Mitsuhiro Endoh

Genomewide Analysis of PRC1 and PRC2 Occupancy Identifies Two Classes of Bivalent Domains

In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2,000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb-repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation, followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes—the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes, and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes in pluripotent cells.

Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity

The Polycomb group (PcG) proteins mediate heritable silencing of developmental regulators in metazoans, participating in one of two distinct multimeric protein complexes, the Polycomb repressive complexes 1 (PRC1) and 2 (PRC2). Although PRC2 has been shown to share target genes with the core transcription network, including Oct3/4, to maintain embryonic stem (ES) cells, it is still unclear whether PcG proteins and the core transcription network are functionally linked. Here, we identify an essential role for the core PRC1 components Ring1A/B in repressing developmental regulators in mouse ES cells and, thereby, in maintaining ES cell identity. A significant proportion of the PRC1 target genes are also repressed by Oct3/4. We demonstrate that engagement of PRC1 at target genes is Oct3/4-dependent, whereas engagement of Oct3/4 is PRC1-independent. Moreover, upon differentiation induced by Gata6 expression, most of the Ring1A/B target genes are derepressed and the binding of Ring1A/B to their target loci is also decreased. Collectively, these results indicate that Ring1A/B-mediated Polycomb silencing functions downstream of the core transcriptional regulatory circuitry to maintain ES cell identity.

Histone H2A Mono-Ubiquitination Is a Crucial Step to Mediate PRC1-Dependent Repression of Developmental Genes to Maintain ES Cell Identity

Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.

H2A.Z landscapes and dual modifications in pluripotent and multipotent stem cells underlie complex genome regulatory functions

BackgroundThe histone variant H2A.Z has been implicated in nucleosome exchange, transcriptional activation and Polycomb repression. However, the relationships among these seemingly disparate functions remain obscure.ResultsWe mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers, and correlates strongly with H3K4 methylation. Accordingly, H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation, but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z, including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin.ConclusionsOur findings associate H2A.Z with functionally distinct genomic elements, and suggest that post-translational modifications may reconcile its contrasting locations and roles.

The Hbo1-Brd1/Brpf2 complex is responsible for global acetylation of H3K14 and required for fetal liver erythropoiesis

The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.

SCL/tal‐1‐dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation

Hematopoiesis in most vertebrate species occurs in two distinct phases, primitive and definitive, which diverge from FLK1+VE‐cadherin− mesoderm and FLK1+VE‐cadherin+ endothelial cells (EC), respectively. This study aimed at determining the stage at which hematopoietic lineage fate is determined by manipulating the SCL/tal‐1 expression that is known to be essential for the early development of the primitive and definitive hematopoietic systems. We established SCL‐null ES cell lines in which SCL expression is rescued by tamoxifen‐inducible Cre recombinase‐loxP site‐mediated recombination. While no hematopoietic cells (HPC) were detected in SCL‐null ES cell differentiation cultures, SCL gene reactivation from day 2 to day 4 after initiation of differentiation could rescue both primitive and definitive hematopoiesis. SCL reactivation at later phases was ineffective. Moreover, generation of VE‐cadherin+ EC that can give rise to definitive HPC required SCL reactivation prior to VE‐cadherin expression. These results indicated that the competence to become HPC is acquired at the mesodermal stage by a SCL‐dependent process that takes place independently of determination of endothelial fate.

Stem cells primed for action: polycomb repressive complexes restrain the expression of lineage-specific regulators in embryonic stem cells.

Stem cells are characterised by a capacity to self renew and generate progenycapable of differentiating along several defined lineage paths. Embryonic Stem (ES) cellsare derived from the inner cell mass (ICM) of early-stage embryos and can contribute toall tissues of the developing embryo. Discovering how ES cell pluripotency and lineageinduction is achieved is important for understanding normal development and forsuccessfully applying stem cell-based therapies. A series of recent studies have shownthat the chromatin profile of ES cells is unusual and have revealed a critical role for thePolycomb Repressive Complexes (PRCs) in maintaining pluripotency. In human andmouse ES cells many genes that encode transcription factors that are required for lineagespecification bind PRC2 and carry bivalent (or opposing) histone signatures, beingenriched for conventional indicators of active chromatin such as acetylated H3K9 andmethylated H3K4, while lying within domains of repressive trimethylated H3K27.Mutant ES cells that lack H3K27 methylation inappropriately expressed these genesshowing that PRC2 represses lineage-specific gene programs in ES cells. Here wediscuss the implications of these new discoveries and explore the interdependence ofPRC1 and PRC2 in regulating lineage-specific gene expression in ES cells.

Role of SOX17 in hematopoietic development from human embryonic stem cells

To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34(+)CD43(-) endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested, only Sox17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34(+)CD43(+)CD45(-/low) cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34(+)CD43(-) ECs compared with low levels in CD34(+)CD43(+)CD45(-) pre-hematopoietic progenitor cells (pre-HPCs) and CD34(+)CD43(+)CD45(+) HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34(+)CD43(+)CD45(-/low) cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation directly. Depletion of SOX17 in CD34(+)CD43(-) ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs, thereby regulating hematopoietic development from hESCs/iPSCs.

Mammalian Polycomb-Like Pcl2/Mtf2 Is a Novel Regulatory Component of PRC2 That Can Differentially Modulate Polycomb Activity both at the Hox Gene Cluster and at Cdkn2a Genes.

ABSTRACT The Polycomb group of proteins forms at least two distinct complexes designated the Polycomb repressive complex-1 (PRC1) and PRC2. These complexes cooperate to mediate transcriptional repression of their target genes, including the Hox gene cluster and the Cdkn2a genes. Mammalian Polycomb-like gene Pcl2/Mtf2 is expressed as four different isoforms, and the longest one contains a Tudor domain and two plant homeodomain (PHD) fingers. Pcl2 forms a complex with PRC2 and binds to Hox genes in a PRC2-dependent manner. We show that Pcl2 is a functional component of PRC2 and is required for PRC2-mediated Hox repression. Pcl2, however, exhibits a profound synergistic effect on PRC1-mediated Hox repression, which is not accompanied by major alterations in the local trimethylation of histone H3 at lysine 27 (H3K27me3) or PRC1 deposition. Pcl2 therefore functions in collaboration with both PRC2 and PRC1 to repress Hox gene expression during axial development. Paradoxically, in embryonic fibroblasts, Pcl2 is shown to activate the expression of Cdkn2a and promote cellular senescence, presumably by suppressing the catalytic activity of PRC2 locally. Taken together, we show that Pcl2 differentially regulates Polycomb-mediated repression of Hox and Cdkn2a genes. We therefore propose a novel role for Pcl2 to modify functional engagement of PRC2 and PRC1, which could be modulated by sensing cellular circumstances.

RYBP Represses Endogenous Retroviruses and Preimplantation- and Germ Line-Specific Genes in Mouse Embryonic Stem Cells

ABSTRACT Polycomb repressive complexes (PRCs) are important chromatin regulators of embryonic stem (ES) cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B and has been suggested to assist PRC localization to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Using ES cells conditionally deficient in RYBP, we found that RYBP is not required for maintenance of the ES cell state, although mutant cells differentiate abnormally. Genome-wide chromatin association studies showed RYBP binding to promoters of Polycomb targets, although its presence is dispensable for gene repression. We discovered, using Eed-knockout (KO) ES cells, that RYBP binding to promoters was independent of H3K27me3. However, recruiting of PRC1 subunits Ring1B and Mel18 to their targets was not altered in the absence of RYBP. In contrast, we have found that RYBP efficiently represses endogenous retroviruses (murine endogenous retrovirus [MuERV] class) and preimplantation (including zygotic genome activation stage)- and germ line-specific genes. These observations support a selective repressor activity for RYBP that is dispensable for Polycomb function in the ES cell state. Also, they suggest a role for RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.