Nucleosome repeat length and linker histone stoichiometry determine chromatin fiber structure

Abstract

To understand how nuclear processes involving DNA are regulated, knowledge of the determinants of chromatin condensation is required. From recent structural studies it has been concluded that the formation of the 30-nm chromatin fiber does not require the linker histone. Here, by comparing the linker histone-dependent compaction of long, reconstituted nucleosome arrays with different nucleosome repeat lengths (NRLs), 167 and 197 bp, we establish that the compaction behavior is both NRL- and linker histone-dependent. Only the 197-bp NRL array can form 30-nm higher-order chromatin structure. Importantly for understanding the regulation of compaction, this array shows a cooperative linker histone-dependent compaction. The 167-bp NRL array displays a limited linker histone-dependent compaction, resulting in a thinner and topologically different fiber. These observations provide an explanation for the distribution of NRLs found in nature.

Fig. 1.

The linker histone is essential for obtaining a native-like compaction. Sedimentation velocity analysis of the 197-bp NRL array (197 bp × 61) reconstituted without (red triangles) or with (green squares) stoichiometric concentrations of linker histone H5 and folded in increasing concentrations of NaCl and 10 mM TEA, pH 7.4 is shown. For comparison, the sedimentation coefficients (S_20,w) are plotted against those of rat liver chromatin fragments containing a full complement of H1 and an average size of 61.5 nucleosomes (black circles) (24). The raw data from which the sedimentation coefficients were derived (35) are shown in Fig. S2 and are those of the monomer peaks. The sedimentation coefficients for the array folded in 5 mM MgCl₂, 100 mM KCl, and 10 mM NaCl (green diamond) and folded in 1.6 mM MgCl₂ alone (green cross) are also shown.

Fig. 2.

Differential linker histone-dependent binding and compaction for the 167- and 197-bp NRL arrays. (a) Gel electrophoretic analysis of the amount of linker histone H5 bound the 167- and 197-bp NRL arrays. The 167-bp × 80 and 197-bp × 61 nucleosome arrays were reconstituted with histone octamer and increasing concentrations of H5 and fixed in 0.1% (vol/vol) glutaraldehyde. To permit the direct visualization of array-bound linker histone, H5 was labeled with ³²P to trace levels. Analysis was by electrophoresis in native 0.8% agarose gels. (b) Quantification of the amount of H5 bound to the nucleosome arrays in each band in the gel in a. The quantification was by densitometer tracing. Plots for the H5 saturation of the 167-bp NRL arrays (orange triangles) and the 197-bp NRL arrays (green squares) are shown. After H5 saturation, the arrays precipitate (dotted lines). (c) Sedimentation velocity analysis of the 167- and 197-bp NRL arrays reconstituted with histone octamer and increasing concentration of H5 as in a and all samples folded in 1.6 mM MgCl₂ and 20 mM TEA pH 7.4 is shown. The plots show the sedimentation coefficients (S_20,w) for the 167-bp (orange triangles) 197-bp NRL arrays (green squares). The raw data from which the sedimentation coefficients were derived (35) are shown in Fig. S3.

Fig. 3.

The 167- and 197-bp NRL arrays have different linker histone-dependent compaction pathways and different structures. (a and b) For EM analysis, the 167-bp × 80 and 197-bp × 61 nucleosome arrays were reconstituted with different concentrations of linker histone as in Fig. 2 and folded in 1.6 mM MgCl₂. The percentage of linker histone H5 saturation (as derived from Fig. 2b) is indicated above each panel. The particles seen in the background of the EM micrographs are individual nucleosomes resulting from excess histone octamer bound to competitor DNA. (a) EM micrographs showing the folding pathway of the 197-bp NRL array from disordered puddles in [H5] = 0%, to the formation of regular 30-nm chromatin fibers at saturation of H5 binding ([H5] = 100%). (b) EM micrographs showing the folding pathway of the 167-bp NRL arrays from thin ladder-like structures formed by the stacking of nucleosome cores in [H5] = 0%, to the formation of thin, more twisted, fibers at saturation of H5 binding ([H5 = 100%]). (c) Histogram representations of the diameter and mass per unit length for the 197-bp × 61 (green) and 167-bp × 80 (orange) fully folded chromatin fibers saturated with H5. Ninety-nine measurements were taken from EM micrographs for each array. The average diameter and mass per unit length for the 167-bp NRL fibers is 21.3 nm (SD = 3.0) and 6.1 nucleosomes per 11 nm (SD = 0.74), and the average diameter and mass per unit length for the 197-bp NRL fibers is 34.3 nm (SD = 2.7) and 11.2 nucleosomes per 11 nm (SD = 1.0). (Scale bar: 100 nm.)

Fig. 4.

The NRL and the linker histone determine chromatin higher-order structure. (Upper) Without linker histone. (Lower) With linker histone. Selected regions of the EM micrographs shown in Fig. 3 a and b are shown next to schematic representations to illustrate the folding pathway. The schematic representations representing different folding states are taken from Monte Carlo simulations performed by Kepper et al. (37) on long nucleosome arrays to explore their conformation dependence on nucleosome geometry and internucleosomal interactions. (Upper Left) Unfolded 167-bp NRL fiber showing the two-start helix arrangement typified by the stacking of nucleosome cores in the absence of linker histone. This nucleosome arrangement is very similar to that seen by Richmond and colleagues (7) in the crystal structure of the 167-bp NRL array containing four nucleosome cores. (Lower Left) Fully folded 167-bp NRL fiber in the presence of saturating linker histone concentrations. (Upper Right) Unfolded 197-bp NRL array showing the formation of puddles in the absence of linker histone. (Lower Right) Folded 197-bp NRL fibers in the presence of saturating linker histone concentrations. This nucleosome packing is in agreement with the model for the 30-nm chromatin fiber proposed by Rhodes and colleagues (8) for NRLs centered on 197 bp with stoichiometric concentrations of linker histone. (Scale bar: 50 nm.)