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First published online 13 March 2008
doi: 10.1242/dev.014340

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

¹ RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan.
² RIKEN Genomic Sciences Center, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan.
³ Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 406, 1098 SM Amsterdam, The Netherlands.
⁴ RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 6500047, Japan.
⁵ Developmental Epigenetics Group, MRC Clinical Sciences Centre, ICFM, Hammersmith Hospital, DuCane Road, London W12 ONN, UK.
⁶ Centro de Investigaciones Biologicas, Department of Developmental and Cell Biology, Ramiro de Maeztu 9, 28040 Madrid, Spain.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Ring1A/B are required for the maintenance of mouse ES cell identity. (A) Western blot analysis showing the kinetics of Ring1B depletion at 0, 3, 6, 12, 24 and 48 hours after treatment of Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2 ES cells with 4-hydroxy tamoxifen (OHT). Lamin B served as a loading control. (B) Western blot showing Ring1B and mono-ubiquitylated H2A (H2Aub1) depletion 2 days after treatment with OHT in Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2 ES cells. OHT was present in (+) or absent from (-) the ES cell culture medium. Coomassie Brilliant Blue (CBB) staining for histones was used as a loading control. (C) Morphology of conditional Ring1A/B-dKO ES cells. Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2 ES cells were cultured in the absence (-OHT) or presence (+OHT) of OHT, which represent the single Ring1A-KO or Ring1A/B-dKO cells, respectively. At day 2, Ring1A/B-dKO ES cells retain ES-cell-like morphology; however, from day 3-4, Ring1A/B-dKO ES cells begin to lose ES-cell-like morphology. (D) Gene ontology (GO) analysis of genes more than 2-fold derepressed 4 days after OHT treatment of Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2 ES cells. The significance (P-value) of the enrichment of each GO term is indicated for each category of biological process. For details, see Table S1 in the supplementary material. (E) Changes in expression levels of Hoxa9, Hoxb4, Hoxb8, Gata6, Cdx2, Zic1 and T at 2 and 4 days after OHT treatment (+OHT) of Ring1Bfl/fl;Rosa26::CreERT2 or Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2 ES cells as determined by real-time PCR. Expression levels were normalized to an Actb control and are depicted as fold changes relative to the OHT-untreated (-OHT) ES cells. Error bars are based on the s.d. as derived from triplicate PCR reactions. (F) Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2 ES cells were cultured in the absence (-OHT, upper panels) or presence (+OHT, day 4, lower panels) of OHT, and were immunostained with antibodies to Oct3/4 (green) and Gata4 (red). The left-most panels show nuclei stained with Hoechst 33342 (blue); the right-most panels show merged images. Arrowheads indicate differentiated cells that express Gata4 but not Oct3/4. Arrows indicate feeder cells. Scale bars: 200 µm in C; 45 µm in F.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Ring1A/B mediate repression of developmental regulators by inhibiting chromatin remodeling via direct binding. (A) Loss of Ring1B and Phc1 binding to the selected target promoter regions upon depletion of Ring1B in ES cells. Kinetics of local levels of Ring1B binding and Phc1 binding after OHT administration in Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2 ES cells 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 s.d. determined from at least three independent experiments. (B) Quantitative representation of the correlation between Ring1B binding and degree of derepression. Genes bound by Ring1B in their promoter regions in wild-type ES cells were identified by a ChIP-on-chip approach. Fold enrichment values for respective genes were calculated against the input and binned (each bin containing 2.5-fold enrichment). The number of genes in a bin (yellow bar) and the average change in expression from microarray analysis of Ring1B-KO (blue) and Ring1A/B-dKO (red) are indicated. Expression changes were statistically evaluated using Student's t-test under the null hypothesis that derepression was not observed. Significantly (P<0.05) derepressed bins and insignificant bins are indicated by solid and open circles, respectively. For actual values used to derive the graph and a list of Ring1B-bound genes, see Tables S4 and S5, respectively, in the supplementary material. (C) Changes in PRC2 binding and histone modification at Ring1B target loci following Ring1A/B depletion in ES cells. Kinetics of local levels of Eed, histone H3 lysine 27 trimethylation (H3K27me3), lysine 4 trimethylation (H3K4me3), lysine 9/14 acetylation (H3Ac), and non-phosphorylated RNA polymerase II (RNAPII) binding at the selected targets for Ring1B after OHT administration in Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2 ES cells were determined by ChIP and site-specific real-time PCR. The relative amount of immunoprecipitated DNA is depicted as a percentage of input. Error bars represent s.d. determined from at least three independent experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Significant overlap of derepressed genes in Ring1A/B-dKO and Oct3/4-KO ES cells. (A) Scatter diagrams representing the correlation for changes in gene expression between respective KO ES cells. Each dot represents a specific probe. Fold changes of expression given by each probe in each KO against parental ES cells are dotted in the scatter diagram. Pearson's correlation coefficient (r) in each comparison is indicated in each panel. The distribution of dots is approximated by dotted ellipses. We prepared RNA from Oct3/4-KO ES cells 1 day after gene deletion was induced; Ring1A/B-dKO RNA was isolated at 4 days. Diagrams indicate the correlation of gene expression in Eed-KO versus Ring1A/B-dKO (green), Oct3/4-KO versus Ring1AB-dKO (red), Dnmt1-KO versus Ring1A/B-dKO (yellow), and Dnmt1-KO versus Oct3/4-KO (blue). (B) Pearson's correlation of expression changes for probes that belong to specific GO classifications are shown by bars. The same color codes are used as in A. Annotations are indicated above each graph. Error bars represent 95% confidence intervals of correlation coefficients calculated by Z transformation. (C) Graphical representation of correlation of derepressed genes in Oct3/4-KO and Ring1A/B-dKO ES cells in terms of the degree of Ring1B binding. Based on the genome-wide gene expression profiling, we first identified groups of genes more than 2-fold derepressed (red circles) or repressed (blue circles) in Oct3/4-KO ES cells. Average expression changes in Ring1A/B-dKO cells among those genes were plotted according to the degree of Ring1B binding determined by ChIP-chip and compared with the average of total genes (yellow circles). Where average expression changes in respective groups were statistically significant, relative to the total, the circles are solid; the open blue circles indicate that the difference from total genes is not statistically significant. P-values over 5-, 10- and 15-fold enrichment for Ring1B binding are shown. For actual values, see Table S6 in the supplementary material. (D) A significant fraction of the genes repressed by Oct3/4 is bound by Ring1B. The number of genes in each category of the Venn diagram is indicated.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Oct3/4 is required to engage PRC1 and PRC2 at target gene promoters. (A) Changes in expression levels for the selected Ring1B target genes after tetracycline treatment of ZHBTc4 ES cells were determined as described in Fig. 1E. (B) ChIP analysis showing binding of Ring1B and Eed and levels of H3K4me3 at the promoter regions of the selected target genes after tetracycline treatment of ZHBTc4 ES cells. The relative amount of immunoprecipitated DNA is depicted as a percentage of input. Error bars represent s.d. determined from at least three independent experiments. (C) ChIP-on-chip analysis showing the average Ring1B binding to the promoter regions (from -8 kb to +2 kb relative to the transcription start sites) of the target genes before and after conditional deletion of Oct3/4. (D) ChIP analysis showing binding of Oct3/4 at the promoter regions of the selected target genes after OHT treatment of Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2 ES cells. The relative amount of immunoprecipitated DNA is depicted as a percentage of input. Error bars represent s.d. determined from at least three independent experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Possible mechanisms for the reduction in Ring1B binding upon Oct3/4 depletion or differentiation cues. (A) Western blot demonstrating changes in the levels of Oct3/4, Ring1B, Phc1, Eed, Suz12 and H3K27me3 after conditional deletion of Oct3/4 by tetracycline (Tc) treatment of ZHBTc4 ES cells. (B) Changes in gene expression levels for Eed, Suz12, Ezh2, Ring1B, Phc1 and Bmi1 after tetracycline treatment (+Tc) of ZHBTc4 ES cells were determined by real-time PCR, normalized to an Actb control and depicted as fold changes relative to the tetracycline-untreated (-Tc) ES cells. Error bars are based on the s.d. derived from triplicate PCR reactions. (C) Physical interaction of Ring1B and Oct3/4 in wild-type ES cells demonstrated by reciprocal immunoprecipitation/immunoblot analyses. Antibodies used for immunoprecipitation (IP, top) and immunoblotting (IB, side) are indicated. The association between Ring1B and Oct3/4 proteins remains intact in the presence of ethidium bromide (+EtBr), a DNA-intercalating drug that can disassociate proteins from DNA.

View larger version (19K): [in this window] [in a new window]   Fig. 6. The engagement of PRC1 and PRC2 is gradually decreased upon Gata6-mediated differentiation of ES cells into primitive endoderm lineages. (A) Western blot analysis demonstrating changes in the levels of Oct3/4, Ring1B, Phc1, Ezh2, Eed, Suz12 and H3K27me3 after conditional activation of Gata6 by dexamethasone (Dex) treatment of G6GR ES cells. Lamin B and CBB staining confirmed equal loading. (B) Significant overlap of expression profiles in Ring1A/B-dKO (day 4), Oct3/4-KO (day 1) and Gata6-differentiated (day 2) ES cells. Pearson's correlation of expression changes for probes that belong to specific GO classifications are shown by bars. Functional groupings are indicated above each graph. Error bars represent 95% confidence intervals of correlation coefficients calculated by Z transformation. (C) ChIP analysis showing binding of Ring1B, Phc1 and Eed at the promoter regions of the selected target genes after Dex treatment in G6GR ES cells. The relative amount of immunoprecipitated DNA is depicted as a percentage of input. Error bars represent s.d. determined from at least three independent experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 7. Schematic representation of the interaction between Ring1A/B and the core transcriptional regulatory circuitry in mouse ES cells. Ring1A/B-mediated PcG silencing functions downstream of the core transcriptional regulatory circuitry including Oct3/4. Developmental cues, such as Gata6 activation, downregulate the activity of the core transcriptional regulatory circuitry, which accompanies a global decrease in PcG silencing.

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