The structure of a chromatin binding (chromo) domain of the chromatin modifier protein 1 (MoMOD1) was determined using nuclear magnetic resonance (NMR) spectroscopy. MoMOD1 is a murine HP1-like heterochromatin-associated protein. The protein consists of an N-terminal three-stranded anti-parallel beta-sheet that folds against a C-terminal alpha-helix. The C-terminal "shadow" domain is likely to fold into the same structure as the N-terminal chromo domain. The structure reveals an unexpected homology to two archaebacterial DNA binding proteins, which are also involved in chromatin structure. Structural comparisons suggest that chromo domains, of which more than 40 are now known, act as protein interaction motifs, and that the MoMOD1 protein acts as an adaptor mediating interactions between different proteins (Ball, 1997).

Three clones for chicken chromobox proteins were obtained from liver and ovary cDNA libraries. pCHCB1 and pCHCB2 encode polypeptides showing 96% and 95% identity with mouse M31 and M32, respectively, which are homologs of Drosophila heterochromatin protein 1 (HP1); pCHCB3 encodes a polypeptide whose sequences of chromobox and C-terminal region show high-level similarities with those of mouse M33, Drosophila polycomb (Pc) protein, and Xenopus Pc homolog. When these cDNAs are expressed in female chicken embryonic fibroblasts (CEFs) as GFP-fused or HA-tagged proteins, all three proteins are found to be localized in nuclei. Among them, CHCB1 associates with brightly stained spots with 4', 6-diamidino-2-phenylindole (DAPI), suggesting CHCB1 accumulation on heterochromatins. One of these spots was identified as W-heterochromatin, known to consist of XhoI and EcoRI family repetitive sequences, constituting the majority of sequences in the chicken W chromosome. When CHCB1 lacking the N-terminal basic/acidic region or a part of the chromobox region is overexpressed in CEFs, W-heterochromatin becames partially or extensively decondensed in the majority of nuclei. Overexpression of CHCB3 lacking a part of the chromobox does not cause decondensation of W-heterochromatin. Specific antisera raised against a part of CHCB1 or CHCB2, produced in Escherichia coli, detects protein species having apparent molecular masses of 25 kDa or 22 plus 23 kDa, respectively, in the subnuclear fraction containing the majority of chromatin from female chicken MSB-1 cells (Yamaguchi, 1998).

The murine gene CHD1 (MmCHD1; see Drosophila Chd1) was previously isolated in a search for proteins that would bind a DNA promoter element. The presence of chromo (chromatin organization modifier) domains (such as those found in Drosophila Brahma and Imitation SWI) and an SNF2-related helicase/ATPase domain (present also in Drosophila HP1 and Polycomb) led to speculation that this gene might regulate chromatin structure or gene transcription. Three novel human genes are related to MmCHD1. Examination of sequence databases produce several more related genes, most of which are not known to be similar to MmCHD1, yielding a total of 12 highly conserved CHD genes from organisms as diverse as yeast and mammals. A Drosophila homolog, DmCHD1, contains all the domains found in the human sequence; another homolog, DmCHD3, lacks the DNA-binding domain sequence. MmCHD1 preferentially binds via minor groove interactions to DNA that contains (A+T)-rich tracts including those in a matrix attachment region. The major region of sequence variation in CHD proteins is in the C-terminal part of the protein, a region with DNA-binding activity in MmCHD1. Targeted deletion of ScCHD1, the sole Saccharomyces cerevesiae CHD gene, was performed with deletion strains being less sensitive than wild type to the cytotoxic effect of 6-azauracil. This finding suggests that enhanced transcriptional arrest at RNA polymerase II pause sites (due to 6-azauracil-induced nucleotide pool depletion) is reduced in the deletion strain and that ScCHD1 inhibits transcription. This observation, along with the known roles of other proteins with chromo or SNF2-related helicase/ATPase domains, suggests that alteration of gene expression by CHD genes might occur by modifications of chromatin structure, with altered access of the transcriptional apparatus to its chromosomal DNA template (Woodage, 1997).

A mammalian chromatin-associated protein, CHD1 (chromo-ATPase/helicase-DNA-binding domain), might have an important role in the modification of chromatin structure. The Drosophila melanogaster CHD1 homolog (dCHD1) encodes an 1883-aa open reading frame that is 50% identical and 68% similar to the mouse CHD1 sequence, including conservation of the three signature domains for which the protein was named. dCHD1 is related to both Drosophila Brahma and Imitation SWI as well as to Polycomb and HP1, based on the presence of both a helicase domain (found in Brahma and ISWI) and a chromo domain (found in Polycomb and HP1). When the chromo and ATPase/helicase domain sequences in various CHD1 homologs are compared with the corresponding sequences in other proteins, certain distinctive features of the CHD1 chromo and ATPase/helicase domains are revealed. This suggests that CHD constitutes a distinct subgroup that diverged early in evolution from HP1 and PC subgroups as well as from ISWI type proteins. The dCHD1 gene maps to position 23C-24A on chromosome 2L. Western blot analyses with antibodies raised against a dCHD1 fusion protein specifically recognize an approximately 210-kDa protein in nuclear extracts from Drosophila embryos and cultured cells. Most interestingly, these antibodies reveal that dCHD1 localizes to sites of extended chromatin (interbands) and regions associated with high transcriptional activity (puffs) on polytene chromosomes from salivary glands of third instar larvae. These observations strongly support the idea that CHD1 functions to alter chromatin structure in a way that facilitates gene expression (Stokes, 1996).

M33, a mouse homolog of the Drosophila Polycomb protein, can substitute for Polycomb in transgenic flies. Polycomb protein is thought to join with other Polycomb-group proteins to build a complex that silences selector genes. Since members of this group of proteins have their homologs in mice, these results suggest that the molecular mechanism of cell determination is widely conserved (Muller, 1995).

To assess its function during development, M33, the murine counterpart of Drosophila Polycomb was targeted in mice by homologous recombination in embryonic stem (ES) cells. Homozygous M33 mutant mice show greatly retarded growth, homeotic transformations of the axial skeleton, sternal and limb malformations and a failure of several cell types to expand in vitro including lymphocytes and fibroblasts. Mutant mice show a posteriorisation of the thoracic vertebra T7 into T8, resulting in the presence of six vertebrosternal ribs instead of seven in nonmutated mice. In addition, 15% of the mutant mice show a transformation of the lumbar vetebra L6 into a first sacral vertebra. Hoxa-3, a homolog of Drosophila proboscipedia, is detected over the basioccipital bone anlage in mutant mice but not in wild-type mice. This anterior shift in the Hoxa-3 boundary is made wider by the fusion of the basioccipital and the first prevertebra. Other Hox genes show a similar anterior transformation. M33 null mutant mice show an aggravation of the skeletal malformations when treated with retinoic acid at embryonic day 7.5, leading to the hypothesis that during development, the M33 gene might play a role in defining access to retinoic acid response elements localised in the regulatory regions of several Hox genes, For example retinoic acid response genes have been found in Hoxa-1, Hoxb-1 (both labial homologs) and Hoxd-4 (a Deformed homolog) (Coré, 1997).

In Drosophila and mouse, Polycomb group genes are involved in the maintenance of homeotic gene expression patterns throughout development. Skeletal phenotypes are found in mouse compound mutants for two Polycomb group genes bmi1 and M33. Mice deficient for both bmi1 and M33 present stronger homeotic transformations of the axial skeleton as compared to each single Polycomb group mutant, indicating strong dosage interactions between those two genes. These skeletal transformations are accompanied with an enhanced shift of the anterior limit of expression of several Hox genes in the somitic mesoderm. These results demonstrate that in mice the Polycomb group genes act in synergy to control the nested expression pattern of some Hox genes in somitic mesodermal tissues during development (Bel, 1998).

When Pc-G mutant mice are compared, loss of each Pc-G gene shows a unique subset of affected Hox genes. For example, in M33 mutant mice, only anterior shifts for Hoxa3 can be detected and, in some cases, for Hoxc8. mel18 -/-, bmi1 -/- and rae28 -/- mice present a more extensive overlap in affected Hox genes, encompassing one prevertebrae anterior shifts of Hoxa5 and Hoxc8. However, Hoxc6 and Hoxc5 are uniquely affected in bmi1 -/- mice, while Hoxa7 and Hoxd4 are only affected in mel18 -/- mice and Hoxb5 is unaffected in all those mutant mice. In M33 bmi1 double mutant mice, the anterior limit of expression of at least two Hox genes, Hoxc9 and Hoxc8, is significantly more severely shifted as compared to both single mutants, demonstrating the additive effect of Pc-G products in maintaining the boundaries of selected Hox genes. One striking observation is that deleting three gene doses of these Pc-G genes does not increase the derepressive effect of Hoxc8 or Hoxc9 expression; full deficiency of M33 and bmi1 is required to induce extensive ectopic expression of these two genes in mesodermal tissues. Nevertheless, according to the differential expression of Hoxc8 and Hoxc9 in both single mutants, it seems that Hoxc8 is more sensitive to M33 regulation since a one-segment anterior shift is observed in M33 -/- bmi1 +/- (prevertebrae 10), whereas that shift is not visible in bmi1 -/- M33 +/- (prevertebrae 11); reciprocally, Hoxc9 is more sensitive to bmi1 regulation. In contrast, some other Hox genes like Hoxb1, Hoxd4 and Hoxd11 are not affected, either in single or in double mutant mice. This suggests that several murine multimeric Pc-G protein complexes of different composition might exist that differ in their affinities for specific Hox genes. Alternatively, since in mammals Pc-G genes exist as highly related gene pairs (such as mel18/bmi1, Enx1/Enx2, M33/MPc2, hPc1/hPc2 and Hph1/rae28/Hph2), a potential redundancy likely exists. This suggests that the homologous gene complements part of the function and thus maintains the boundaries of expression for a subset of Hox genes. Analysis of double mutant mice for a related pair such as mel18 and bmi1 should clarify the degree of redundancy. Differential effects of Pc-G mutations on Hox gene expression in different tissues like the somitic mesoderm and the neural tube, also suggest that different Pc-G complexes may regulate a specific subset of Hox genes in a tissue-dependent manner (Bel, 1998).

The Polycomb group genes are required for the correct expression of the homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. In mouse, inactivation studies of several Polycomb group genes indicate that they are also involved in Hox gene regulation. M33 mutants have been used to study the function of M33, the mouse homolog of the Drosophila Polycomb gene. In the absence of M33, the window of Hoxd4 retinoic acid (RA) responsiveness is opened earlier and Hoxd11 gene expression is activated earlier in development This indicates that M33 antagonizes the RA pathway and has a function in the establishment of the early temporal sequence of activation of Hox genes. Despite the early activation, A-P boundaries are correct in later stages, indicating a separate control mechanism for early aspects of Hox regulation. This raises a number of interesting issues with respect to the roles of both Pc-G proteins and Hox regulatory mechanisms. It is proposed that a function of the M33 protein is to control the accessibility of retinoic acid response elements in the vicinity of Hox genes regulatory regions by direct or indirect mechanisms or both. This could provide a means for preventing ectopic transactivation early in development and be part of the molecular basis for temporal colinearity of Hox gene expression (Bel-Vialar, 2000).

A model proposes that each gene within a Hox cluster would show a graded sensitivity to RA through a repression imposed by Pc-G genes: with 3' genes being accessible earlier than the adjacent 5' gene. This differential repression contributed by M33 can be hypothesized since, in two different backgrounds in M33-/- mice, the skeletal transformations are found more frequently in the anterior (due to the deregulation of more 3' Hox genes) than in the posterior regions. This would imply that the Pc-G repression is more dependent on the M33 product at the 3' end of the cluster than at the 5' end, or/and that the nature of the complex may vary from the 3' end to the 5' end of a Hox cluster. This repression could again be mediated by direct or indirect mechanisms (Bel-Vialar, 2000).

Bmi1-interacting proteins are constituents of a multimeric mammalian Polycomb complex. Bmi, and the closely related Mel18 gene share sequence homology to Drosophila PSC and SU(Z)2. M33 is a murine homolog of Drosophila Polycomb protein. A Bmi binding protein, Mph1 has similarity to Drosophila Polyhomeotic. Although the overall identity of the Mph1 protein to Polyhomeotic is only 31%, Mph1 and PH share significant similarity at the C-terminal region. A C-terminal homology domain shares weak similarity to members of the Ets family of transcription factors. This domain is most similar to the Drosophila protein Lethal(3)malignant brain tumor, Bmi1 and Mph1, as well as the Mel18 and M33 proteins are constituents of a multimeric protein complex in mouse embryos and human cells. A central domain of Bmi1 interacts with the carboxyl terminus of Mph1, a conserved alpha-helical domain in the Mph1 protein is required for its homodimerization. Transgenic mice overexpressing various mutant Bmi1 proteins demonstrate that the central domain of Bmi1 is required for the induction of anterior transformations of the axial skeleton. Bmi1, M33 and Mph1 show an overlapping speckled distribution in interphase nuclei. Localization of Bmi1 to subnuclear domains depends on an intact RING finger (Alkema, 1997a).

The proteins of the Polycomb group (PcG) are required for maintaining regulator genes, such as the homeotic selectors, stably and heritably repressed in appropriate developmental domains. It has been suggested that PcG proteins silence genes by creating higher-order chromatin structures at their chromosomal targets, thus preventing the interaction of components of the transcriptional machinery with their cis-regulatory elements. An unresolved issue is how higher order-structures are anchored at the chromatin base, the nucleosomal fiber. A direct biochemical interaction of a PcG protein -- the Polycomb (PC) protein -- with nucleosomal core particles has been demonstrated in vitro. The main nucleosome-binding domain coincides with a region in the C-terminal part of PC previously identified as the repression domain. These results suggest that PC, by binding to the core particle, recruits other PcG proteins to chromatin. This interaction could provide a key step in the establishment or regulation of higher-order chromatin structures (Breiling, 1999).

The evolutionarily conserved polycomb and trithorax-group genes are required to maintain stable expression patterns of homeotic genes and other target genes throughout development. A novel mouse polycomb homolog, MPc2, was cloned and characterized in addition to the previously described M33 polycomb gene. Co-immunoprecipitations and subnuclear co-localization studies show that MPc2 interacts with the mouse polycomb-group oncoprotein Bmi1 and is a new member of the mouse polycomb multiprotein complex. Either Gal4DB-MPc2 or Gal4DB-M33 fusion proteins mediate a five- to ten-fold repression of stably integrated reporter constructs carrying GAL4 binding sites, demonstrating that these proteins are transcriptional repressors. The MPc2 gene is localized on chromosome 11, in close proximity to the classical mouse mutations tail short (Ts) and rabo torcido (Rbt). Ts and Rbt hemizygous mice display anemia and transformations of the axial skeleton reminiscent of phenotypes observed in mice with mutated polycomb or trithorax-group genes, suggesting that MPc2 is a candidate gene for Ts and Rbt (Alkema, 1997b).

Xenopus homologs of the Drosophila Polycomb gene and the vertebrate bmi-1 gene have been isolated. bmi-1 is a proto-oncogene which has sequence homology with the Polycomb group gene Posterior Sex Combs. XPolycomb and Xbmi-1 genes are expressed in overlapping patterns in the central nervous system of Xenopus embryos. However, XPolycomb is also expressed in the somites, whereas Xbmi-1 is not. The XPolycomb and Xbmi-1 proteins are able to interact with each other via conserved sequence motifs. These data suggest that vertebrate Polycomb group proteins form multimeric complexes (Reijnen, 1995).

In Drosophila, the Polycomb-group constitutes a set of structurally diverse proteins that act together to silence target genes. Many mammalian Polycomb-group proteins have also been identified and show functional similarities with their invertebrate counterparts. To begin to analyze the function of Polycomb-group proteins in Xenopus development, a Xenopus homolog of Drosophila Polycomblike, XPcl1, has been cloned. XPcl1 mRNA is present both maternally and zygotically, with prominent zygotic expression in the anterior central nervous system. Misexpression of Pcl1 by RNA injection into embryos produces defects in the anterior central nervous system. The forebrain and midbrain contain excess neural tissue at the expense of the ventricle and include greatly thickened floor and roof plates. The eye fields are present but Rx2A, an eye-specific marker, is completely repressed. Overexpression of Pcl1 in Xenopus embryos alters two hindbrain markers, repressing En-2 and shifting it and Krox-20 in a posterior direction. Similar neural phenotypes and effects on the En-2 expression pattern were produced by overexpression of three other structurally unrelated Polycomb-group proteins: M33 (homolog of Drosophila Polycomb), XBmi-1 (homolog of Drosophila Polycomb), and mPh2 (homolog of Drosophila Polyhomeotic). These observations indicate an important role for the Polycomb-group in regulating gene expression in the developing anterior central nervous system (Yoshitake, 1999).

The bmi-1 gene was first isolated as an oncogene that cooperates with c-myc in the generation of mouse lymphomas. Subsequently Bmi-1 was identified as a transcriptional repressor belonging to the mouse Polycomb group. The Polycomb group comprises an important, conserved set of proteins that are required to maintain stable repression of specific target genes, such as homeobox-cluster genes, during development. In mice, the absence of bmi-1 expression results in neurological defects and severe proliferative defects in lymphoid cells, whereas bmi-1 overexpression induces lymphomas. bmi-1-deficient primary mouse embryonic fibroblasts are impaired in their progression into the S phase of the cell cycle and they undergo premature senescence. In these fibroblasts and in bmi-1-deficient lymphocytes, the expression of the tumor suppressors p16 and p19Arf, which are encoded by ink4a, is raised markedly. Conversely, overexpression of bmi-1 allows fibroblast immortalization, downregulates expression of p16 and p19Arf and, in combination with H-ras, leads to neoplastic transformation. Removal of ink4a dramatically reduces the lymphoid and neurological defects seen in bmi-1-deficient mice, indicating that ink4a is a critical in vivo target for Bmi-1. These results connect transcriptional repression by Polycomb-group proteins with cell-cycle control and senescence (Jacobs, 1999).

The murine Polycomb homolog M33 is implicated in mesoderm patterning in the mouse; it acts as a transcriptional repressor in transiently transfected cells. Two murine proteins, Ring1A and Ring1B, have been identified that interact directly with the repressor domain of M33. Ring1A and Ring1B display blocks of similarity throughout their sequences, including an N-terminal RING finger domain. However, the interaction with M33 occurs through a region at the C-terminus. Ring1A represses transcription through sequences not involved in M33 binding. Ring1A protein co-localizes in nuclear domains with M33 and other Pc-G homologs, such as Bmi1. The expression of Ring1A at early stages of development is restricted to the neural tube, whereas M33 is expressed ubiquitously. Within the neural tube, Ring1A RNA is located at the rhombomere boundaries of the hindbrain. Taken together, these data suggest that Ring1A may contribute to a tissue-specific function of Pc-G-protein complexes during mammalian development (Schoorlemmer, 1997).

Polycomb (Pc) is part of a Pc group (PcG) protein complex that is involved in repression of gene activity during Drosophila and vertebrate development. To identify proteins that interact with vertebrate Pc homologs, two-hybrid screens were performed with Xenopus Pc (XPc) and human Pc2 (HPC2). C-terminal binding protein (CtBP) interacts with XPc and HPC2; CtBP and HPC2 coimmunoprecipitate, and CtBP and HPC2 partially colocalize in large PcG domains in interphase nuclei. CtBP is a protein with unknown function that binds to a conserved 6-amino-acid motif in the C terminus of the adenovirus E1A protein. Also, the Drosophila CtBP homolog interacts, through this conserved amino acid motif, with several segmentation proteins that act as repressors. Similarly, it is found that CtBP binds with HPC2 and XPc through the conserved 6-amino-acid motif. Importantly, CtBP does not interact with another vertebrate Pc homolog, M33, which lacks this amino acid motif, indicating specificity among vertebrate Pc homologs. Finally, CtBP is shown to be a transcriptional repressor. The results are discussed in terms of a model that brings together PcG-mediated repression and repression systems that require corepressors such as CtBP (Sewalt, 1999).

Transcriptional repression mediated by Polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators

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 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).

Polycomb group (PcG) proteins form large multimeric complexes (PcG bodies) that are involved in the stable repression of gene expression. The human PcG protein, Pc2, has been shown to recruit the transcriptional corepressor, CtBP, to PcG bodies. CtBP is sumoylated at a single lysine. In vitro, CtBP sumoylation minimally requires the SUMO E1 and E2 (Ubc9) and SUMO-1 (see Drosophila SUMO). However, Pc2 dramatically enhances CtBP sumoylation. In vivo, this is likely due to the ability of Pc2 to recruit both CtBP and Ubc9 to PcG bodies, thereby bringing together substrate and E2, and stimulating the transfer of SUMO to CtBP. These results demonstrate that Pc2 is a SUMO E3, and suggest that PcG bodies may be sumoylation centers (Kagey, 2003).

Asymmetric expression of sonic hedgehog (Shh) in the left side of Hensen's node, a crucial step for specifying the left-right (LR) axis in the chick embryo, is established by the repression of Shh expression in the right side of the node. The transcriptional regulator that mediates this repression has not been identified. A novel chick Polycomblike 2 gene, chick Pcl2, has been isolated and characterized that encodes a transcription repressor and displays an asymmetric expression, downstream from Activin-ßB and Bmp4, in the right side of Hensen's node in the developing embryo. In vitro mapping studies define the transcription repression activity to the PHD finger domain of the chick Pcl2 protein. Repression of chick Pcl2 expression in the early embryo results in randomized heart looping direction, which is accompanied by the ectopic expression of Shh in the right side of the node and Shh downstream genes in the right lateral plate mesoderm (LPM), while overexpression of chick Pcl2 represses Shh expression in the node. The repression of Shh by chick Pcl2 is also supported by studies in which chick Pcl2 was overexpressed in the developing chick limb bud and feather bud. Similarly, transgenic overexpression of chick Pcl2 in the developing mouse limb inhibits Shh expression in the ZPA. In vitro pull-down assays demonstrate a direct interaction of the chick Pcl2 PHD finger with EZH2, a component of the ESC/E(Z) repressive complex. Taken together with the fact that chick Pcl2 directly represses Shh promoter activity in vitro, these results demonstrate a crucial role for chick Pcl2 in regulating LR axis patterning in the chick by silencing Shh in the right side of the node (Wang, 2004).

Polycomb interacts with other proteins to form a complex

A novel human Pc homolog, hPc2, is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, a mutant protein, delta hPc2, was generated, lacking an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the delta hPc2 mutant protein that lacks the C-terminal domain is unable to repress gene activity. Expression of the delta hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in delta hPc2-transformed cells, the expression of the c-myc proto-oncogene (See Drosophila Myc) is strongly enhanced; serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. These data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation (Satijn, 1997).

In a two-hybrid screen with a vertebrate Polycomb homolog as a target (Xenopus Pc), the human RING1 (Drosophila homolog Sex combs extra/Ring) protein was identified as interacting with Pc. RING1 is a protein that contains the RING finger motif, a specific zinc-binding domain, which is found in many regulatory proteins. So far, the function of the RING1 protein has remained enigmatic. RING1 coimmunoprecipitates with a human Pc homolog, the vertebrate PcG protein BMI1, and HPH1, a human homolog of the PcG protein Polyhomeotic (Ph). The human polycomb homolog, (hPc2) shows an overall identity of 70% with XPc but a mere 24% identity with M33, a murine Pc homolog closely related to hPc1. Interaction between RING1 and XPc does not involve the RING finger motif. RING1 colocalizes with the vertebrate PcG proteins in nuclear domains of SW480 human colorectal adenocarcinoma and Saos-2 human osteosarcoma cells. RING1, like Pc, is able to repress gene activity when targeted to a reporter gene. These findings indicate that RING1 is associated with the human PcG protein complex and that RING1, like PcG proteins, can act as a transcriptional repressor. It is possible that RING1 is a vertebrate homolog of the product of a Drosophila PcG gene that has not yet been characterized. No Drosophila RING1 homolog has yet been described (Satijn, 1997).

Polycomb-group (PcG) proteins form large multimeric protein complexes that are involved in maintaining the transcriptionally repressive state of genes. RING1 interacts with vertebrate Polycomb (Pc) homologs and is associated with or is part of a human PcG complex. However, very little is known about the role of RING1 as a component of the PcG complex. A detailed characterization of RING1 protein-protein interactions has been undertaken. By using directed two-hybrid and in vitro protein-protein analyses, it has been demonstrated that RING1, in addition to interacting with the human Pc homolog HPC2, can also interact with itself and with the vertebrate PcG protein BMI1. Distinct domains in the RING1 protein are involved in the self-association and in the interaction with BMI1. Further, the BMI1 protein can also interact with itself. To better understand the role of RING1 in regulating gene expression, the protein was overexpressed in mammalian cells and differences in gene expression levels were analyzed. This analysis shows that overexpression of RING1 strongly represses En-2, a mammalian homolog of the well-characterized Drosophila PcG target gene engrailed. Furthermore, RING1 overexpression results in enhanced expression of the proto-oncogenes c-jun and c-fos. The changes in expression levels of these proto-oncogenes are accompanied by cellular transformation, as judged by anchorage-independent growth and the induction of tumors in athymic mice. These data demonstrate that RING1 interacts with multiple human PcG proteins, indicating an important role for RING1 in the PcG complex. Further, deregulation of RING1 expression leads to oncogenic transformation by deregulation of the expression levels of certain oncogenes (Satijn, 1999).

The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. A detailed characterization of the human PcG complex is described in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins (RING1, BMI1, and hPc2) it has been demonstrated in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that is termed the PcG body. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered in defined areas within the nucleus. In three different human cell lines the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG-heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions (Saurin, 1998).

Polycomb group (PcG) proteins were first described in Drosophila as factors responsible for maintaining the transcriptionally repressed state of Hox/homeotic genes in a stable and heritable manner throughout development. A growing number of vertebrate genes related to the Drosophila PcG proteins have been identified, including two Polycomb orthologues, Pc2 and M33. PcG proteins form multiprotein complexes, termed PcG bodies, that are thought to repress transcription by altering chromatin structure. HPC3 (human Polycomb 3), a novel PcG protein, was isolated in a yeast two-hybrid screen using human RING1 as bait. HPC3 shows strong sequence similarity to Drosophila Pc and also to vertebrate Pc2 and M33, particularly within the chromodomain and C-box. M33 and human Pc2 (HPC2) can interact with RING1, and HPC3 also binds to RING1. This interaction is dependent upon the HPC3 C-box but, only partially on the RING finger of RING1. In contrast to HPC2, HPC3 interactions with RING1 are only observed in vivo with covalently modified forms of RING1. HPC3 also colocalizes with other PcG proteins in human PcG bodies. Consistent with its role as a PcG member, HPC3 is able to act as a long range transcriptional silencer when targeted to a reporter gene by a heterologous DNA-binding domain. Taken together, these data suggest that HPC3 is part of a large multiprotein complex that also contains other PcG proteins and is involved in repression of transcriptional activity (Bardos, 2000).

Association between Rb and PcG proteins forms a repressor complex that blocks entry of cells into mitosis. Also, evidence is provided that Rb colocalizes with nuclear PcG complexes and is important for association of PcG complexes with nuclear targets. The Rb-PcG complex may provide a means to link cell cycle arrest to differentiation events leading to embryonic pattern formation (Dahiya, 2001).

Evidence is presented that a polycomb group (PcG) protein, HPC2, in association with its RING finger protein binding partner, Ring1, serves as an HDAC-independent corepressor for Rb, which specifically represses expression of cyclin A and cdc2 and arrests cells in G2. PcG proteins classically repress Hox genes, and this repression coupled with transcriptional activation by trithorax proteins imposes the patterns of Hox expression required for proper embryonic development. However, in addition to their role in repression of Hox genes, PcG proteins are critical regulators of the cell cycle. Mice lacking one of the PcG family members, Bmi-1 (BMI-1 in humans), have severe defects in lymphoid cell proliferation. And, overexpression of Bmi-1 as a result of viral insertion stimulates cell proliferation and is oncogenic. The molecular basis for this has recently been uncovered. Bmi-1 represses expression of the INK4a/ARF locus encoding the cdk4/6 inhibitor p16 and the ARF protein. Also, crossing the Bmi-1-/- mice into an INK4a/ARF-/- background reverses much of the proliferative defect in the Bmi-1-/- mice. p16 is a central component of the Rb pathway in human cells, and in the absence of Bmi-1, p16 accumulates, blocking cyclin D/cdk4 activity and leading to accumulation of hypophosphorylated (active) Rb and growth arrest (Dahiya, 2001).

As with Bmi-1, another PcG family member, M33 (CBX2/HPC1 in humans), is required for normal proliferation of fibroblasts and lymphocytes. However, it is not yet clear how M33 is involved in regulation of cell proliferation. In contrast to Bmi-1 and M33, other PcG family members, such as eed (EED in humans and Esc in Drosophila) and HPC2 (Mpc2 in mice), are negative regulators of the cell cycle. Expression of eed blocks cell proliferation, and expression of a dominant-negative hpc2 leads to transformed morphology and growth of cells in suspension. Eed+/- mice have a myelo- and lympho-proliferative syndrome associated with lymphoid tumor formation. A cross of the eed+/- mice into a Bmi-1-/- background largely reverses the lymphoproliferative defect in the Bmi-1-/- mice, indicating that the family members have opposing activities on cell proliferation and leading the authors to conclude that Bmi-1 is epistatic to eed (Dahiya, 2001 and references therein).

Both HPC2 and Bmi-1 interact with the RING finger protein Ring1, and the Ring1 binding domain is required for Bmi-1 to repress the INK4a/ARF locus and for HPC2 to arrest cells. Ring1 associates with Rb in vivo, and Ring1 coimmunoprecipitates in a complex with E2F in an Rb-dependent fashion. These results provide evidence that Ring1 is present in the E2F-Rb-CtBP-HPC2 complex. When Ring1 is expressed in HPC2(-) cells, there is little detectable effect on the cell cycle profile; however, when a subthreshold level (a level where little or no increase in cells in S or G2/M is evident) of HPC2 (or Rb and HPC2) is expressed, Ring1 synergizes with HPC2 to trigger accumulation of cells in G2 (Dahiya, 2001).

Ring1 binds to the C-terminal end of HPC2. Therefore, an HPC2 mutant was used with the Ring1 binding site deleted (deletion of the C-terminal 30 amino acids). CtBP binds to the PIDLRS sequence N-terminal to this deletion, such that this C-terminal deletion of HPC2 does not affect its binding to CtBP, nor does it affect the ability of HPC2 to interact with Rb in coimmunoprecipitation assays. Therefore, this C-terminal truncation mutant of HPC2 competes with wild-type HPC2 for binding to CtBP in the E2F-Rb-CtBP-HPC2 complex, but it should act as a dominant negative because it is unable to recruit Ring1, which is essential for PcG function. Indeed, expression of this HPC2 mutant has been shown to produce a transformed morphology in cells, and to allow growth in suspension; these phenotypes were reversed by coexpression of wild-type HPC2. Rb- and HPC2-dependent repression is blocked by this DN-HPC2 in transfection assays (Dahiya, 2001).

PcG proteins such as Bmi and HPC2 are present in a single large complex. It is then unclear how Bmi and HPC2 might target distinct sets of genes and have opposing effects on the cell cycle if they are in a common complex. And further, it is unclear why there are substantial differences in homeotic transformations seen in mice lacking different PcG proteins. It is suggested that the relative concentration of a given PcG protein in the complex may direct the targeting of the complex. Such a model may allow for an 'all or none' targeting effect. For example, an excess of Bmi would lead to the complex being directed toward the p16/ARF locus and away from cyclin A and cdc2 genes. And vice versa, excess HPC2 would target the complex toward cyclin A and cdc2 genes and away from the INK4a/ARF locus (Dahiya, 2001).

Mammalian Polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1

Polycomb group (PcG) proteins are responsible for the stable repression of homeotic (Hox) genes by forming multimeric protein complexes. Physical interaction is shown between components of the U2 small nuclear ribonucleoprotein particle (U2 snRNP), including Sf3b1 and PcG proteins Zfp144 and Rnf2. Sf3b1 heterozygous mice exhibit skeletal transformations concomitant with ectopic Hox expressions. These alterations are enhanced by Zfp144 mutation but repressed by Mll mutation (a trithorax homolog). Importantly, the levels of Sf3b1 in PcG complexes are decreased in Sf3b1-heterozygous embryos. These findings suggest that Sf3b1-PcG protein interaction is essential for true PcG-mediated repression of Hox genes (Isono, 2005).

These results show a significant and novel mechanistic link between Sf3b1 (together probably with other U2 snRNP components) and PcG repressive complexes on Hox loci. This idea is strongly supported by the observation that heterozygous mutant mice for Sf3b2, another U2 snRNP component, exhibit skeletal abnormality similar to Sf3b1 phenotypes. However, although with respect to the PcG-mediated repression in the transcriptional-competent regions, these findings are in general accord with previous reports, nevertheless they indicate the presence a different gene silencing mechanism. Evidence that the level of Sf3b1 in PcG complexes affects the expressional boundary of Hox genes implies that Sf3b1 supports the activity of PcG complexes. The simplest explanation is that Sf3b1/U2 snRNP might be a PcG protein and could form repressive PcG complexes together with other PcG proteins. A more interesting hypothesis is that this interaction constitutes part of a mechanism that is designed to maintain the amount of Hox transcripts required to confer the appropriate positional identities. Regulation of Hox expressions in the vicinity of their boundaries is thought to be loose, because even wild type occasionally exhibits homeotic transformations. RNAs, mistranscribed beyond loose repression, may be tethered by Sf3b1/U2 snRNP bound to PcG complex, leading to the arrest of splicing and a normal Hox boundary as a consequence. However, in Sf3b1+/- cells, because of the decrease of PcG complex-bound Sf3b1, such mistranscribed RNAs become easily associated with splicing-active nucleoplasmic Sf3b1/U2 snRNP. This association leads to the achievement of a splicing reaction, which results in the anterior shift of Hox expression. In support of this model is the important evidence that the Mll mutation completely suppresses Sf3b1 phenotypes, indicating that the PcG-like function of Sf3b1 is very susceptible to levels of Mll; in other words the acting points of both proteins are spatially very close. Of further note is the fact that the human MLL supercomplex includes the 116-kDa protein specific to the U5 snRNP, which acts on pre-mRNA following the U2 snRNP. Finally, it appears that there are multiple interacting surfaces between PcG complexes and gene expression machineries. It might be that, through this interaction, PcG complexes act as a part of the modules that sense the transcriptional status in transcriptional competent regions of the Hox cluster (Isono, 2005).

Novel functions of chromatin-bound IkappaBalpha in oncogenic transformation

The nuclear factor-kappaB (NF-kappaB) signalling pathway participates in a multitude of biological processes, which imply the requirement of a complex and precise regulation. IkappaB (for Inhibitor of kappaB) proteins, which bind and retain NF-kappaB dimers in the cytoplasm, are the main contributors to negative regulation of NF-kappaB under non-stimulation conditions. Nevertheless, increasing evidences indicate that IkappaB proteins exert specific nuclear roles that directly contribute to the control of gene transcription. In particular, hypophosphorylated IkappaBbeta can bind the promoter region of TNFalpha leading to persistent gene transcription in macrophages and contributing to the regulation of the inflammatory response. Recently, it was demonstrated that phosphorylated and SUMOylated IkappaBalpha resides in the nucleus of the cells where it binds to chromatin leading to specific transcriptional repression. Mechanistically, IkappaBalpha associates and regulates Polycomb Repressor Complex activity, a function that is evolutionary conserved from flies to mammals, as indicate the homeotic phenotype of Drosophila mutants. The implications of chromatin-bound IkappaBalpha function is discussed in the context of tumorigenesis (Espinosa, 2014).

Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin

The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. The five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8 have been investigated. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, it was found that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. It is proposed that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites (Bernstein, 2005).

The founding member of the PcG genes is Drosophila melanogaster Polycomb (Pc) -- mutations in Pc result in body segment transformations. Pc is encoded by a single gene in Drosophila, while the mouse homologs have expanded into five family members known as Chromobox 2 (Cbx2) (mPc1 or M33), Cbx4 (mPc2), Cbx6, Cbx7, and Cbx8 (mPc3) (34). Importantly, these proteins contain a highly conserved N-terminal chromodomain (CD), a module first identified in the Drosophila proteins heterochromatin protein 1 (HP1) and Pc. The CD is found in a wide range of chromatin-associated proteins, most with transcriptionally repressive functions. The CD binds to methylated histones: the CD of Drosophila HP1 binds histone H3K9me2 and me3, while that of Pc specifically binds K27me3 on H3. Besides methyl-lysine binding, several reports have also suggested that certain CDs bind nucleic acids (Bernstein, 2005).

Based on findings with Cbx7, it is concluded that although the H3K27me3 mark seems to be important for the recruitment of Cbx7 via its CD, other components, including RNAs, must also be required for its recruitment and maintenance on the Xi. Indeed, recent reports have described the recruitment of several PRC1 proteins to the Xi, which may be part of one or multiple complexes. It will be interesting to identify Cbx7-interacting proteins at different stages of development in order to understand the mechanism(s) by which Cbx7 associates with the inactive X (Xi) chromosome, and with chromatin in general, during ES cell differentiation. For example, in addition to representing a potential mechanism by which Ring1 is able to ubiquitylate H2A on the Xi, the association of Cbx7 with chromatin during ES cell differentiation may be linked to the rather sudden appearance of macroH2A on the Xi. Finally, the role of RNA in Cbx7 chromatin association is of particular interest in light of recent evidence in fission yeast, flies, and mammals suggesting that noncoding RNAs can impact the chromatin template. Moreover, TAP-tag purification of Cbx7 from human cells has demonstrated that this Pc protein interacts with an RNA-helicase, suggesting the involvement of RNA in Cbx7-mediated repression. The ability of particular CDs to potentially bind both methylated histone tails and RNA suggests that a cooperative binding mechanism may mediate the enrichment of particular CD-containing proteins in chromatin. Future functional and structural studies will be required to determine the nature of this potential synergy, particularly in the case of Cbx7 and Xist RNA in the context of the inactive X chromosome (Bernstein, 2005).

Polycomb complexes repress developmental regulators in murine embryonic stem cells

The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. This study shows that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, it was found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed (homolog of Drosophila Extra sexcombs), and were preferentially activated on induction of differentiation. These results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development (Boyer, 2006).

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. The Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 was mapped across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. It was found SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. PRC2 target genes are preferentially activated during ES cell differentiation and the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation (Lee, 2006).

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ~40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, 'bivalent' ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation (Min, 2011).

Determining the presence and status of Pol II at bivalent genes is important for understanding the mechanisms that govern their transcription. Bivalent genes that are targets of the PRC2 complex, but not PRC1, retain promoter-proximal engaged Pol II that is more tightly confined to a region near the TSS and also display dramatically reduced levels of elongating Pol II. These results suggest that transcription at bivalent genes bound by PRC2 is regulated after the initiation step but very early in elongation. Although PRC2 activity has been shown to function in recruiting PRC1 and in DNA looping, no connections to distinct steps in transcription have been made previously (Min, 2011).

The bivalent genes that are occupied by both the PRC2 and the PRC1 complexes display much less promoter-proximal Pol II, suggesting a preinitiation block to earlier steps in transcription at these genes. This is consistent with the ability of PRC1 to compact nucleosomes, interfere with specific functions of the transcriptional apparatus, or both. The stronger repression of bivalent genes through the additional activity of PRC1 is also evident in that these genes show more robust retention of repressive chromatin through differentiation than PRC1-,2+ bivalent genes. Nonetheless, these genes are not completely silent. Both bivalent gene classes were found to exhibit significant levels of productive elongation that are higher than at PRC-bound genes with H3K27me3 but not H3K4me3 (Min, 2011).

The transcriptional activities of most genes are proportional to the levels of H3K4me3 on their promoter regions. In marked contrast, the transcriptional activity and divergent transcription of both classes of bivalent genes can vary widely, but the amount of H3K4me3 on the promoter remains at a level that is almost equal to the genome-wide average. This constant presence of H3K4me3 modifications across the promoter region may suggest that bivalent genes are poised for further activation. Recently, H3K4me3 modifications have been shown to evoke a dynamic cycle of histone acetylation and deacetylation at the promoters of inactive genes, which may facilitate the cross-talk between different histone modifications to prepare for activation. In addition, the high affinity of the TAF3 subunit of TFIID for the H3K4me3-modified histone tail may assist activation for bivalent genes that are highly enriched for CpG islands but lacking a TATA box (Min, 2011).

Many developmental regulatory genes are targets of PRC2 and PRC1 in ESCs, and most are transcribed at a modest level but do not feature a significant peak of paused Pol II. Interestingly, another study has shown that these genes retain a high level of Ser5-phosphorylated Pol II at the promoter relative to the gene body. Although paused Pol II is Ser5-phosphorylated, these promoters could possibly contain a form of Pol II that either has not fully entered elongation or is backtracked and unable to elongate in a run-on assay (Min, 2011).

Most developmental regulatory genes are not highly expressed in ESCs; however, it is noted that regulators of multiple lineages show some 'promiscuous transcription,' and this shares some similarities with what has been observed in the hematopoietic system. Interestingly, the regulators of neural and neuroectodermal lineages are among the highly expressed regulators in ESCs, supporting the hypothesis that ESCs in culture have an 'aptitude' for neural differentiation (Min, 2011).

OCT4, SOX2, and NANOG play important roles in preventing activation of specific lineage differentiation pathways, as well as forming the positive feedback transcription network for maintaining and establishing the pluripotent and self-renewal potentials in ESCs. Oct4 and Nanog are actively transcribed in ESCs but still exhibit a rate-limiting step at pausing. Rapid, synchronous, and high levels of activation correlate better with genes that possess paused Pol II over genes that do not. Taken together, it is speculated that pausing provides a responsive transcriptional regulatory step for controlling the level of critical core pluripotency transcription factors in ESCs (Min, 2011).

The profile of engaged RNA polymerases provides both a measure of transcription and a means of identifying those steps that are slow and regulated. This comparison of ESCs and MEFs establishes that transcription elongation is often controlled by dynamically tuning the release of the paused Pol II. However, an important class of regulated genes in ESCs that show bivalent histone modifications is modulated at both elongation and stages prior to elongation. Identification of the molecular targets of upstream activators or repressors and the role of these targets in modulating the rate-limiting steps of transcription will be essential to fully elucidate the mechanisms governing the regulation of the ESC state (Min, 2011).

Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression

A key step in gene repression by Polycomb is trimethylation of histone H3 K27 by PCR2 to form H3K27me3. H3K27me3 provides a binding surface for PRC1. This study shows that monoubiquitination of histone H2A by PRC1-type complexes to form H2Aub creates a binding site for Jarid2-Aebp2-containing PRC2 and promotes H3K27 trimethylation on H2Aub nucleosomes. Jarid2, Aebp2 and H2Aub thus constitute components of a positive feedback loop establishing H3K27me3 chromatin domains (Kalb, 2014).

Targeting polycomb to pericentric heterochromatin in embryonic stem cells reveals a role for H2AK119u1 in PRC2 recruitment

The mechanisms by which the major Polycomb group (PcG) complexes PRC1 and PRC2 are recruited to target sites in vertebrate cells are not well understood. Building on recent studies that determined a reciprocal relationship between DNA methylation and Polycomb activity, This study demonstrates that, in methylation-deficient embryonic stem cells (ESCs), CpG density combined with antagonistic effects of H3K9me3 and H3K36me3 redirects PcG complexes to pericentric heterochromatin and gene-rich domains. Surprisingly, it was found that PRC1-linked H2A monoubiquitylation is sufficient to recruit PRC2 to chromatin in vivo, suggesting a mechanism through which recognition of unmethylated CpG determines the localization of both PRC1 and PRC2 at canonical and atypical target sites. These data are discussed in light of emerging evidence suggesting that PcG recruitment is a default state at licensed chromatin sites, mediated by interplay between CpG hypomethylation and counteracting H3 tail modifications (Cooper, 2014).

Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes

The product of the Scmh1 gene, a mammalian homolog of Drosophila Sex comb on midleg, is a constituent of the mammalian Polycomb repressive complexes 1 (Prc1). Scmh1 has been identified as an indispensable component of the Prc1. During progression through pachytene, Scmh1 was shown to be excluded from the XY body at late pachytene, together with other Prc1 components such as Phc1 and Phc2 (Polyhomeotic homologs), Rnf110 (Pcgf2), Bmi1 [Drosophila homologs Psc and Su(z)2] and Cbx2 (homolog of Polycomb). The role of Scmh1 in mediating the survival of late pachytene spermatocytes has been identified. Apoptotic elimination of Scmh1-/- spermatocytes is accompanied by the preceding failure of several specific chromatin modifications at the XY body, whereas synapsis of homologous autosomes is not affected. It is therefore suggested that Scmh1 is involved in regulating the sequential changes in chromatin modifications at the XY chromatin domain of the pachytene spermatocytes. Restoration of defects in Scmh1-/- spermatocytes by Phc2 mutation indicates that Scmh1 exerts its molecular functions via its interaction with Prc1. Therefore, for the first time, it is possible to indicate a functional involvement of Prc1 during the meiotic prophase of male germ cells and a regulatory role of Scmh1 for Prc1, which involves sex chromosomes (Takada, 2007).

Based on the present observations, it is postulated that Scmh1 could primarily promote the exclusion of Prc1 components from the XY body in the pachytene spermatocytes because Scmh1 itself is a functional component of Prc1. By contrast, failure to maintain exclusion of trimethylated H3-K27 and to undergo H3-K9 methylation at the XY body in Scmh1-/- spermatocytes may occur secondarily to the failure to exclude Prc1 from the XY body. At many loci, epistatic engagement of Prc1 by Prc2 has been shown to be essential for the mediation of transcriptional repression. Preceding exclusion of trimethylated H3-K27, which represents Prc2 actions, for Prc1 exclusion from the XY body, is consistent with epistatic roles of Prc2 for Prc1 at the XY body. Therefore, Scmh1 may affect H3-K27 trimethylation at the XY body through the Prc1-Prc2 engagement. It is noteworthy that H3-K27 trimethylation has been shown to be regulated by Prc1 at the XY body. This may imply that Prc1-Prc2 engagement is a reciprocal rather than epistatic process at the XY body. This possibility should be addressed by using conditional mutants for Prc2 components. A functional correlation between Prc1 exclusion and H3-K9 methylations at the XY body is also hypothesized because the indispensable H3-K9 methyltransferase complex, composed of G9a and GLP, is constitutively associated with E2F6 complexes, which share at least Rnf2 and Ring1 components with Prc1. Moreover, several components of respective complexes are structurally related to each other. Intriguingly, although Prc1 components, apart from Rnf2, have been shown to be excluded from the XY body at late pachytene stage, components of E2F6 complexes including Rnf2, RYBP, HP1gamma and G9a are retained. The most attractive scenario would be that exclusion of Prc1 is a prerequisite for the functional manifestation of E2F6 complexes to mediate the hypermethylation of H3-K9 at the XY body. It is thus proposed that Scmh1-mediated exclusion of Prc1 from the XY body might be a prerequisite for maintaining appropriate chromatin structure to undergo subsequent sequential chromatin remodeling of the XY chromatin in pachytene spermatocytes (Takada, 2007).

It is also suggested that sequential changes in chromatin modifications of the sex chromosomes in the pachytene spermatocytes might be monitored by some meiotic checkpoint mechanisms. This is supported by the temporal concurrence of Prc1 exclusion from the XY body and apoptotic depletion of meiotic spermatocytes, their coincidental restorations by Phc2 mutation, and normal oogenesis and fertility in Scmh1-/- females. In addition, defects in the XY body formations have been shown to correlate with apoptotic depletion of meiotic spermatocytes by studies using H2A.X and Brca1 mutants, although developmental arrests occurred by early pachytene stage. However, this link has not been substantially demonstrated (Takada, 2007).

Although Scmh1 has been shown to act together with Prc1, the role of Scmh1 for Prc1 might be modified in a tissue- or locusspecific manner because spermatogenic defects by Scmh1 mutation are restored by Phc2 mutation, whereas premature senescence of MEFs is enhanced mutually by both mutations. This is supported by an immunofluorescence study revealing the co-localization of Scmh1 with other class 2 PcG proteins in subnuclear speckles in U2OS cells, whereas in female trophoblastic stem (TS) cells it is excluded from the inactive X chromosome domain, which is intensely decorated by Rnf2, Phc2 and Rnf110. It may be possible to postulate some additional factors that modify the molecular functions or subnuclear localization of Scmh1. Indeed, most of the soluble pool of SCM in Drosophila embryos is not stably associated with Prc1, although SCM is capable of assembling with the Polyhomeotic protein by their respective SPM domains in the Polycomb core complex. As the SPM domain is shared, not only by polyhomeotic homologs, but also by multiple paralogs of the Drosophila Scm gene, namely Scml1, Scml2, Sfmbt, l(3)mbt3 and others in mammals, these structurally related gene products may potentially interact with Scmh1 and modulate its functions. Conservations of crucial amino acid residues required for the mutual interaction of SPM domains and multiple mbt repeats in these proteins may further suggest functional overlap with Scmh1. It is notable that phenotypic expressions of Scmh1 mutation are quite variable during spermatogenesis and axial development even after more than five times backcrossing to a C57Bl/6 background. This incomplete penetrance might involve multiple paralogs of the canonical Scm proteins, which may act in compensatory manner for Scmh1 mutation, as revealed between Rnf110 and Bmi1 or Phc1 and Phc2 (Takada, 2007).

A vertebrate Polycomb response element governs segmentation of the posterior hindbrain

Chromatin remodeling by Polycomb group (PcG) and trithorax group (trxG) proteins regulates gene expression in all metazoans. Two major complexes, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), are thought to mediate PcG-dependent repression in flies and mammals. In Drosophila, PcG/trxG protein complexes are recruited by PcG/trxG response elements (PREs). However, it has been unclear how PcG/trxG are recruited in vertebrates. This study identified a vertebrate PRE, PRE-kr, that regulates expression of the mouse MafB/Kreisler gene. PRE-kr recruits PcG proteins in flies and mouse F9 cells and represses gene expression in a PcG/trxG-dependent manner. PRC1 and 2 bind to a minimal PRE-kr region, which can recruit stable PRC1 binding but only weak PRC2 binding when introduced ectopically, suggesting that PRC1 and 2 have different binding requirements. Thus, evidence is provided that similar to invertebrates, PREs act as entry sites for PcG/trxG chromatin remodeling in vertebrates (Sing, 2009).

Analyses of the kr inversion suggest that PRE-kr directs PcG-protein-dependent repression in the anterior hindbrain and trxG-protein-dependent activation in the posterior hindbrain. A combination of factors might influence such position-specific function. (1) Analyses in Drosophila have shown that PREs associate robustly with promoters nearest to them. Thus, PRE-kr could interact with the regulatory elements or promoters from Nnat or MafB closest to it. (2) PRE-kr function could depend on the composition of the PcG protein complexes that bind it. Various studies suggest selective interactions of PREs with specific PcG subunits. For example, whereas redundancy of M33/Cbx2 with its homologs Cbx4, 6, 7, and 8 might contribute to the variable effects of M33 dosage on MafB, Cbx/Polycomb family members also have distinct roles in governing the cell cycle. Cbx4 does not affect replicative senescence of fibroblasts, a Cbx8-Bmi1 complex binds the INK4a-ARF locus to overcome senescence, and Cbx7-mediated bypass of senescence is Bmi1 independent. (3) Interactions of PcG proteins with other transcription factors could provide additional specificity. For example, Bmi1 interacts with the E2F6 transcription factor to repress Hox genes but acts independently of E2F6 to repress the Ink4a-Arf locus. (4) PRE-kr might be sensitive to signals governing anterior-posterior patterning since an extrinsic RARE can overcome PRE-kr-mediated repression in transgenic mice. Whether PRE-kr responds selectively to RA or also to other signaling pathways remains to be determined (Sing, 2009).

In Drosophila, trxG proteins interact selectively with PRE-kr. Only trxl/GAGA factor affected PRE-kr-directed repression in flies. Considering that vertebrates lack Trxl/GAGAF orthologs, the effect of Trxl on PRE-kr-dependent repression in flies suggests that a GAGAF-containing trxG complex, which also contains other trxG proteins, is conserved between flies and mice and promotes PRE-kr dependent activation. The SWI/SNF-related ATP-dependent Brahma chromatin-remodeling complex components, Brahma and Moira, did not affect PRE-kr-dependent repression in flies. This potentially reflects the selective action of SWI/SNF-containing complexes seen also in vertebrate neural development. Future studies will be needed to investigate if PRE-kr serves as a substrate for specific SWI/SNF-containing complexes that interact selectively with PREs governing early neural patterning genes (Sing, 2009).

In Drosophila, PcG complexes bind to discrete sequence platforms. Similarly, a distinct peak of SUZ12 and Bmi1 binding was observed within PRE-kr in F9 cells. By contrast, the H3K27 signature, which is thought to be laid down by Ezh1/2-Eed complex(es), covered the MafB locus. However, RA-induced decrease of the H3K27 mark and loss of PcG binding were highly localized. These observations suggest that two distinct H3K27 pools, only one of which depends directly on the presence of PRC1 and 2 binding, exist. Consistent with current observations, it has been proposed that distinct PRC2 complexes exist and only a subset specifically target PREs. Thus, other cues, such as nucleosomal modifications, could collaborate with the H3K27 mark to flag and re-enforce distinct SUZ12- and PRC1-binding platforms at PRE-kr (Sing, 2009).

A distinct difference was uncovered in sequence requirements for PRC1 and 2 binding. The minimal hcPRE-kr region could recruit Bmi1, but not SUZ12, binding as effectively in an exogenous context as in an endogenous context. Only stable binding of PRC1 appears to be required for PRE-kr to repress reporter gene expression at ectopic sites. Notably, in flies, recruitment of the PRC1 components Pc and Ph to PRE transgenic insertion sites has served as a criterion for validating ectopically introduced Drosophilid PREs, but binding of PRC2 components has not been similarly examined. Perhaps improved PRC2-specific antibodies will elucidate if the requirements for stable PRC1 and PRC2 occupancy differ in flies, as suggested by findings in F9 cells. Other studies in mammals have found that PRC1 is associated with repressive activity even in the absence of PRC2. CBX8 and Bmi1 exhibit similar levels of binding to many genes even in the absence of detectable H3K27 methylation in Suz12-/- ES cells. In Eed null cells, several PRC1 components are recruited to the inactive X, whereas maternally provided PRC1 components show Ezh2-independent targeting to paternal heterochromatin. Furthermore, PRC1 in vitro is able to repress transcription and inhibit ATP-dependent chromatin remodeling mediated by the human SWI/SNF complex -- a complex related to the Drosophila TrxG Brahma complex (Sing, 2009).

The observation that knockdown of SUZ12 affects Bmi1 binding to endogous and ectopic PRE-kr fits well with the 'hierarchical recruitment' model, which proposes sequential action of PRC2 and 1. Given that SUZ12 binding to ectopic hcPRE-kr was very low, it is proposed that a transient, unstable SUZ12 association with ectopic hcPRE-kr is sufficient to stabilize PRC1 binding -- perhaps by introducing the necessary methylation signature. An observation not directly in line with the 'hierarchical recruitment' model is that Bmi1 knockdown reduced SUZ12 binding to endogenous but not ectopic PRE-kr. This observation could be explained by a sequence-dependent role for PRC1 in supporting stable PRC2 binding or indirect effects of PRC1 on other PRC2 components, for example by reducing their levels. Thus, a possible interdependence of PRC1 and 2 binding and function still remains to be elucidated for PRE-kr and PREs (Sing, 2009).

These results have wide-ranging implications for PcG mechanisms, but also possibily for organization of transcriptional neighborhoods and human disorders. The finding that the kr inversion translocates a PRE from one rhombomere-specific gene to another might be a coincidence or could suggest that PREs are involved in long-range organization of transcriptional neighborhoods (Sing, 2009).

Polycomb eviction as a new distant enhancer function

Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. This study analyzed the influence of a remote enhancer on the balance between repression (Polycomb-PcG) and activation (Trithorax-TrxG) of a developmentally regulated gene associated with a CpG island. Its essential, nonredundant role in clearing the PcG complex and H3K27me3 from the CpG island is revealed. In the absence of the enhancer, the H3K27me3 demethylase (JMJD3) is not recruited to the CpG island. A new role is proposed for long-range regulatory elements in removing repressive PcG complexes (Vernimmen, 2011).

There is increasing evidence that CpG islands, such as those associated with the α-globin promoter, constitute at least one element that can mediate recruitment of PcG and TrxG complexes to mammalian promoters (Mendenhall et al. 2010). As previously noted, PcG-binding sites are dynamic, are nucleosome-depleted, and have a rapid histone turnover (the residency time of PcG is in the order of a few minutes). PcG binding is therefore thought to be dynamic and sensitive to the antagonistic action of TrxG proteins together with positive and negative input from other TFs and cofactors. However, it is not known whether the eviction of PcG silencing complex from its targets, seen during development and differentiation, depends on the presence of distal regulatory elements or only on (co)factors acting at proximal cis elements. This study used a mouse experimental model to analyze the CpG island associated with the human α-globin promoter in two states: without and with its interacting distant enhancer, both in terminally differentiated erythroid cells. The recruitment was compared of CGBP in nonexpressing versus expressing human cells. In nonerythroid cells, the unmethylated, nuclease-insensitive CpG island associated with the α-globin gene is bound by PcG and is transcriptionally silenced (referred to as the 'silent state'). In erythroid cells without MCS-R2 (referred to as 'basal state'), and in contrast to nonerythroid cells, the promoter becomes accessible to some TFs and is associated with some active chromatin modifications (e.g., H3K4me3) with relatively low levels of transcription (~2% of normal). Nevertheless, the PcG complex with its associated modification (H3K27me3) is still prominent at the α-globin CpG island. It was also demonstrates that PcG and CGBP binding are mutually exclusive. In erythroid cells with MCS-R2 (referred to as 'active state'), PcG complexes are completely removed from the CpG island. Furthermore, the histone H3K27 demethylase JMJD3, which may remove H3K27me3 and thereby facilitate transcription, is also recruited at high levels. Recruitment of the SAGA complex (e.g., PCAF and GCN5) becomes prominent and the downstream effects (e.g., deposition of H2Bub and H3K79 methylation) are established. At this stage, high levels of transcription are associated with binding of CGBP. This study thus shows that the recruitment of the demethylase JMJD3 and full clearance of the PcG-repressive complex (including PRC2 and HDAC1) at the α-globin CpG island depend on one or more activities mediated by the remote regulatory element and are associated with the transition between basal and fully activated transcription (Vernimmen, 2011).

A model is presented for long-range control of epigenetic regulation. In nonerythroid cells, the CpG island is entirely silenced by PcG and HDAC1, associated with the repressive histone mark H3K27me3. The promoter 'P' is not sensitive to DNaseI, and transcription does not occur. In erythroid cells lacking the enhancer, the gene remains repressed by PcG and marked by H3K27me3. At this basal level of expression, the promoter becomes accessible to some TFs and chromatin-modifying enzymes and is marked by moderate levels of H3K4me3, which reflect very low levels of transcription. In the presence of the enhancer, PcG is evicted and the H3K27me3 histone mark is erased by recruitment of demethylase JMJD3. Acetylation (H3ac and H4ac), H3K79me3, and H2Bub increases with spreading of HAT and Bre (SAGA) along the coding sequence. At this activated stage, the remaining TFs, including Pol II, are now fully recruited, and a high rate of transcription occurs. The CpG island at this stage is also bound by CGBP (Vernimmen, 2011).

These findings demonstrate for the first time that the pattern of PcG binding at a CpG island may be affected by cis-acting elements located far away from the associated promoter. In contrast, the chromatin modification associated with TrxG activity (H3K4me3) appears to be more dependent on local changes at the CpG island that occur in the context of basal transcription. Future studies will address how long-range enhancers exert these effects. It is possible that transcriptional activation per se competes with the competitive binding of PcG complexes and is responsible for the clearance of these complexes. The second is that upstream elements also deliver new proteins (e.g., JMJD3) or modify proteins (e.g., histones) that facilitate the removal of PcG. In the past, detailed analysis of the globin genes has established many of the general principles underlying mammalian gene regulation, and it therefore seems probable that this new role of distal regulatory elements in removing PcG from their target promoters will be of considerable general importance (Vernimmen, 2011).

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).

Polycomb: Biological Overview | Regulation | Protein interactions | Developmental Biology | Effects of Mutation | References

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