Genome regulation by polycomb and trithorax proteins

Polycomb group (PcG) and trithorax group (trxG) proteins are critical regulators of numerous developmental genes. To silence or activate gene expression, respectively, PcG and trxG proteins bind to specific regions of DNA and direct the posttranslational modification of histones. Recent work suggests that PcG proteins regulate the nuclear organization of their target genes and that PcG-mediated gene silencing involves noncoding RNAs and the RNAi machinery.     

Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila

In Drosophila melanogaster, Hox genes are organized in an anterior and a posterior cluster, called Antennapedia complex and bithorax complex, located on the same chromosome arm and separated by 10 Mb of DNA. Both clusters are repressed by Polycomb group (PcG) proteins. Here, we show that genes of the two Hox complexes can interact within nuclear PcG bodies in tissues where they are corepressed. This colocalization increases during development and depends on PcG proteins. Hox gene contacts are conserved in the distantly related Drosophila virilis species and they are part of a large gene interaction network that includes other PcG target genes. Importantly, mutations on one of the loci weaken silencing of genes in the other locus, resulting in the exacerbation of homeotic phenotypes in sensitized genetic backgrounds. Thus, the three-dimensional organization of Polycomb target genes in the cell nucleus stabilizes the maintenance of epigenetic gene silencing.     

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.     

Insulators, not Polycomb response elements, are required for long-range interactions between Polycomb targets in Drosophila melanogaster

The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb "bodies" or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.     

Structure of a polycomb response element and in vitro binding of polycomb group complexes containing GAGA factor

Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.     

Drosophila chromosome condensation proteins Topoisomerase II and Barren colocalize with Polycomb and maintain Fab-7 PRE silencing

Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. We have investigated whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. We show that Topoisomerase II (TOPOII) and Barren (BARR) interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, we find that the PcG protein Polyhomeotic (PH) interacts physically with TOPOII and BARR and that BARR is required for Fab-7-regulated homeotic gene expression. Conversely, we find defects in chromosome segregation associated with ph mutations. We propose that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression.     

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

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

The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3

Polycomb group (PcG) proteins are important for maintaining the silenced state of homeotic genes. Biochemical and genetic studies in Drosophila and mammalian cells indicate that PcG proteins function in at least two distinct protein complexes: the ESC-E(Z) or EED-EZH2 complex, and the PRC1 complex. Recent work has shown that at least part of the silencing function of the ESC-E(Z) complex is mediated by its intrinsic activity for methylating histone H3 on lysine 27. In addition to being involved in Hox gene silencing, the complex and its associated histone methyltransferase activity are important in other biological processes including X-inactivation, germline development, stem cell pluripotency and cancer metastasis.     

Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants

In both Drosophila and vertebrates, spatially restricted expression of HOX genes is controlled by the Polycomb group (PcG) repressors. Here we characterize a novel Drosophila PcG gene, Suppressor of zeste 12 (Su(z)12). Su(z)12 mutants exhibit very strong homeotic transformations and Su(z)12 function is required throughout development to maintain the repressed state of HOX genes. Unlike most other PcG mutations, Su(z)12 mutations are strong suppressors of position-effect variegation (PEV), suggesting that Su(z)12 also functions in heterochromatin-mediated repression. Furthermore, Su(z)12 function is required for germ cell development. The Su(z)12 protein is highly conserved in vertebrates and is related to the Arabidopsis proteins EMF2, FIS2 and VRN2. Notably, EMF2 is a repressor of floral homeotic genes. These results suggest that at least some of the regulatory machinery that controls homeotic gene expression is conserved between animals and plants.     

The DNA-binding polycomb group protein pleiohomeotic mediates silencing of a Drosophila homeotic gene.

Polycomb group (PcG) proteins repress homeotic genes in cells where these genes must remain inactive during development. This repression requires cis-acting silencers, also called PcG response elements. Currently, these silencers are ill-defined sequences and it is not known how PcG proteins associate with DNA. Here, we show that the Drosophila PcG protein Pleiohomeotic binds to specific sites in a silencer of the homeotic gene Ultrabithorax. In an Ultrabithorax reporter gene, point mutations in these Pleiohomeotic binding sites abolish PcG repression in vivo. Hence, DNA-bound Pleiohomeotic protein may function in the recruitment of other non-DNA-binding PcG proteins to homeotic gene silencers.     

O-GlcNAc cycling: Emerging roles in development and epigenetics

The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan. Surprisingly, from flies to man, the O-GlcNAcase (OGA, MGEA5) gene is embedded within the NK cluster, the most evolutionarily ancient of three homeobox gene clusters regulated by PcG repression. PcG repression also plays a key role in maintaining stem cell identity, recruiting the DNA methyltransferase machinery for imprinting, and in X-chromosome inactivation. Intriguingly, the Ogt gene resides near the Xist locus in vertebrates and is subject to regulation by PcG-dependent X-inactivation. OGT is also an enzymatic component of the human dosage compensation complex. These ‘evo-devo’ relationships linking O-GlcNAc cycling to higher order chromatin structure provide insights into how nutrient availability may influence the epigenetic regulation of gene expression. O-GlcNAc cycling at promoters and PcG repression represent concrete mechanisms by which nutritional information may be transmitted across generations in the intra-uterine environment. Thus, the nutrient-sensing hexosamine signaling pathway may be a key contributor to the metabolic deregulation resulting from prenatal exposure to famine, or the ‘vicious cycle’ observed in children of mothers with type-2 diabetes and metabolic disease.