H2A.Z acidic patch is necessary for regulation of bivalent genes during ESC differentiation.

<p>(A) Heat map displaying genes with absolute fold-changes of >1.5 fold at D0 or D3 in H2A.Z^AP3 EBs relative to H2A.Z^WT. Log2-fold changes in expression of these genes were hierarchically clustered using a Euclidean distance metric. Gene Ontology analysis of the clusters was performed using DAVID. (B) Venn diagrams representing overlap of differentially-regulated genes (>1.5 fold) in H2A.Z^AP3 relative to H2A.Z^WT (green) and genes with the bivalent chromatin mark within +/−2 kb of its TSS (blue) at D0 (top) and D3 (bottom). The p values were calculated by hypergeometric tests. (C) Heat map showing changes in transcript levels of a representative subset of genes (including developmental regulators, pluripotency factors, chromatin regulators and house keeping genes) using Nanostring in H2A.Z^WT, H2A.Z^KD, and H2A.Z^AP3 ESCs at D0 and D3 of EB differentiation. The heat map was generated using log counts from each experiment, which were normalized to the geometric mean of the expression of housekeeping genes <i>Gapdh</i>, <i>Tubb5</i>, and <i>Cltc</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003725#pgen.1003725.s008" target="_blank">Table S3</a>). Genes were then normalized across the 6 experiments. Yellow represents genes that are up-regulated 2 fold or over and blue represents genes down-regulated by 2 fold or more. (D) Boxplots representing the median expression levels of H2A.Z^WT, H2A.Z^KD, and H2A.Z^AP3 D0 and D3 EBs were generated using log transformed, normalized gene expression values of all genes represented in the Nanostring probe set (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003725#pgen.1003725.s008" target="_blank">Table S3</a>). P values were calculated by unpaired student t-test. *p<0.008, **p<0.0008, and ***p<0.0002. The false discovery rate for a p value 0.0074 (<0.008) was generated using Monte Carlo simulation and found to be 3.9% from 10,000 random iterations, suggesting a small but significant change in median expression levels between Day 3 H2A.Z^WT and H2A.Z^KD EBs. qPCR was performed on ChIP DNA generated in H2A.Z^WT and H2A.Z^AP3 ESCs using H3K4me3 (E) and H3K27me3 (F) antibodies. (G) Quantitative qPCR was performed on ChIP-DNA enriched for H3.3 and H3.1 (Flag-HA tagged) using a mixture of Flag and HA antibodies in H2A.Z^WT and H2A.Z^AP3 containing HA-Flag tagged H3.3 and H3.1 transgenes independently. The ratio of H3.3 enrichment relative to H3.1 was plotted for active (<i>Zfpm2, NagK, Thra</i>) and bivalent (<i>Hoxa11, Foxa2, Bmp2</i>) H2A.Z target promoters. Relative enrichment ratios were also plotted for control regions- <i>Pou5f1/Oct4</i> promoter, coding regions for <i>Pou5f1</i> (Pou5f1-CR) and <i>Bmp2</i> (Bmp2-CR) and heterochromatin elements (LINE L1, SINE B2). Student's t-test was performed to generate indicated p values. *p<0.05. Error bars represent standard deviations from a triplicate set of experiments. NS indicates p values >0.5.