H2A.Z acidic patch is incorporated at lower levels at target genes.

<p>ChIP-Seq analysis of H2A.Z in ESCs shows that the divergent acidic patch residues are required for stable incorporation of H2A.Z (A) Density map of H2A.Z^WT (dark blue), H2A.Z^AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) enrichment at all H2A.Z target genes ordered from most H3K27me3 enriched genes to least H3K27me3 enriched genes in ESCs within the region −4 kb to +4 kb relative to the TSS. The right panel representing the expression levels of the corresponding genes in ESCs generated from RNA-Seq data. Red to white gradient represents genes with high to low expression levels respectively. (B) Average enrichment patterns of H2A.Z^WT, H2A.Z^AP3, H3K4me3, H3K27me3, and RNAP2-Ser5P +/−2 kb around the TSS at bivalent (top) and H3K4me3 (H3K27me3 negative) only promoters (bottom). H2A.Z^WT, H2A.Z^AP3, and H3K27me3 are plotted on the primary axis (right). H3K4me3 and RNAP2-Ser5P are plotted on the secondary axis (left). (C) Average read density plots comparing binding profiles of H2A.Z^WT, H2A.Z^AP3, and input at all H2A.Z target gene promoters in ESCs plotted +/−2 kb relative to TSS. The ChIP-Seq datasets for H2A.Z^WT and H2A.Z^AP3 were generated using GFP antibodies against the YFP transgene. (D) Genome profile of ChIP-Seq reads showing the distribution of H2A.Z^WT (dark blue), H2A.Z^AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) across the HoxA locus- a representative set of H2A.Z target genes. (E) Semi-quantitative western blot of H2A.Z^WT and H2A.Z^AP3 chromatin fractions probed with GFP and H3 (load control) using a range of DNA concentrations (top). Graph quantifying the ratio of transgene levels relative to H3 at the indicated DNA concentrations shows ∼1.85 fold more H2A.Z^WT in chromatin fractions compared to H2A.Z^AP3 (bottom). Fold change was calculated from the average ratio of each transgene to H3. Ratios for H2A.Z^WT/H3 (0.439) and H2A.Z^AP3/H3 (0.255) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) for replicate 1 (R1) were used to calculate the 1.72 (0.439/0.255) fold change between H2A.Z^WT and H2A.Z^AP3. Similar results were obtained for an independent replicate (R2). Ratios for H2A.Z^WT/H3 (0.439) and H2A.Z^AP3/H3 (0.219) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) were used to calculate the 2.0 (0.439/0.219) for R2. Thus, the levels of H2A.Z^WT were on average 1.85-fold higher in chromatin-associated fractions relative to H2A.Z^AP3. (F) Graph showing the ratio of SRCAP and RUVBL1 signal to their respective input signal, from co-immunoprecipitation analyses performed in H2A.Z^WT and H2A.Z^AP3 ESCs (in the endogenous H2A.Z knockdown background). Densitometric measurements of the western blots were performed in ImageJ. The standard deviations were generated from triplicates data points. (G) Nuclei isolated from H2A.Z^WT and H2A.Z^AP3 expressing ESCs were subjected to increasing salt concentrations as indicated. Histones were extracted at these salt concentrations and resolved by SDS-PAGE. Histones were detected by immunoblotting with GFP antibodies.