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

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▿

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

A schematic summary of this study demonstrating that PRC1-dependent repression of developmental genes in ES cells is mediated by multiple effector mechanisms.

<p>A schematic summary of this study demonstrating that PRC1-dependent repression of developmental genes in ES cells is mediated by multiple effector mechanisms.</p

H2A ubiquitination activity of Ring1B is essential for the maintenance of ESC identity and repression of target gene expression.

<p>(A) Morphology of OHT-untreated and –treated (day 8) <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines expressing the indicated transgene. The images were acquired under a phase-contrast microscope. Scale bars indicate 200 µm. (B) Histograms showing the expression changes of H2AK119u1+ and H2AK119u1− genes in <i>Ring1A^−/−; Ring1B^fl/fl; Rosa26::CreERT2</i> ESCs expressing mock (blue line), WT Ring1B (red line), or mutant Ring1B (green dotted line) following OHT treatment. (C) Expression levels of <i>Hoxa9</i>, <i>Hoxb13</i>, <i>Hoxd11</i>, <i>Zic1</i>, <i>Pax3</i> and <i>Pou5f1</i> in <i>Ring1A^−/−; Ring1B^fl/fl; Rosa26::CreERT2</i> ESCs expressing mock, WT, I53S, or I53A Ring1B before (−) or after (+) OHT treatment (day 2). Expression levels were normalized to a <i>Gapdh</i> control and are depicted as fold changes relative to mock (OHT-untreated) ESCs. Error bars represent standard deviation determined from at least three independent experiments. (D) Local levels of trimethylated H3K4 (H3K4me3) at promoter regions of representative target genes in <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESCs stably expressing mock, WT, I53S, or I53A Ring1B before (−) or after (+) OHT treatment (day 2) were determined by ChIP and site-specific real-time PCR. The relative amount of immunoprecipitated DNA is depicted as a percentage of input DNA. Error bars represent standard deviation determined from at least three independent experiments. (E) As in (D), but showing local levels of RNA polymerase II (RNAP) detected with the 8WG16 antibody.</p

Generation of ESCs expressing catalytically inactive Ring1B.

<p>(A) Schematic representation of 3xFlag-tagged Ring1B, showing wild-type and point-mutant derivatives. Each of these construct was stably transfected into <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESCs. (B) Immunoblot analysis of Ring1A, Ring1B, Flag, H2AK119u1 and Lamin B protein levels in whole cell lysates of <i>wild-type</i> and <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines expressing mock, WT, I53S, or I53A Ring1B with or without OHT treatment (OHT+ and −, respectively). (C) Immunoprecipitation (IP) analysis showing the association of exogenous Ring1B WT, I53S or I53A with an endogenous PRC1 component Mel18. Extracts of OHT-untreated (−) and -treated [(+); day 2] <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines expressing each of the constructs were immunoprecipitated with anti-Flag antibody. Resulting precipitates (IP) and lysates (Input) were immunoblotted with antibodies against Flag, Ring1B and Mel18. (D) Association of Flag-tagged proteins in <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines stably expressing mock, Flag-tagged Ring1B WT, I53S, or I53A with promoter regions of their representative target genes before (−) or after (+) OHT treatment (day 2) as determined by ChIP and site-specific real-time PCR. Error bars represent standard deviations determined from three independent experiments. (E) 3D FISH with probe pairs at <i>Hoxb</i> locus (<i>Hoxb1</i> and <i>Hoxb13</i>) in PFA-fixed nuclei of <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines stably expressing mock, WT, I53S, or I53A Ring1B before (−) or after (+) OHT treatment (day 2). Scale bars indicate 1 µm. The boxes show the median and interquartile range of interprobe distances (µm) in the indicated cells. Open circles indicate outliers. The statistical significance of differences between the indicated two data was examined by the Mann-Whitney U test.</p

Global mapping of Ring1B-dependent H2AK119u1 deposition in ESCs reveals that genes occupied by H2AK119u1 represent central targets of PRC1.

<p>(A) ChIP-on-chip analysis showing the average of H2AK119u1 distributions at the promoter regions (from −5 kb to +5 kb relative to TSS) of Ring1B-bound and –unbound genes in <i>Ring1A^−/−</i> (OHT−: green line) and <i>Ring1A/B</i>-dKO (OHT+: red line) ESCs. Enrichment of H2AK119u1 (obtained by E6C5 mAb) and H2A is expressed relative to input DNA, and H2AK119u1 is normalized to H2A. (B) Venn diagram representing the overlap among genes occupied by Ring1B, H2AK119u1 and H3K27me3. Numbers in parentheses represent the total number of genes occupied by each one. (C) Graphic representation of expression changes induced by <i>Ring1B</i> depletion (2 days after OHT treatment) for each subset of genes classified by the presence (+) or absence (−) of Ring1B, H2AK119u1 and H3K27me3 is shown. The average, deviation and distribution of the expression changes for the respective subsets of genes determined by microarray analysis are shown. The 95% Confidence interval (CI) and standard deviation (SD) for the average value of the expression change are indicated. Significant (<i>P</i><0.001) and insignificant (<i>P</i>≥0.01) expression changes were determined by the Student's <i>t</i>-test and are indicated in orange and grey, respectively. <i>P</i>-values for the difference of expression changes between the indicated 2 groups are calculated by the Student's <i>t</i>-test and are indicated above each graph.</p

Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells

SummaryPolycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation