Heritable Repression Research Articles

Genomic context- and H2AK119 ubiquitination-dependent inheritance of human Polycomb silencing.

Polycomb repressive complexes 1 and 2 (PRC1 and 2) are required for heritable repression of developmental genes. The cis- and trans-acting factors that contribute to epigenetic inheritance of mammalian Polycomb repression are not fully understood. Here, we show that, in human cells, ectopically induced Polycomb silencing at initially active developmental genes, but not near ubiquitously expressed housekeeping genes, is inherited for many cell divisions. Unexpectedly, silencing is heritable in cells with mutations in the H3K27me3 binding pocket of the Embryonic Ectoderm Development (EED) subunit of PRC2, which are known to disrupt H3K27me3 recognition and lead to loss of H3K27me3. This mode of inheritance is less stable and requires intact PRC2 and recognition of H2AK119ub1 by PRC1. Our findings suggest that maintenance of Polycomb silencing is sensitive to local genomic context and can be mediated by PRC1-dependent H2AK119ub1 and PRC2 independently of H3K27me3 recognition.

Small-RNA-guided histone modifications and somatic genome elimination in ciliates.

Transposable elements and other repeats are repressed by small-RNA-guided histone modifications in fungi, plants and animals. The specificity of silencing is achieved through base-pairing of small RNAs corresponding to the these genomic loci to nascent noncoding RNAs, which allows the recruitment of histone methyltransferases that methylate histone H3 on lysine 9. Self-reinforcing feedback loops enhance small RNA production and ensure robust and heritable repression. In the unicellular ciliate Paramecium tetraurelia, small-RNA-guided histone modifications lead to the elimination of transposable elements and their remnants, a definitive form of repression. In this organism, germline and somatic functions are separated within two types of nuclei with different genomes. At each sexual cycle, development of the somatic genome is accompanied by the reproducible removal of approximately a third of the germline genome. Instead of recruiting a H3K9 methyltransferase, small RNAs corresponding to eliminated sequences tether Polycomb Repressive Complex 2, which in ciliates has the unique property of catalyzing both lysine 9 and lysine 27 trimethylation of histone H3. These histone modifications that are crucial for the elimination of transposable elements are thought to guide the endonuclease complex, which triggers double-strand breaks at these specific genomic loci. The comparison between ciliates and other eukaryotes underscores the importance of investigating small-RNAs-directed chromatin silencing in a diverse range of organisms. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action.

Small RNA-Mediated De Novo Silencing of Ac/Ds Transposons Is Initiated by Alternative Transposition in Maize.

Although transposable elements (TEs) comprise a major fraction of many higher eukaryotic genomes, most TEs are silenced by host defense mechanisms. The means by which otherwise active TEs are recognized and silenced remains poorly understood. Here we analyzed two independent cases of spontaneous silencing of the active maize Ac/Ds transposon system. This silencing is initiated by alternative transposition, a type of aberrant transposition event that engages the termini of two nearby separate TEs. Alternative transposition during DNA replication can generate Composite Insertions that contain inverted duplications of the transposon sequences. We show that the inverted duplications of two Composite Insertions are transcribed to produce double-stranded RNAs that trigger the production of two distinct classes of small interfering RNAs: a 24-nt class complementary to the TE terminal inverted repeats and noncoding subterminal regions, and a 21- to 22-nt class corresponding to the TE transcribed regions. Plants containing these small interfering RNA-generating Composite Insertions exhibit decreased levels of Ac transcript and heritable repression of Ac/Ds transposition. Further, we demonstrate that Composite Insertions can heritably silence otherwise active elements in trans This study documents the first case of transposon silencing induced by alternative transposition and may represent a general initiating mechanism for silencing of DNA transposons.

CRISPR/Cas9-Targeted De Novo DNA Methylation Is Maintained and Impacts the Colony Forming Potential of Human Hematopoietic CD34+ Cells

Introduction The epigenome is significantly perturbed in hematological malignancies with global DNA hypomethylation and localized hypermethylation of gene promoter CpG islands. Whether specific gene promoter hypermethylation can contribute to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) in humans by affecting HSPC biology, independently of genetic mutations, has not previously been investigated due to the lack of appropriate tools. We show for the first time that it is possible to target de novo DNA methylation using CRISPR/Cas9 in human CD34+ cells isolated from cord blood (CB). DNA methylation targeted to key cell cycle control gene promoters, INK4b (p15) and ARF (p14), is permanently maintained after dCas9 3A3L degradation and inherited as cells differentiate; inhibiting gene expression and affecting the colony forming potential of CD34+ cells. This demonstrates that specific DNA hypermethylation events can permanently change HSPC biology and impact differentiation, potentially contributing to pre-malignant processes. Methods Human CD34+ HSPCs were isolated from human CB and maintained in liquid culture for 24 hours before nucleofection with mRNA encoding an adapted form of CRISPR/Cas9 which has no nuclease activity (dCas9) and is fused to the catalytic domain of DNA methyltransferase 3A (DNMT3A) and 3L (3A3L). The nucleofection cocktail contained dCas9 3A3L or dCas9 3A3L-mut (lacks methyltransferase activity) and 1 to 3 guide RNAs to target DNA methylation to combinations of the INK4a-ARF-INK4b locus. Cells were then seeded into methylcellulose for a primary colony forming assay (CFU). Colonies were scored after 14 days and cells were either harvested and pooled or individual colonies were picked for single-colony molecular analyses. The DNA was extracted and methylation at the INK4a-ARF-INK4b promoters was quantified using targeted bisulfite sequencing; target gene expression was measured using qPCR. The remaining cells from the primary CFU were re-plated a second (secondary CFU) and third (tertiary CFU) time and colonies were again scored after 14 days. Results and Conclusions Targeting DNA methylation to the INK4a-ARF-INK4b locus or INK4b individually in human CD34+ cells resulted in maintenance of hypermethylation at ARF and/or INK4b gene promoters in individual BFU-E (burst-forming unit-erythroid) and CFU-GM (granulocyte, macrophage) colonies as measured by single-colony targeted bisulfite sequencing after the primary CFU; causing heritable repression of INK4b gene expression in the differentiated cells. Some CpGs were up to 90% methylated, indicating that DNA methylation added at these gene promoters is highly stable as cells differentiate. Hypermethylation of ARF and INK4b was found in some colonies even after the tertiary CFU, demonstrating long-term maintenance of promoter hypermethylation. Unexpectedly, no DNA hypermethylation was detected at INK4a in differentiated cells, but whether this is the case for all subpopulations of HSPCs (i.e. HSCs or lymphoid progenitors) is under investigation. Hypermethylation of INK4b and ARF increased the colony forming potential of CD34+ cells in primary, secondary and tertiary CFUs, compared to the control. Conversely, methylation targeted to INK4b alone did not significantly affect the number of colonies in the first CFU, and decreased the number of colonies in the secondary CFU. This suggests a complex interplay between key cell cycle regulators ARF and INK4b in CD34+ cells and during differentiation which can be disrupted by DNA hypermethylation and gene repression. These findings demonstrate the novel insights we can gain by using CRISPR/Cas9 tools to target DNA methylation and these investigations will reveal how gene promoter hypermethylation can impact HSPC function. Furthermore, studying this locus may uncover an important role for DNA hypermethylation in the development of myeloid malignancies, since INK4b is frequently hypermethylated, but rarely mutated, in myeloid dysplastic/proliferative neoplasms and acute myeloid leukemia. Disclosures Gribben: Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation

Master regulatory genes require stable silencing by the polycomb group (PcG) to prevent misexpression during differentiation and development. Some PcG proteins covalently modify histones, which contributes to heritable repression. The role for other effects on chromatin structure is less understood. We characterized the organization of PcG target genes in ESCs and neural progenitors using 5C and super-resolution microscopy. The genomic loci of repressed PcG targets formed discrete, small (20-140 Kb) domains of tight interaction that corresponded to locations bound by canonical polycomb repressive complex 1 (PRC1). These domains changed during differentiation as PRC1 binding changed. Their formation depended upon the Polyhomeotic component of canonical PRC1 and occurred independently of PRC1-catalyzed ubiquitylation. PRC1 domains differ from topologically associating domains in size and boundary characteristics. These domains have the potential to play a key role in transmitting epigenetic silencing of PcG targets by linking PRC1 to formation of a repressive higher-order structure.

Trithorax and Polycomb group-dependent regulation: a tale of opposing activities

Intricate layers of regulation determine the unique gene expression profiles of a given cell and, therefore, underlie the immense phenotypic diversity observed among cell types. Understanding the mechanisms that govern which genes are expressed and which genes are silenced is a fundamental focus in biology. The Polycomb and Trithorax group chromatin proteins play important roles promoting the stable and heritable repression and activation of gene expression, respectively. These proteins, which are conserved across metazoans, modulate post-translational modifications on histone tails and regulate nucleosomal structures. Here, we review recent advances that have shed light on the mechanisms by which these two classes of proteins act to maintain epigenetic memory and allow dynamic switches in gene expression during development.

Transgenerational epigenetics in the germline cycle of Caenorhabditis elegans.

Epigenetic mechanisms create variably stable changes in gene expression through the establishment of heritable states of chromatin architecture. While many epigenetic phenomena are, by definition, heritably passed through cell division during animal and plant development, evidence suggests that ‘epigenetic states’ may also be inherited across multiple generations. Work in the nematode Caenorhabditis elegans has uncovered a number of mechanisms that participate in regulating the transgenerational passage of epigenetic states. These mechanisms include some that establish and maintain heritable epigenetic information in the form of histone modifications, as well as those that filter the epigenetic information that is stably transmitted. The information appears to influence and help guide or regulate gene activity and repression in subsequent generations. Genome surveillance mechanisms guided by small RNAs appear to be involved in identifying and directing heritable repression of genomic elements, and thus may participate in filtering information that is inappropriate for stable transmission. This review will attempt to summarize recent findings that illustrate this simple nematode to be a truly elegant resource for defining emerging biological paradigms.As the cell lineage that links generations, the germline is the carrier of both genetic and epigenetic information. Like genetic information, information in the epigenome can heritably affect gene regulation and phenotype; yet unlike genetic information, the epigenome of the germ lineage is highly modified within each generation. Despite such alterations, some epigenetic information is highly stable across generations, leading to transgenerationally stable phenotypes that are unlinked to genetic changes. Studies in the nematode C. elegans have uncovered mechanisms that contribute to transgenerational repression as well as to the expression of genes that rely on histone modifying machinery and/or non-coding RNA-based mechanisms. These studies indicate that epigenetic mechanisms operating within the germ cell cycle of this organism filter and maintain an epigenetic memory that is required for germ cell function and can also influence gene expression in somatic lineages.

Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Epigenetic silencing of genes in association with aberrant promoter DNA hypermethylation has emerged as a significant mechanism in the development of human cancers. Such genes are also often targets of the polycomb group repressive complexes in embryonic cells. The polycomb repressive complex 2 (PRC2) has been best studied in this regard. We now examine a link between PRC1 and cancer-specific gene silencing. Here, we show a novel and direct association between a constituent of the PRC1 complex, CBX7, with gene repression and promoter DNA hypermethylation of genes frequently silenced in cancer. CBX7 is able to complex with DNA methyltransferase (DNMT) enzymes, leading us to explore a role for CBX7 in maintenance and initiation of gene silencing. Knockdown of CBX7 was unable to relieve suppression of deeply silenced genes in cancer cells; however, in embryonal carcinoma (EC) cells, CBX7 can initiate stable repression of genes that are frequently silenced in adult cancers. Furthermore, we are able to observe assembly of DNMTs at CBX7 target gene promoters. Sustained expression of CBX7 in EC cells confers a growth advantage and resistance to retinoic acid-induced differentiation. In this setting, especially, there is increased promoter DNA hypermethylation for many genes by analysis of specific genes, as well as through epigenomic studies. Our results allow us to propose a potential mechanism through assembly of novel repressive complexes, by which the polycomb component of PRC1 can promote the initiation of epigenetic changes involving abnormal DNA hypermethylation of genes frequently silenced in adult cancers.

Silent chromatin at the middle and ends: lessons from yeasts

Eukaryotic centromeres and telomeres are specialized chromosomal regions that share one common characteristic: their underlying DNA sequences are assembled into heritably repressed chromatin. Silent chromatin in budding and fission yeast is composed of fundamentally divergent proteins tat assemble very different chromatin structures. However, the ultimate behaviour of silent chromatin and the pathways that assemble it seem strikingly similar among Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe) and other eukaryotes. Thus, studies in both yeasts have been instrumental in dissecting the mechanisms that establish and maintain silent chromatin in eukaryotes, contributing substantially to our understanding of epigenetic processes. In this review, we discuss current models for the generation of heterochromatic domains at centromeres and telomeres in the two yeast species.

The MUT9p kinase phosphorylates histone H3 threonine 3 and is necessary for heritable epigenetic silencing in Chlamydomonas

Changes in chromatin organization are emerging as key regulators in nearly every aspect of DNA-templated metabolism in eukaryotes. Histones undergo many, largely reversible, posttranslational modifications that affect chromatin structure. Some modifications, such as trimethylation of histone H3 on Lys 4 (H3K4me3), correlate with transcriptional activation, whereas others, such as methylation of histone H3 on Lys 27 (H3K27me), are associated with silent chromatin. Posttranslational histone modifications may also be involved in the inheritance of chromatin states. Histone phosphorylation has been implicated in a variety of cellular processes but, because of the dynamic nature of this modification, its potential role in long-term gene silencing has remained relatively unexplored. We report here that a Chlamydomonas reinhardtii mutant defective in a Ser/Thr protein kinase (MUT9p), which phosphorylates histones H3 and H2A, shows deficiencies in the heritable repression of transgenes and transposons. Moreover, based on chromatin immunoprecipitation analyses, phosphorylated H3T3 (H3T3ph) and monomethylated H3K4 (H3K4me1) are inversely correlated with di/trimethylated H3K4 and associate preferentially with silenced transcription units. Conversely, the loss of those marks in mutant strains correlates with the transcriptional reactivation of transgenes and transposons. Our results suggest that H3T3ph and H3K4me1 function as reinforcing epigenetic marks for the silencing of euchromatic loci in Chlamydomonas.

Heritable Gene Repression through the Action of a Directed DNA Methyltransferase at a Chromosomal Locus

The ability to exogenously impose targeted epigenetic changes in the genome represents an attractive route for the simulation of genomic de novo epigenetic events characteristic of some diseases and for the study of their downstream effects and also provides a potential therapeutic approach for the heritable repression of selected genes. Here we demonstrate for the first time the ability of zinc finger peptides to deliver DNA cytosine methylation in vivo to a genomic integrated target promoter when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular genomic de novo methylation events and allowing a direct analysis of the mechanics of de novo DNA methylation-mediated gene silencing at a genomic locus. We show that targeted methylation leads to gene silencing via the initiation of a repressive chromatin signature at the targeted genomic locus. This repression is maintained after the loss of targeted methyltransferase enzyme from the cell, confirming epigenetic maintenance purely through the action of cellular enzymes. The inherited DNA methylation pattern is restricted only to targeted sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. As well as demonstrating the potential of these enzymes as tools for the exogenous, heritable control of cellular gene expression, this work also provides the most definitive confirmation to date for a transcriptionally repressive role for de novo DNA methylation in the cell and lends some weight to the hypothesis that the aberrant methylation associated with certain diseases may well be a cause rather than a consequence of transcriptional gene repression.

Directed De Novo DNA Methylation of a Genomic Locus Leads to Heritable Transcriptional Repression.

The ability to impose exogenous targeted epigenetic changes in the genome represents an attractive goal in gene therapy for the heritable repression of target genes, while potentially enabling the generation and subsequent study of the downstream effects of de novo epigenetic events, which are known to occur in disease. Here we demonstrate the ability of zinc-finger peptides to deliver DNA cytosine methylation in vivo to a genomic target promoter, when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular de novo methylation events. We show for the first time targeted gene silencing in response to directed DNA cytosine methylation via initiation of a repressive chromatin signature at a targeted genomic locus, characterised by elevation of histone H3K9Me2 and reduction of H3K4Me3 levels at that region. This transcriptional repression is maintained in the absence of sustained targeted methyltransferase action, confirming epigenetic maintenance by the cells own machinery. The inherited DNA methylation pattern is restricted to specific target sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. Therefore, we demonstrate for the first time, targeted DNA methyltransferases as potential tools for the exogenous and heritable control of gene expression at the chromosomal level, while providing the clearest and most direct confirmation to date of the functional and mechanistic consequences of de novo DNA methylation in the cell. This work represents an important step towards the longer term goal of controlling gene expression through the inheritance of a repressive DNA state, as well as providing a valuable tool for studying spatial and temporal issues associated with ‘genuine' de novo methylation, on transcription and chromatin structure.

The Polycomb group protein EZH2 directly controls DNA methylation: Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, Bernard D, Vanderwinden JM, Bollen M, Esteller M, Di Croce L, de Launoit Y, Fuks F, Free University of Brussels, Faculty of Medicine, Laboratory of Molecular Virology, 808 route de Lennik, 1070 Brussels, Belgium

The establishment and maintenance of epigenetic gene silencing is fundamental to cell determination and function. The essential epigenetic systems involved in heritable repression of gene activity are the Polycomb group (PcG) proteins and the DNA methylation systems. Here we show that the corresponding silencing pathways are mechanistically linked. We find that the PcG protein EZH2 (Enhancer of Zeste homolog 2) interacts-within the context of the Polycomb repressive complexes 2 and 3 (PRC2/3)-with DNA methyltransferases (DNMTs) and associates with DNMT activity in vivo. Chromatin immunoprecipitations indicate that binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, we show by bisulphite genomic sequencing that EZH2 is required for DNA methylation of EZH2-target promoters. Our results suggest that EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems.

The Polycomb group protein EZH2 directly controls DNA methylation

The establishment and maintenance of epigenetic gene silencing is fundamental to cell determination and function. The essential epigenetic systems involved in heritable repression of gene activity are the Polycomb group (PcG) proteins and the DNA methylation systems. Here we show that the corresponding silencing pathways are mechanistically linked. We find that the PcG protein EZH2 (Enhancer of Zeste homolog 2) interacts-within the context of the Polycomb repressive complexes 2 and 3 (PRC2/3)-with DNA methyltransferases (DNMTs) and associates with DNMT activity in vivo. Chromatin immunoprecipitations indicate that binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, we show by bisulphite genomic sequencing that EZH2 is required for DNA methylation of EZH2-target promoters. Our results suggest that EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems.

In Vivo HP1 Targeting Causes Large-Scale Chromatin Condensation and Enhanced Histone Lysine Methylation

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.

Propagation of Silencing: Recruitment and Repression of Naive Chromatin In trans by Polycomb Repressed Chromatin

The Polycomb group (PcG) proteins maintain stable and heritable repression of homeotic genes. Typically, Polycomb response elements (PRE) that direct PcG repression are located at great distances (10s of kb) from the promoters of PcG-repressed genes, and it is not known how these PREs can communicate with promoters over such distances. Using Class II mouse PRC core complexes (mPCCs) assembled from recombinant subunits, we investigated how PcG complexes might bridge distant chromosomal regions. Like native and recombinant Drosophila Class II complexes, mPCC represses chromatin remodeling and transcription. Interestingly, mPCC bound to one polynucleosome template can recruit a second template from solution and renders it refractory to transcription and chromatin remodeling. A Drosophila PRC core complex (dPCC) also is able to recruit a second template. Posterior sex combs (PSC), a subunit of dPCC, inhibits chromatin remodeling and transcription efficiently but requires assembly with dRING1 to recruit chromatin. Thus, repression and template bridging require different subunits of PcG complexes, suggesting that long-range effects may be mechanistically distinct from repression.

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.

RNAi triggered by symmetrically transcribed transgenes in Drosophila melanogaster.

Specific silencing of target genes can be induced in a variety of organisms by providing homologous double-stranded RNA molecules. In vivo, these molecules can be generated either by transcription of sequences having an inverted-repeat (IR) configuration or by simultaneous transcription of sense-antisense strands. Since IR constructs are difficult to prepare and can stimulate genomic rearrangements, we investigated the silencing potential of symmetrically transcribed sequences. We report that Drosophila transgenes whose sense-antisense transcription was driven by two convergent arrays of Gal4-dependent UAS sequences can induce specific, dominant, and heritable repression of target genes. This effect is not dependent on a mechanism based on homology-dependent DNA/DNA interactions, but is directly triggered by transcriptional activation and is accompanied by specific depletion of the endogenous target RNA. Tissue-specific induction of these transgenes restricts the target gene silencing to selected body domains, and spreading phenomena described in other cases of post-transcriptional gene silencing (PTGS) were not observed. In addition to providing an additional tool useful for Drosophila functional genomic analysis, these results add further strength to the view that events of sense-antisense transcription may readily account for some, if not all, PTGS-cosuppression phenomena and can potentially play a relevant role in gene regulation.

RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1.

The products of the Polycomb group (PcG) of genes are necessary for the maintenance of transcriptional repression of a number of important developmental genes, including the homeotic genes. A two-hybrid screen was used to search for putative new members of the PcG of genes in mammals. We have identified a new Zn finger protein, RYBP, which interacts directly with both Ring1 proteins (Ring1A and Ring1B) and with M33, two mutually interacting sets of proteins of the mammalian Polycomb complex. Ring1 binds RYBP and M33 through the same C-terminal domain, whereas the RYBP-M33 interaction takes place through an M33 domain not involved in Ring1 binding. RYBP also interacts directly with YY1, a transcription factor partially related to the product of the Drosophila pleiohomeotic gene. In addition, we show here that RYBP acts as a transcriptional repressor in transiently transfected cells. Finally, RYBP shows a dynamic expression pattern during embryogenesis which initially overlaps partially that of Ring1A in the central nervous system, and later becomes ubiquitous. Taken together, these data suggest that RYBP may play a relevant role in PcG function in mammals.

Isolation and characterization of a novel cDNA clone belonging to polycomb group (PcG) gene

The Polycomb group (PcG) proteins maintain stable and heritable repression of homeotic genes. Typically, Polycomb response elements (PRE) that direct PcG repression are located at great distances (10s of kb) from the promoters of PcG-repressed genes, and it is not known how these PREs can communicate with promoters over such distances. Using Class II mouse PRC core complexes (mPCCs) assembled from recombinant subunits, we investigated how PcG complexes might bridge distant chromosomal regions. Like native and recombinant Drosophila Class II complexes, mPCC represses chromatin remodeling and transcription. Interestingly, mPCC bound to one polynucleosome template can recruit a second template from solution and renders it refractory to transcription and chromatin remodeling. A Drosophila PRC core complex (dPCC) also is able to recruit a second template. Posterior sex combs (PSC), a subunit of dPCC, inhibits chromatin remodeling and transcription efficiently but requires assembly with dRING1 to recruit chromatin. Thus, repression and template bridging require different subunits of PcG complexes, suggesting that long-range effects may be mechanistically distinct from repression.