A nucleosome-free region locally abrogates histone H1-dependent restriction of linker DNA accessibility in chromatin

Abstract

Eukaryotic genomes are packaged into linker-oligonucleosome assemblies, providing compaction of genomic DNA and contributing to gene regulation and genome integrity. To define minimal requirements for initial steps in the transition of compact, closed chromatin to a transcriptionally active, open state, we developed a model in vitro system containing a single, unique, "target" nucleosome in the center of a 25-nucleosome array and evaluated the accessibility of the linker DNA adjacent to this target nucleosome. We found that condensation of H1-lacking chromatin results in ∼60-fold reduction in linker DNA accessibility and that mimics of acetylation within all four core histone tail domains of the target nucleosome synergize to increase accessibility ∼3-fold. Notably, stoichiometric binding of histone H1 caused >2 orders of magnitude reduction in accessibility that was marginally diminished by histone acetylation mimics. Remarkably, a nucleosome-free region (NFR) in place of the target nucleosome completely abrogated H1-dependent restriction of linker accessibility in the immediate vicinity of the NFR. Our results suggest that linker DNA is as inaccessible as DNA within the nucleosome core in fully condensed, H1-containing chromatin. They further imply that an unrecognized function of NFRs in gene promoter regions is to locally abrogate the severe restriction of linker DNA accessibility imposed by H1s.

Keywords: chromatin; chromatin regulation; chromatin structure; epigenetics; gene regulation; histone; histone H1; histone acetylation; linker histone; nucleosome; nucleosome free region.

Figure 1.

Generation and analysis of 25-nucleosome arrays. A, the T_Nuc and 12-mer arrays are independently reconstituted and then ligated via directional DraIII half-sites (arrowheads) to generate 25-mer arrays in which the T_Nuc is flanked by linker regions containing the DraIII sites (red arrows). REA, restriction enzyme accessibility. B, representative self-association assay showing that arrays remain saturated with nucleosomes after ligation. Arrays were incubated with increasing MgCl₂, then samples were centrifuged, and supernatants were loaded on SDS-agarose gels (see “Experimental procedures”). Loss of solubility in the range of ∼3 mm MgCl₂ indicates saturation of the array. C, example of quantitative DraIII digestion assay. Arrays were mixed with an equal mass of a naked control DNA containing a single DraIII site and then digested, and the products were analyzed on SDS-agarose gels followed by ethidium bromide staining. Bands corresponding to the 5.3-kb 25-mer array template, the 4.5-kb naked DNA control, and residual 12/13-mer arrays and other digestion products (bracket) are indicated. D, plot of digestion data quantified as described under “Experimental procedures.” Lines represent linear regression fits with R² indicated. k_rel was calculated with correction for inherent rate of digestion of chromatin template versus naked DNA control (see “Experimental procedures”). The small fraction of the chromatin that was digested with naked DNA kinetics (typically 10–20%) was excluded from the fits.

Figure 2.

Acetylation mimics in all core histone tail domains increase accessibility of central target nucleosome linker DNA within a condensed 25-mer array. A, location of Lys → Gln substitutions as acetylation mimics (ac^m) in each of the core histone N-terminal tail domains (blue lines). B, representative SDS-agarose gels showing the DraIII digestion time course for 25-mer arrays with T_Nuc containing the indicated modified histones. Digestions were carried out for the times indicated above the lanes, and then products were separated on 0.7% SDS-agarose gels and stained with ethidium bromide. Gels were quantified, and rates of digestions were determined as described under “Experimental procedures.” The relative rate of digestion for each construct, normalized to the WT T_Nuc, is shown below the gel. See also Table 1. Bands corresponding to the 5.3-kb 25-mer array template (array) and the 4.5-kb naked control DNA (DNA) are indicated.

Figure 3.

Stoichiometric binding of H1 drastically decreases linker DNA accessibility. A, deposition of H1 onto nucleosome arrays via Nap1. Arrays were incubated with increasing amounts of H1–Nap1 complex, and binding was analyzed on a native agarose gel stained with ethidium bromide. The white lines indicate a species with a distinct mobility that arises over a range of H1–Nap1:nucleosome ratios (numbers above the lanes) consistent with one H1 bound to each nucleosome (white lines). B, self-association assay showing H1-bound arrays are soluble in 0.5 mm Mg²⁺. Nucleosome arrays were incubated as in A in H1 binding buffer (50 mm KCl) and 0.5 mm Mg²⁺ and centrifuged, and the DNA content of the supernatant was assessed on SDS-agarose gels. C, stoichiometric binding of H1 drastically reduces accessibility of T_Nuc linker DNA. 25-mer arrays containing a WT nucleosome at the central (T_Nuc) position were digested with DraIII either in the absence or presence of a 1.3:1 ratio of H1–Nap1:nucleosome. Digestions were carried out in buffer containing 0.5 mm Mg²⁺. Note that chromatin in the absence or presence of H1 was digested with either 10 or 100 units of DraIII, respectively. Digestion rates were determined as described, and relative rates were calculated and adjusted for amount of enzyme in each reaction. D, as in C except that the central nucleosome contained histones with acetylation mimics in all four histone proteins (All-ac^m). E, data from C and D were plotted as described in the text. The relative digestion rates in the absence/presence of H1 for this experiment are shown. See also Table 1. F, rates of DraIII digestion were determined for WT and All-ac^m arrays in the absence or presence of increasing H1–Nap1. Plotted is the rate of digestion normalized to the rate in the absence of H1 (100%).

Figure 4.

The NFR overrides the H1-dependent decrease in linker DNA accessibility. A, digests of nucleosome arrays containing WT T_Nuc in 10 mm MgCl₂ in the absence and presence of H1. Units of DraIII used in each digestion are indicated above the gels. B, plot of data taken from gel shown in A. C and D, as in A and B except arrays contained an NFR in place of the T_Nuc.

Figure 5.

The effect of the NFR on H1-dependent restriction of linker DNA accessibility does not extend to the entire array. A, schematic showing the 25-mer array, EcoRV sites (vertical arrows) and the T_Nuc (center; shaded). B, 25-mer arrays containing either a WT nucleosome or an NFR at the central (T_Nuc) position were incubated with or without H1 in buffer containing 10 mm Mg²⁺ as indicated and digested with EcoRV (2 units) for the indicated times, and then products were separated on SDS-agarose gels.

Figure 6.

Factors affecting linker DNA accessibility. A pioneer factor (red) binds the target nucleosome (orange) in closed chromatin and recruits histone acetyltransferases (1), resulting in ∼3–4-fold increase in accessibility to the linker DNA (2). The acetylation and increased accessibility allow the recruitment of additional factors, resulting in nucleosome (Nuc) and H1 displacement (3) and a ∼200-fold increase in accessibility to the DNA. Note that some pioneer factors displace H1 directly, resulting in accessible nucleosomes (68). Note that the NFR was still digested 4 times slower than naked DNA or a nucleosome ligated to two naked 25-mer templates, indicating that the folding of the remainder of the nucleosome arrays still provides significant impediment (21).