(A) A schematic illustrating the Rybpfl/fl;Yaf2−/− mouse embryonic stem cell model in which the addition of tamoxifen (OHT) leads to deletion of RYBP. (B) Western blot analysis demonstrating that treatment of Rybpfl/fl;Yaf2−/− cells with tamoxifen for 96 hr results in loss of RYBP protein. The levels of the PRC1 component, RING1B, and the PRC2 component, SUZ12, are unchanged. TATA Box Binding Protein (TBP) is included as a loading control. (C) A genomic snapshot from RYBP ChIP-seq analysis in Rybpfl/fl;Yaf2−/− cells before (UNT) and after 96 hr of tamoxifen treatment (OHT) showing that RYBP occupancy on chromatin is lost. (D) Western blot analysis of H2AK119ub1 in Rybpfl/fl ESCs, compared with Rybpfl/fl;Yaf2−/− cells before (UNT) and after 96 hr tamoxifen treatment (OHT). Western blot with a histone H3-specific antibody is shown as a loading control. (E) A quantitation of western blot analysis for H2AK119ub1 relative to histone H3 in Rybpfl/fl ESCs, compared with RYBP/YAF2 deletion indicating no global reduction in H2AK119ub1. Errors bars indicate standard deviation for three biological replicate experiments. (F) Genome-wide correlation of wildtype H2AK119ub1, RYBP, RING1B and H3K4me3 ChIP-seq using 10 kb windows. H2AK119ub1 ChIP-seq correlates well with RYBP and RING1B genome-wide. (G) A genomic snapshot of calibrated H2AK119ub1 ChIP-seq in the Rybpfl/fl;Yaf2−/− mouse embryonic stem cells before (UNT) and after 96 hr tamoxifen (OHT) treatment at two polycomb-occupied target sites indicating significant reductions in H2AK119ub1 in the absence of RYBP. (H) A metaplot illustrating calibrated H2AK119ub1 ChIP-seq read density at peaks of H2AK119ub1 (n = 2407) before (UNT-solid line) and after (OHT- dotted line) tamoxifen treatment. Removal of RYBP has a broad effect on H2AK119ub1. (I) A scatter plot of normalised read densities for calibrated H2AK119ub1 ChIP-seq illustrates reduced H2AK119ub1 signal at nearly all H2AK119ub1 peaks in OHT-treated Rybpfl/fl;Yaf2−/− cells (OHT) when compared to untreated cells (UNT). Red dots correspond to statistically significant reductions in H2AK119ub1.