Linking the Rb and polycomb pathways

Polycomb group (PcG) proteins associate to form complexes that repress Hox genes, thereby imposing the patterning of Hox expression required for development. However, these proteins have a second Hox-independent role in regulating cell proliferation. Our results suggest that association between Rb and PcG proteins forms a repressor complex that blocks entry of cells into mitosis. Also, we provide evidence 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.     

Molecular and genetic analysis of the Polycomb group gene Sex combs extra/Ring in Drosophila

Polycomb group (PcG) proteins repress homeotic genes and other developmental regulatory genes in cells where these genes must remain inactive during development. In Drosophila and in vertebrates, PcG proteins exist in two distinct multiprotein complexes, the Esc/Eed-E(z) complex and PRC1. Drosophila PRC1 contains Polycomb, Posterior sexcombs and Polyhomeotic, the products of three PcG genes that are critically needed for PcG silencing. Formation of stable PRC1 requires Ring, the product of a gene for which no mutations have been described. Here, we show that Sex combs extra (Sce) encodes Ring and that Sce/Ring function is critically required for PcG silencing.     

The core of the polycomb repressive complex is compositionally and functionally conserved in flies and humans

The Polycomb group (PcG) genes are required to maintain homeotic genes in a silenced state during development in drosophila and mammals and are thought to form several distinct silencing complexes that maintain homeotic gene repression during development. Mutations in the PcG genes result in developmental defects and have been implicated in human cancer. Although some PcG protein domains are conserved between flies and humans, substantial regions of several PcG proteins are divergent and humans contain multiple versions of each PcG gene. To determine the effects of these changes on the composition and function of a PcG complex, we have purified a human Polycomb repressive complex from HeLa cells (hPRC-H) that contains homologues of PcG proteins found in drosophila embryonic PRC1 (dPRC1). hPRC-H was found to have fewer components than dPRC1, retaining the PcG core proteins of dPRC1 but lacking most non-PcG proteins. Preparations of hPRC-H contained either two or three different homologues of most of the core PcG proteins, including a new Ph homologue we have named HPH3. Despite differences in composition, dPRC1 and hPRC-H have similar functions: hPRC-H is able to efficiently block remodeling of nucleosomal arrays through a mechanism that does not block the ability of nucleases to access and cleave the arrays.     

Reconstitution of a functional core polycomb repressive complex

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

Programming of gene expression by Polycomb group proteins

Polycomb group (PcG) complexes maintain epigenetically repressed states that need to be reprogrammed when cells become committed to differentiation. In contrast to the previously held belief that PcG complexes regulate only a few selected genes, recent efforts have revealed hundreds of potential PcG targets in mammals, insects and plants. These results have changed our perception about PcG recruitment and function on chromatin. Both in animals and plants, evolutionarily conserved PcG complexes mark the chromatin of their target genes by methylation at histone H3 lysine 27. Surprisingly, however, both the proteins recognizing this mark and the mechanisms causing gene repression differ between both kingdoms. This suggests that different developmental strategies used in plant and animal development entailed the evolution of different repressive maintenance mechanisms.     

Are we there yet? Initial targeting of the Male-Specific Lethal and Polycomb group chromatin complexes in Drosophila

Chromatin-binding proteins must navigate the complex nuclear milieu to find their sites of action, and a constellation of protein factors and other properties are likely to influence targeting specificity. Despite considerable progress, the precise rules by which binding specificity is achieved have remained elusive. Here, we consider early targeting events for two groups of chromatin-binding complexes in Drosophila: the Male-Specific Lethal (MSL) and the Polycomb group (PcG) complexes. These two serve as models for understanding targeting, because they have been extensively studied and play vital roles in Drosophila, and their targets have been documented at high resolution. Furthermore, the proteins and biochemical properties of both complexes are largely conserved in multicellular organisms, including humans. While the MSL complex increases gene expression and PcG members repress genes, the two groups share many similarities such as the ability to modify their chromatin environment to create active or repressive domains, respectively. With legacies of in-depth genetic, biochemical and now genomic approaches, the MSL and PcG complexes will continue to provide tractable systems for understanding the recruitment of multiprotein chromatin complexes to their target loci.     

Association of BMI1 with polycomb bodies is dynamic and requires PRC2/EZH2 and the maintenance DNA methyltransferase DNMT1

Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in heritable gene repression. Two main PcG complexes have been characterized. Polycomb repressive complex 2 (PRC2) is thought to be involved in the initiation of gene silencing, whereas Polycomb repressive complex 1 (PRC1) is implicated in the stable maintenance of gene repression. Here, we investigate the kinetic properties of the binding of one of the PRC1 core components, BMI1, with PcG bodies. PcG bodies are unique nuclear structures located on regions of pericentric heterochromatin, found to be the site of accumulation of PcG complexes in different cell lines. We report the presence of at least two kinetically different pools of BMI1, a highly dynamic and a less dynamic fraction, which may reflect BMI1 pools with different binding capacities to these stable heterochromatin domains. Interestingly, PRC2 members EED and EZH2 appear to be essential for BMI1 recruitment to the PcG bodies. Furthermore, we demonstrate that the maintenance DNA methyltransferase DNMT1 is necessary for proper PcG body assembly independent of DNMT-associated histone deacetylase activity. Together, these results provide new insights in the mechanism for regulation of chromatin silencing by PcG proteins and suggest a highly regulated recruitment of PRC1 to chromatin.     

The Drosophila pleiohomeotic mutation enhances the Polycomblike and Polycomb mutant phenotypes during embryogenesis and in the adult

In Drosophila, the spatially restricted expression of the homeotic genes is controlled by Polycomb group (PcG) repression. PcG proteins appear to form different complexes to repress this gene expression. Although the pleiohomeotic gene (pho) shares mutational phenotypes with other PcG mutations, which demonstrates that PHO binds directly with a Polycomb (Pc)-containing complex, the genetic interactions of pho with other PcG genes have not been examined in detail. Here we investigated whether pho interacts with Polycomblike (Pcl) and Polycomb (Pc) during embryonic and adult development using developmental and genetic approaches. Pcl and Pc strongly enhanced pho phenotypes in the legs and tergite of the adult fly. Embryonic cuticle transformation was also greatly enhanced in Pcl; pho or Pc; pho double mutant embryos. The double mutant phenotypes were more severely affected by the pho maternal effect mutation than in zygotic mutant background, suggesting dosage-dependent processes. Taken together, these results provide genetic evidence of an interaction between PHO with other Polycomb group proteins at the embryonic and adult stages, and of the functioning of PHO as a component of the PcG complex.     

Epigenetic chromatin modifiers in barley: IV. The study of barley Polycomb group (PcG) genes during seed development and in response to external ABA

Background  

Epigenetic phenomena have been associated with the regulation of active and silent chromatin states achieved by modifications of chromatin structure through DNA methylation, and histone post-translational modifications. The latter is accomplished, in part, through the action of PcG (Polycomb group) protein complexes which methylate nucleosomal histone tails at specific sites, ultimately leading to chromatin compaction and gene silencing. Different PcG complex variants operating during different developmental stages have been described in plants. In particular, the so-called FIE/MEA/FIS2 complex governs the expression of genes important in embryo and endosperm development in Arabidopsis. In our effort to understand the epigenetic mechanisms regulating seed development in barley (Hordeum vulgare), an agronomically important monocot plant cultivated for its endosperm, we set out to characterize the genes encoding barley PcG proteins.     

The role of Polycomb Group Proteins in Cell Cycle Regulation During Development

Polycomb group (PcG) and trithorax group (trxG) proteins are evolutionarilyconserved chromatin modifiers that have well known roles in the maintenance ofsilent and active expression states of homeotic genes. PcG proteins may also beinvolved in the control of cellular proliferation, as several PcG complexes have beenshown to act either as proto-oncogenes or as tumor suppressors in vertebrates. InDrosophila, PcG factors associate with specific DNA regions termed PcG responseelements (PREs), and a PRE was recently identified in the gene encoding Cyclin A.Still, it is not yet clear how PcG complexes could control cell cycle progression.Beyond acting as stable silencers of cell cycle genes during the differentiationprocess, PcG complexes might also be integrators and/or modulators of cell cyclecheckpoints in dividing cells. Here, we discuss this dual aspect of PcG involvement inepigenetic cell cycle control.     

Variable Requirements for DNA-Binding Proteins at Polycomb-Dependent Repressive Regions in Human HOX Clusters

Polycomb group (PcG)-mediated repression is an evolutionarily conserved process critical for cell fate determination and maintenance of gene expression during embryonic development. However, the mechanisms underlying PcG recruitment in mammals remain unclear since few regulatory sites have been identified. We report two novel prospective PcG-dependent regulatory elements within the human HOXB and HOXC clusters and compare their repressive activities to a previously identified element in the HOXD cluster. These regions recruited the PcG proteins BMI1 and SUZ12 to a reporter construct in mesenchymal stem cells and conferred repression that was dependent upon PcG expression. Furthermore, we examined the potential of two DNA-binding proteins, JARID2 and YY1, to regulate PcG activity at these three elements. JARID2 has differential requirements, whereas YY1 appears to be required for repressive activity at all 3 sites. We conclude that distinct elements of the mammalian HOX clusters can recruit components of the PcG complexes and confer repression, similar to what has been seen in Drosophila. These elements, however, have diverse requirements for binding factors, which, combined with previous data on other loci, speaks to the complexity of PcG targeting in mammals.     

Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins

Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.