The first relates to whether all bivalent domains have the same regulatory structure. of Polycomb complexes in pluripotent cells. == Author Summary == Polycomb-group (PcG) proteins play essential functions in the epigenetic regulation of gene expression during development. PcG proteins are repressors that catalyze lysine 27 tri-methylation on histone H3. They are antagonized by trithorax-group proteins that catalyze lysine 4 tri-methylation. Recent studies of ES cells revealed a novel chromatin pattern consisting of overlapping lysine 27 and lysine 4 tri-methylation. Genomic regions with these opposing modifications were termed bivalent domains and proposed to silence developmental regulators while keeping them poised for alternate fates. However, our understanding of PcG regulation and bivalent domains remains limited. For instance, bivalent domains impact over 2,000 promoters with diverse functions, which suggests that they may function in diverse cellular processes. Moreover, the mechanisms that underlie the targeting of PcG complexes to specific genomic regions remain completely unknown. To gain insight into these issues, we used ultra high-throughput sequencing to map PcG complexes and related modifications genomewide in human and mouse ES cells. The data identify two classes of bivalent domains with unique regulatory properties. They also reveal striking associations between genome sequence and chromatin state that suggest a prominent role for the DNA sequence in dictating the genomewide localization of PcG complexes and, consequently, bivalent domains in ES cells. == Introduction == Increasing evidence suggests that Polycomb- (PcG) and trithorax-group (trxG) proteins and associated histone modifications are critical for the plasticity of the pluripotent state, for the dynamic changes in gene expression that accompany ES cell differentiation, and for subsequent maintenance of lineage-specific gene expression programs[1][4]. PcG proteins are transcriptional repressors that function by modulating chromatin structure[2][4]. Fulvestrant R enantiomer They reside in two main complexes, termed Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 contains Ezh2, which catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), as well as Eed and Suz12. PRC1 contains Ring1, an E3 ubiquitin ligase that mono-ubiquitinylates histone H2A at lysine 119 (H2Aub1)[5],[6]. Other PRC1 components include Bmi1, Mel-18, and Cbx family proteins with Fulvestrant R enantiomer affinity for H3K27me3[2],[3]. Interplay between PcG complexes and altered histones has been proposed to mediate stable transcriptional repression[2],[3]. In the prevailing model, PRC2 is usually recruited to specific genomic locations where it catalyzes H3K27me3. The altered histones in turn recruit PRC1, which catalyzes H2Aub1 and thereby impedes RNA polymerase II elongation[7],[8]. PRC1 may also affect PRC2 function through as yet undefined mechanisms[2],[3]. Several groups have combined chromatin immunoprecipitation (ChIP) with microarrays to examine the genomic localizations of individual PcG subunits[9][13]. Lee et al used tiling arrays to map the PRC2 subunit Suz12 in human ES cells, identifying nearly 2000 gene targets. Boyer Fulvestrant R enantiomer et al used promoter arrays to identify 512 genes co-occupied by PRC2 and PRC1 components in mouse ES cells. In both studies, the implicated gene units were highly enriched for developmental transcription factors (TFs), many of which become de-repressed upon ES cell differentiation or in a PRC2-deficient background. Concurrent studies of histone methylation in ES cells led to the unexpected finding that virtually all sites of PcG activity not only carry the repressive H3K27me3 modification, but are also strongly enriched for the activating, trxG-associated H3 lysine 4 tri-methylation (H3K4me3) mark[14],[15]. Genomic regions with the two opposing modifications were termed bivalent domains and proposed to silence developmental regulators while keeping them poised for alternate fates. Upon ES IFNB1 cell differentiation, most bivalent promoters handle to a univalent state. Induced genes become further enriched for H3K4me3 and drop H3K27me3, while many non-induced genes retain H3K27me3 but drop H3K4me3[15],[16]. Despite this progress, our understanding of PcG regulation and bivalent domains remains limited. In the current study we sought to address two outstanding issues. The first relates to whether all bivalent domains have the same regulatory structure. The.