Histone H1 binding is inhibited by histone variant H3.3

Abstract

Linker histones are involved in the formation of higher-order chromatin structure and the regulation of specific genes, yet it remains unclear what their principal binding determinants are. We generated a genome-wide high-resolution binding map for linker histone H1 in Drosophila cells, using DamID. H1 binds at similar levels across much of the genome, both in classic euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites for active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes; rather, all regions with low binding of H1 show enrichment of the histone variant H3.3. Knockdown of H3.3 causes H1 levels to increase at these sites, with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1, providing a mechanism to keep diverse genomic sites in an open chromatin conformation.

Figure 1

Characteristics of the H1 binding profile. (A) Profiles of H1 and HP1 (de Wit et al, 2007) at the boundary of pericentric heterochromatin on chromosome 2R. Red bars indicate H1 dips as defined in Materials and Methods section. Hatched bar indicates heterochromatin according to Release 5 of the Drosophila genome (Hoskins et al, 2007). Black and white bars indicate genes on the top and bottom strand, respectively. Profiles are depicted as three-probe running median. (B) H1 and Pc (Tolhuis et al, 2006) across a region of chromosome 2L with Pc domains. Hatched bars indicate Pc domain as annotated in the study by Tolhuis et al (2006). Other details as in (A). (C) DamID binding values of H1, Pc (Tolhuis et al, 2006), and HP1 (de Wit et al, 2007) outside of annotated H1 dips in pericentric heterochromatin (defined as in the study by Hoskins et al (2007) and including the whole of chromosome 4), Pc domains, or euchromatin (remaining parts of the genome). Only probes overlapping with the design regions of the microarray used in the previous studies (Tolhuis et al, 2006; de Wit et al, 2007) were analysed. Lower end, thick line, and upper end of the boxes indicate 1st quartile, median, and 3rd quartile, respectively; whiskers extend to the most extreme data points with a distance to the box at most 1.5 times the box height. (D) Frequency of genes and H1 dips in the same genomic regions as defined in (C). (E), Detail of genomic profiles. Red, H1 DamID; black, RpII18 DamID; grey, nucleosome occupancy (MNase digestion); turquoise, enrichment of H3.3 over H3 in biotin ChIP (Mito et al, 2007). Pale red vertical bars highlight H1 dips. Black and white boxes denote genes on the top and bottom strand, respectively, with introns depicted as lines.

Figure 2

H3.3 enrichment is inversely correlated with H1 binding. Average binding profiles aligned to genomic features. Coloured lines represent running means of 2% of the probes in the window region except where indicated otherwise. Datasets are the same as in Figure 1. (A, B), (A) Transcription start sites and (B) transcript 3′ ends of the 40% most active genes. Expression profile taken from the study by Pickersgill et al (2006). Arrow indicates the location of genes. (C) Centres of tRNA genes. (D) 5′ ends of naturally occurring transposable elements (running mean of 0.5% of depicted probes). Arrow indicates the location of TEs. (E) Binding sites of the trxG protein Zeste in Drosophila embryos (Moses et al, 2006). (F) Co-binding sites of the PcG proteins Enhancer of Zeste and Posterior sex combs in S2 cells (Schwartz et al, 2006).

Figure 3

Intergenic H1 dips are reminiscent of regulatory elements. Alignment of FAIRE data (gold), H1 (red), RpII18 (black), and nucleosome occupancy (grey) to genomic features as in Figure 2. (A) TSSs of the 40% most active genes as measured in the study by Pickersgill et al (2006); (B) Embryonic Z-binding sites (Moses et al, 2006); (C) Co-binding sites of E(Z) and PSC (Schwartz et al, 2006); (D) geometric centres of H1 dips that have no overlap with genes.

Figure 4

Depletion of H3.3 causes increased binding of H1. (A) Quantitative RT–PCR of His3.3A and His3.3B mRNA after knockdown of white or simultaneous knockdown of His3.3A and His3.3B. Average of cumulative mRNA concentration (His3.3A+His3.3B) after knockdown was 46%. (B) Examples of H1 levels at H1 dips on H3.3 knockdown. DamID binding values after knockdown of white (black) and H3.3 (red). Black and white boxes denote genes on top and bottom strand, respectively, with introns depicted as lines. (C) Scatter plots for H1 change (log₂ ratio of DamID values of H3.3 knockdown over white knockdown) versus H1 binding after white knockdown for all microarray probes. Colours are according to H3.3 enrichment (log₂ of H3.3 ChIP over H3.1 ChIP (Mito et al, 2007)) re-sampled to the same resolution with blue⩽−1 and yellow ⩾1.5. Blue and yellow lines are regression lines of the points associated with the lowest 20% and highest 20% H3 enrichment, respectively. (D–F) Alignment of H1 DamID values after white knockdown (black) and H3.3 knockdown (red) as in Figure 2. (D) TSSs of the 40% most active genes; (E) geometric centres of intergenic H1 dips; (F) 5′ ends of transposable elements. Asterisks indicate statistical significance of difference in window ±1 kb (P<4.4 × 10⁻²², Mann–Whitney U-test). (G) Western blots for whole-cell extracts after knockdown. (H) Western blots for supernatants (SN) and pellets (PEL) after sequential extraction using 80 mM and 600 mM salt containing buffers. One-fortieth of the sample was loaded in each lane. —indicates whole cell lysate of untreated cells.

Figure 5

Depletion of H3.3 leads to increased nucleosome repeat length. Mononucleosomal DNA from RNAi-treated cells (Supplementary Figure 5C) was hybridized to tiling microarrays together with in vitro-digested control DNA. Signals were subjected to autocorrelation analysis (see Materials and methods and Supplementary Figure 6) to yield average nucleosome spacing. Probes in HP1-bound regions (de Wit et al, 2007) have been excluded because nucleosome spacing is different in these regions. Dotted lines at multiples of the estimated nucleosome repeat length (NRL) indicate positions of peaks and valleys corresponding to nucleosomes and linker DNA in white (black) and H3.3 (grey) knockdown cells.