30 nm chromatin fibre decompaction requires both H4-K16 acetylation and linker histone eviction

Abstract

The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concentration of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.

Figure 1. Recombinant histone octamers containing different N-terminal tail deletions

(a) Schematic representation of N-terminal histone octamer tail deletion. The position of the truncation point in each histone N-terminal tail is indicated with an arrow and the number of the residue. (b) SDS-PAGE analysis of the histone composition of histone octamers containing multiple histone octamer deletions. Purified chicken erythrocyte (CE) (Lane 1) and WT recombinant (Lane 2) histone octamers are shown for reference. Tailless histones are indicated above the lanes (Lanes 3–5) by the prefix “g” which refers to the globular nature of the resulting histones. (c) SDS-PAGE analysis of the histone composition of histone octamers containing single histone tail deletions (Lanes 2–3).

Figure 2. The H4 N-terminal tail is the most important in nucleosome array compaction

(a) Deletion of the H3-H4 tails results in partial inhibition of compaction. Electrophoretic analysis in native 0.9% agarose of the compaction state of 202bp × 61 nucleosome arrays fully reconstituted with histone octamers containing combinations of N-terminal tail deletions as indicated above the lanes: Chicken erythrocyte (CE); wild type recombinant (WT); H3-H4 tailless (gH3gH4); H2A-H2B-tailless (gH2AgH2B); and completely tailless (gAll) histone octamers. The presence of the linker histone H5 is indicated (+). Arrays were folded in 1.6mM Mg₂Cl and 20mM TEA pH7.4 and analysed in both the unfixed (−) or fixed state (+). Fixation was by 0.1% (v/v) glutaraldehyde. The migration in the gel reflects the compaction state of the nucleosome array: the faster migration the more compact the fibre. (b) Deletion of the H4 tail has the most crucial effect on compaction. Electrophoretic analysis in native 0.9% agarose of the compaction state of 202bp × 61 nucleosome arrays fully reconstituted with different histone octamers as indicated above the lanes: tailless H3 (gH3) and tailless H4 (gH4). The presence of the linker histone H5 is indicated (+). The nucleosome arrays were reconstituted, folded and fixed as in (a).

Figure 3. Deletion of the H4 N-terminal leads to decompaction of the `30nm' chromatin fibre

Electron micrographs showing the effects of different N-terminal histone octamer tail deletions on the compaction state of the chromatin fibres. The electron micrographs of the 202bp × 61 nucleosome arrays with combinations of N-terminal tail deletions analysed by gel electrophoresis in Figure 2. Nucleosome arrays were folded as described in Figure 2b and fixed gently in 0.1% (v/v) glutaraldehyde and stained with 2% (w/v) uranyl acetate. In the background, surrounding the folded chromatin fibres, individual nucleosomes formed from excess histones and competitor DNA are seen. For each nucleosome array three representative examples are shown. The bar corresponds to 100nm.

Figure 4. Partial acetylation of H4-K16 disrupts the `30nm' chromatin fibre

(a) Electrophoretic analysis of the effect of 30% H4-K16Ac on the compaction state of the `30nm' chromatin fibre. The 202bp × 61 DNA arrays were reconstituted with wild type recombinant histone octamer (WT), 30% acetylated H4-K16Ac histone octamer or the histone octamer containing the H4-K16Q mutation. The presence of the linker histone H5 is indicated (+). Arrays were folded in 1.6mM Mg<sub>2</sub>Cl and 20mM TEA pH7.4 and analysed in both their unfixed (−) or fixed state (+). Fixation was by 0.1% (v/v) glutaraldehyde. (b) EM analysis of the effect of 30% H4-K16Ac on the compaction state of the `30nm' chromatin fibre. Nucleosome arrays were folded as described in (a) and fixed gently in 0.1% (v/v) glutaraldehyde and stained with 2% (w/v) uranyl acetate. The electron micrographs show four representative examples of the WT and H4-K16Ac chromatin fibres as indicated. In the background, surrounding the folded chromatin fibres, individual nucleosomes formed from excess histones and competitor DNA are seen. The bar corresponds to 100nm.

Figure 5. `30nm' chromatin fibre compaction is additive involving both histone tails and the linker histone: a unique role for H4-K16Ac in fibre decompaction

Summary of the sedimentation coefficients for the 202bp × 61 nucleosome arrays acetylated at K16 of H4 as well as different combinations of histone octamer N-terminal tail deletions folded in the presence (green histograms) and absence (red histograms) of the linker histone H5. Reconstituted arrays were folded side-by-side in the same folding buffer containing 1.6mM Mg²⁺ and the sedimentation analysis was performed without the prior fixation of the nucleosome arrays. The composition of the different nucleosome arrays is indicated below the histograms. The presence of the linker histone H5 is indicated (+) as well as whether the nucleosome arrays were folded in MgCl₂ (+) or not (−). The error bar represents the 95% confidence interval in the fit of to the g(s*) distribution, indicating the high homogeneity of the individual samples.