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^-/-;Ring1B^fl/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^-/-;Ring1B^fl/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.