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

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

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

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