Genome-Wide Analysis of the Chromatin Composition of Histone H2A and H3 Variants in Mouse Embryonic Stem Cells

<div><p>Genome-wide distribution of the majority of H2A and H3 variants (H2A, H2AX, H2AZ, macroH2A, H3.1, H3.2 and H3.3) was simultaneously investigated in mouse embryonic stem cells by chromatin immunoprecipitation sequencing. Around the transcription start site, histone variant distribution differed between genes possessing promoters of high and low CpG density, regardless of their expression levels. In the intergenic regions, regulatory elements were enriched in H2A.Z and H3.3, whereas repeat elements were abundant in H2A and macroH2A, and H3.1, respectively. Analysis of H2A and H3 variant combinations composing nucleosomes revealed that the H2A.Z and H3.3 combinations were present at a higher frequency throughout the genome than the other combinations, suggesting that H2A.Z and H3.3 associate preferentially with each other to comprise the nucleosomes independently of genome region. Finally, we found that chromatin was unstable only in regions where it was enriched in both H2A.Z and H3.3, but strongly quantified stable in regions in which only H3.3 was abundant. Therefore, histone variant composition is an important determinant of chromatin structure, which is associated with specific genomic functions.</p></div

Enrichment of histone variants in repeat elements.

<p>Bars represent the ratios of the number of ChIP reads to that of input reads in repeat elements.</p

Enrichment of histone variants around the TSS with promoters of high and low CpG density.

<p>Line graphs show average enrichment of histone variants around the TSS of HCP & active, HCP & inactive, LCP & active and LCP & inactive genes.</p

Enrichment of histone variants in repeat elements.

<p>Bars represent the ratios of the number of ChIP reads to that of input reads in repeat elements.</p

Enrichment of histone variants in insulator regions.

<p>Profile of the average enrichment of H2A (upper) and H3 (lower) variants in insulator regions defined as CTCF binding sites in genomic regions except for gene body obtained from Chen's ChIPseq data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092689#pone.0092689-Chen1" target="_blank">[35]</a>. The 0 position is the center of the CTCF binding site. CTCF binding sites were ordered based on the amount of CTCF bound; the average enrichment of histone variants in the top (red) and bottom (green) 25% is shown.</p

Enrichment of histone variants in enhancer regions.

<p>Bars represent the ratios (%) of enrichment of the histone H2A (A) and H3 (B) variants ±200 bp from the center of the transcription factor binding site (TFBS) as defined using Chen's TF ChIPseq data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092689#pone.0092689-Chen1" target="_blank">[35]</a> to the average enrichment in 5,000 400-bp regions randomly selected from the whole genome, excluding TFBSs. The dotted line indicates 100%, representing the average value of the enrichment in randomly selected regions.</p

Schematic view of histone variant enrichment in various genome regions.

<p>Relative enrichment of each histone variant in various genome regions is indicated by the ellipse magnitude. Stability (+) and (−) indicate stable and unstable chromatin, respectively. Chromatin was unstable only in regions enriched for both H2A.Z and H3.3.</p

Enrichment of histone variants in active and inactive genes.

<p>Line graphs show the average enrichment (ChIP/input) of each histone variant in active (red) and inactive (dark cyan) genes. TSS and TES are the transcription start and end sites, respectively. The length of genes was normalized. In this analysis, genes of lengths <4 kb were excluded, as overlapping TSS and TES vicinities after length normalization would result in inaccuracies in the data of the gene bodies. In addition, genes whose distance from neighboring genes was <4 kb were also excluded to avoid overlapping of the flanking regions.</p

Preferred combination of H2A and H3 variants in various genome regions.

<p>The genome was divided into 150 nucleotide bins, and the bins in which a particular variant was significantly enriched were determined as described in the Materials and Methods (<i>see </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092689#pone.0092689.s004" target="_blank">Figure S4</a>). The frequency of bins in which two H2A and H3 variants were simultaneously enriched was determined for all combinations of H2A and H3 variants. Bars represent the ratios of the frequencies determined as described above to those of the expected values calculated from the number of bins in which each variant was enriched. Therefore, values >1.0 indicate a preference for that particular H2A and H3 variant combination.</p

H3K4me3 level in H2A.Z- and H3.3-enriched regions.

<p>The genome was divided into 150 nucleotide bins, and the bins in which a particular variant was significantly enriched was determined as described in Materials and Methods (<i>see </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092689#pone.0092689.s004" target="_blank">Figure S4</a>). (A) Boxplot of H3K4me3 levels in H2A-and-H3-variant-enriched bins. (B) Boxplot of H3K4me3 levels in H2A.Z- or H3.3-, and both H2A.Z and H3.3 (H2A.Z/H3.3)-enriched bins. The bottom and top of the box are the 25th and 75th percentile, respectively, and the upper and lower whiskers represent 2.5th and 97.5th percentile, respectively. <i>P</i>-values were calculated using Mann–Whitney U-tests; (*) <i>P</i><0.001.</p