H2A.Z nucleosomes enriched over active genes are homotypic

Abstract

Nucleosomes that contain the histone variant H2A.Z are enriched around transcriptional start sites, but the mechanistic basis for this enrichment is unknown. A single octameric nucleosome can contain two H2A.Z histones (homotypic) or one H2A.Z and one canonical H2A (heterotypic). To elucidate the function of H2A.Z, we generated high-resolution maps of homotypic and heterotypic Drosophila H2A.Z (H2Av) nucleosomes. Although homotypic and heterotypic H2A.Z nucleosomes mapped throughout most of the genome, homotypic nucleosomes were enriched and heterotypic nucleosomes were depleted downstream of active promoters and intron-exon junctions. The distribution of homotypic H2A.Z nucleosomes resembled that of classical active chromatin and showed evidence of disruption during transcriptional elongation. Both homotypic H2A.Z nucleosomes and classical active chromatin were depleted downstream of paused polymerases. Our results suggest that H2A.Z enrichment patterns result from intrinsic structural differences between heterotypic and homotypic H2A.Z nucleosomes that follow disruption during transcriptional elongation.

Figure 1

Broad distribution of homotypic and heterotypic H2Av nucleosomes. (a) Western analyses of two replicates shows that sequential affinity-purified heterotypic and homotypic nucleosomes contain the expected composition of tagged histones and that endogenous epitopes are preserved. From the heterotypic purification (Het PD), antibodies (or streptavidin) recognize endogenous epitopes and tags from Biotag-H2A (16.9kDa) and FLAG-H2Av (18kDa). From the homotypic purification (Hom PD), antibodies [anti-H2A (Upstate 07-146), anti-H2Av or anti-FLAG (Sigma, F3165)] or streptavidin recognize endogenous epitopes and tags from Biotag-H2Av (18.5kDa) and FLAG-H2Av (H2Av). Proteins extracted from streptavidin beads with affinity-purified nucleosomes were resolved on 18% (w/v) SDS-PAGE gels and transferred to nitrocellulose. (b) Agarose gel showing that input DNA (1μg) comprises mostly mononucleosomal fragments for two replicates. Asterisks mark input samples that were sequenced. (c) Normalized counts in 10-bp intervals from representative regions of chromosome 3R, showing two biological replicates for all DNA sizes for input, homotypic, and heterotypic sequenced libraries.

Figure 2

Homotypic H2Av nucleosomes are enriched downstream of gene promoters. (a) Histogram showing the length distribution of mapped paired-end reads from the mean of two biological replicates for input, heterotypic, and homotypic H2Av purifications. The size range of binned reads is indicated at the top of the graph by arrows and the corresponding size class is labeled. (b) Average profiles for input, heterotypic, and homotypic H2Av purifications for the six size-class intervals: 55-bp (35–75bp), 90-bp (76–110bp), 130-bp (111–140bp), 147-bp (141–160bp), 170-bp (161–180bp), >180-bp (181–333bp). Genes were divided into quintiles based on expression level for all 10,997 fully annotated genes, aligned at their TSSs and averaged within 10-bp bins. Averaging was truncated at 5′ or 3′ ends of neighboring genes. The y-axis refers to mapped paired-end counts. See also Supplementary Fig. 4 for 3′ ends as well.

Figure 3

Homotypic H2Av nucleosomes are enriched downstream of intron/exon Junctions and are low-salt-soluble. (a) Average profile for homotypic H2Av nucleosomes within the 147-bp size class aligned at intron/exon and exon/intron junctions and split into quintiles of gene expression as in Figure 2. (b) Single FLAG-H2Av pulldown (total H2Av) profile for comparison to a. (c) Average profile for DNA from 80mM soluble nucleosomes. Profiles in (a–c) represent means of biological replicates over 67,630 intron/exon junctions, divided into 10-bp bins. (d–f) Same as (a–c) except aligned around gene 5′ and 3′ ends. The y-axis refers to mapped single-end reads, where the scales were adjusted slightly to facilitate peak comparisons between the panels.

Figure 4

Depletion of homotypic H2Av nucleosomes at genes with stalled RNA Polymerase II. (a) Average profiles for homotypic and heterotypic H2Av and input nucleosomes within the 147-bp size class for 950 genes that have promoter-proximal enrichment of polymerase (stalled, red) and 4061 genes that have polymerase throughout the genes but are not enriched proximal to the promoter (green). (b) Genes with and without stalled Pol II show displacement of homotypic H2Av nucleosomes with increasing expression level. Average profiles for homotypic H2Av nucleosomes within the 147-bp size class show shifting of peaks downstream with increasing expression for the top three quintiles, and relative depletion of the top expression quintile. Similar results are seen for the 950 stalled and the 4061 unstalled genes. Only the +1 position is shown for stalled genes because there are fewer genes and the resolution is lower than for unstalled genes. The y-axis refers to paired-end counts.

Figure 5

Low-salt extracted chromatin reveals distinctive features of RNA Pol II stalling. Single-end sequence reads from DNA extracted from low-salt-soluble nucleosomes were displayed for 950 stalled and 4061 unstalled genes (a). The mapped ends of plus-strand reads were offset by adding 75 bp, and minus-strand reads were offset by subtracting 75 bp (dotted blue lines), which assumes that all reads represent one end or the other of a protected nucleosome core. Read ends were mapped separately for plus strands (green lines) and minus strands (orange lines) without offset, which assumes that all reads were derived from a small protected fragment. Average profiles are shown for low-salt-soluble nucleosomes mapped over genes in the stalled and unstalled classes. The y-axis refers to mapped single-end reads. As a negative control, we also performed the same analysis at intron/exon junctions, but did not observe evidence of enhanced protection at the corresponding position in comparing stalled and unstalled profiles (Supplementary Fig. 10). (b) A biological replicate sample of low-salt extracted chromatin was subjected to limited paired-end sequencing. The 35–55-bp fraction is displayed in the bottom panels for quintiles of expression for genes in the stalled and unstalled classes. For comparison, the 5′ plots from panel (a) is shown in the panels above on the same x-axis scale. The y-axis refers to paired-end counts.

Figure 6

Model for the generation of H2A.Z enrichment patterns. The cartoon depicts nucleosome disruptions that occur upon RNA Polymerase II transit. Pol II disruption causes dimer loss that is less for homotypic H2A.Z than for either heterotypic, or canonical H2A, with homotypic H2A.Z being the most stable nucleosome to disruption. H2A.Z homotypics are formed with multiple rounds of Pol II transit and remain relatively stable to further disruption. Heterotypics form but are relatively unstable to successive rounds of Pol II transit, leading to their depletion relative to homotypics. H2A.Z recruits remodelers which should exhibit the highest activity on homotypic H2A.Z, in support of transcription.