Loss of Pcgf5 results in reduction in global H2AK119 levels.

<p>(A) H2AK119ub1 levels in LK cells. LK cells from BM of WT and <i>Pcgf5</i>^<i>Δ/Δ</i> mice were analyzed by Western blotting using anti-H2AK119ub1 and anti-H3K27me3 antibodies at 1-month post deletion of <i>Pcgf5</i>. Levels of H2AK119ub1 and H3K27me3 were normalized to the amount of H2A and H3, respectively, and are indicated relative to wild-type control values at the bottom. The representative data from two independent experiments are presented. (B) Scatter plots showing the correlation of the fold enrichment values against the input signals (ChIP/input) (transcription start site ± 2.0 kb) of H2AK119ub1 and H3K27me3 of RefSeq genes (listed in RefSeq ID) between WT and <i>Pcgf5</i>^<i>Δ/Δ</i> GMPs. The light gray lines represent the boundaries for twofold increase and twofold decrease, respectively. The changes of H2AK119ub1 or H3K27me3 levels in genes upregulated and downregulated more than twofold in <i>Pcgf5</i>^<i>Δ/Δ</i> cells compared with WT cells are indicated in blue and red, respectively. (C) Venn diagram showing the overlap between H2AK119ub1 genes (listed in gene symbol) in GMPs and ES cells. <i>Pcgf5</i>^<i>Δ/Δ</i> _Down genes are also depicted. (D) Box-and-whisker plots showing H2AK119ub1 and H3K27me3 levels in all Refseq genes (All genes) and H2AK119ub1 genes (genes with ≥ 2-fold enrichment of H2AK119ub1 ChIP signals over the input signals at 2.0 kb ± TSSs in WT GMPs) which showed reduction in H2AK119ub1 levels ≥ 2-fold in <i>Pcgf5</i>^<i>Δ/Δ</i> GMPs (<i>Pcgf5</i>^<i>Δ/Δ</i>_Down genes). ***<i>p</i> <0.001 (Student <i>t</i> test). (E) Scatter plots showing the correlation of the fold expression and fold enrichment of H2AK119ub1 ChIP signals in <i>Pcgf5</i>^<i>Δ/Δ</i> GMPs compared with those in WT GMPs. The genes showing reduction in H2AK119ub1 levels greater than 2-fold (below dotted line) are indicated in red dots. The score of correlation coefficient between the fold expression and fold enrichment of H2AK119ub1 ChIP signals defined with Pearson’s correlation and the linear regression are shown. (F) Box-and-whisker plots showing the expression levels of all RefSeq genes, H2AK119ub1 genes, and <i>Pcgf5</i>^<i>Δ/Δ</i>_Down genes in WT and <i>Pcgf5</i>^<i>Δ/Δ</i> GMPs in RPKM. Boxes represent 25–75 percentile ranges. Vertical lines represent 10–90 percentile ranges. Horizontal bars represent medians. n.s., not significant. (G) Venn diagram showing the overlap between <i>Pcgf5</i>^<i>Δ/Δ</i>_Down genes (listed in gene symbol) in GMPs and genes upregulated in expression greater than 2-fold in <i>Pcgf5</i>^<i>Δ/Δ</i> GMPs relative to WT GMPs. (H) ChIP-sequence data of 3xFlag-Pcgf1, 3xFlag-Pcgf5, and H2AK119ub1 in MEL cells. Venn diagram showing the overlap between Pcgf1 targets, Pcgf5 targets, and H2AK119ub1 genes (≥ 2-fold enrichment of ChIP signals over the input signals at 2.0 kb ± TSSs) (listed in gene symbol) in MEL cells.