Figure 6. Brd2 is necessary for gene activation and antagonizes PRC2 recruitment at bivalent genes.

A. mRNA expression upon treatment of H2A.Z.1^WT and H2A.Z.1^K3R3 mESCs with BET inhibitor JQ1 at 100nM or DMSO control for 24hrs. Expression is normalized to H2A.Z.1^WT+DMSO using Tubb5. Error bars represent SE of a triplicate set of experiments. B. mRNA and protein levels after 48hrs of Brd2 RNAi mediated knock-down in both H2A.Z.1^WT and H2A.Z.1^K3R3 mESCs. Expression is normalized to a non-targeting siRNA using Tubb5. Error bars represent SE of a triplicate set of experiments. Immunoblots for Brd2 and Gapdh performed on whole cell lysates following 48 hrs siRNA treatment. C. mRNA expression for H2A.Z.1 bivalent and active target genes after Brd2 depletion. Normalized as above. D. mRNA levels after 48 hrs of Brd4 siRNA mediated depletion in both H2A.Z.1^WT and H2A.Z.1^K3R3 mESCs. Expression is normalized to a non-targeting siRNA using Tubb5. Error bars represent SE of a triplicate set of experiments. E. mRNA expression for H2A.Z.1 bivalent and active target genes after treatment with either a negative control siRNA or Brd4 siRNA. Normalized as above. F. Suz12 ChIP-qPCR after treatment of H2A.Z.1^WT or H2A.Z.1^K3R3 mESCs with either 100nM JQ1 or DMSO. Enrichment values are normalized to two gene desert regions. Error bars represent SE of a triplicate set of experiments. G. Suz12 ChIP-qPCR after treatment of H2A.Z.1^WT or H2A.Z.1^K3R3 mESCs with either Brd2 siRNA or negative control siRNA. Enrichment values calculated as above. H. Model for Brd2 recruitment to bivalent genes. Brd2 is localized to active promoters that lack H2A.Z.1ub (indicated by red circles) whereas Brd2 is largely absent from bivalent genes enriched for mono-ubiquitylated H2A.Z.1. H2A.Z.1^K3R3 incorporation leads to Brd2 recruitment and gene activation, which further antagonizes PRC2 at promoters of developmental genes.