealed that, in addition to PRC1 and dRAF subunits, cyclin B (CYC-B), cell division cycle 2 (CDC2, also called cyclin-dependent protein kinase 1), and key subunits of the anaphase-promoting complex (APC) associate with PSC. Although PSC was present in PC, dRING, and PH purifications, CYC-B and the APC were absent. The APC is a multisubunit E3 ubiquitin ligase that is pivotal to cell cycle regulation. CYC-B ubiquitylation by the APC, marking it for destruction by the proteasome, is required for completion of anaphase and cytokinesis. This study confirmed the selective association of PSC with CYC-B and APC by a series of immunoprecipitations (IPs) combined with protein immunoblotting. IPs of PcG proteins showed that only PSC associates with CYC-B; CDC2; and the APC subunits Morula (Mr, also called APC2), CDC23 (APC8), and Lemming (Lmg, also called APC11). Reverse IPs further established the unique association of PSC with CYC-B and the APC. Lmg is a small 85-amino acid protein, comprising mainly a RING domain, that is essential for the ubiquitin ligase activity of the APC. Many RING domain proteins are E3 ubiquitin ligases and frequently function as homo- or heterodimers. For example, PSC and its mammalian homolog BMI1 bind dRING or RING1B, respectively, and stimulate histone H2A ubiquitylation. This study found that the RING domain of PSC was necessary and sufficient to bind LMG, whereas its C-terminal region bound CYC-B. Thus, PSC appears to associate with Lmg and CYC-B directly (Mohd-Sarip, 2012).

To complement these biochemical experiments with a genetic-interaction assay, the GAL4-UAS system was used in Drosophila.The glass multimer reporter (GMR) was used to drive ectopic CYC-B expression (GMR>CYC-B) in the developing eye. Ectopic CYC-B caused a mild rough-eye phenotype, characterized by disorganized ommatidia and loss of bristles. Concomitant expression of dsRNA directed against Psc mRNA [GMR>CYC-B; GMR>PSCRNAi (RNAi, RNA interference)] enhanced the GMR>CYC-B phenotype, consistent with the notion that PSC is a negative regulator of CYC-B. In contrast, expression of dsRNA directed against Pc had no effect on the CYC-B overexpression phenotype. Alone, neither reduction of PSC levels nor PC depletion had an appreciable effect on eye development. Thus, PSC interacts both genetically and biochemically with CYC-B (Mohd-Sarip, 2012).

To test whether PSC regulates abundance of CYC-B in vivo, the Patched (Ptc) driver was used to direct the expression of dsRNA directed against Psc mRNA in a central band across the wing imaginal disc of third instar larvae. Immunostaining of CYC-B (red) and PSC (green) revealed a strong increase in CYC-B, precisely in the area of the disc where PSC was depleted. CYC-B abundance was also reported to increase in cellular clones that lack both Psc and Su(z)2, but not in Pc or dRing mutant clones. The effect of PSC on CYC-B was transcription-independent because expression of cyc-B mRNA was not affected by PSC depletion. Likewise, loss of PSC or LMG in S2 cells caused accumulation of CYC-B, which was even greater when both factors were depleted. However, the abundance of cyc-B mRNA in S2 cells was not affected by depletion of PSC or LMG. Thus, CYC-B accumulation appears to be caused by a transcription-independent mechanism, possibly involving PSC-directed ubiquitylation (Mohd-Sarip, 2012).

To investigate the role of PSC in CYC-B ubiquitylation, CYC-B was immunopurified from cells that were depleted of PSC or LMG and treated with proteasome inhibitors. Immunoblotting revealed that the loss of either PSC or LMG caused decreased levels of polyubiquitylated CYC-B (Ub-CYC-B). Almost no Ub-CYC-B was detectable in cells lacking both PSC and LMG. To test whether failed CYC-B destruction could explain the mitotic defects after the loss of PSC, CYC-B was overexpressed in S2 cells. After ectopic expression of CYC-B, ~70% of mitotic cells displayed a variety of defects. Concomitant overexpression of either PSC or LMG almost completely reversed the CYC-B misexpression phenotype. In contrast, extra PC had no effect. Collectively, these results suggest that PSC-mediated CYC-B ubiquitylation is crucial for normal mitosis (Mohd-Sarip, 2012).

Purified PSC, LMG, and CYC-B were used in a reconstituted ubiquitylation system, which was dependent on E1 and E2 enzymes, to test the ability of PSC to act as a ubiquitin E3 ligase for CYC-B. Approximately equimolar amounts of either PSC or LMG could direct CYC-B ubiquitylation. But together, PSC and LMG generated higher levels of Ub-CYC-B. Determination of the CYC-B ubiquitylation rate revealed a more-than-additive effect of combining PSC and LMG, indicating that they function cooperatively. A substitution mutation replacing a signature cysteine residue of the RING consensus with an alanine [PSC-C287A (C287A: Cys287→Ala287)] abrogated PSC's ability to ubiquitylate CYC-B. PSC-C287A blocked ubiquitylation of CYC-B by LMG, suggesting that it acts as a dominant negative. Indeed, the C287A mutation did not affect PSC binding to LMG or CYC-B. In contrast to ectopic PSC, expression of PSC-C287A caused severe mitotic defects in S2 cells. This mitotic phenotype was relieved by concomitant overexpression of LMG, suggesting that extra LMG squelches the dominant-negative PSC. Whereas ectopic expression of either PSC or LMG in S2 cells did not affect mitosis, overexpression of both PSC and LMG caused mitotic defects. These results suggest that PSC and LMG cooperate in the ubiquitylation of CYC-B, marking it for destruction by the proteasome (Mohd-Sarip, 2012).

Regulated protein destruction is fundamental to cell cycle progression. The work reported in this study shows that, in addition to transcriptional repression, PSC cooperates with LMG in the APC to direct CYC-B degradation. During mitosis, PSC (and its mammalian homologs) and key PRC1 subunits PH and PC dissociate from the chromatin, making a transcriptional function at that time unlikely. Like PSC, other chromatin regulators may also target proteins that are neither involved in chromatin dynamics nor transcription (Mohd-Sarip, 2012).

Posterior sexcombs and Suppressor two of zeste: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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