Reassociation of histone H1 with nucleosomes.

P.P Nelson,S.C Albright,J.M Wiseman,W.T Garrard

doi:10.1016/s0021-9258(19)86547-x

Abstract

The role of histone H1 in nucleosome heterogeneity and structure has been studied using a reconstitution procedure. Histone H1 and non-histone proteins are removed selectively from enzymatically fragmented chromatin by Dowex 50W-X2 treatment. The resulting "stripped" chromatin then is reassociated with purified histone H1 using step gradient dialysis. Material reconstituted in this manner was examined by gel electrophoresis, protein cross-linking, and chromatin fingerprinting. The results demonstrate that the histone H1 molecule efficiently binds to nucleosomes with fidelity in an apparent noncooperative manner. Polynucleosomes possess two specific binding sites for histone H1 per histone octamer; the first binding site is of higher affinity than the second. The 160-base pair nuclease digestion barrier and nucleosome electrophoretic class (MIII)n are established upon binding the 1st histone H1 molecule. Upon binding the 2nd histone H1 molecule, polynucleosomes assume a highly compact conformation. The experimental approach introduced here should permit determining whether nucleosomes possess independent specific binding sites for other chromosomal proteins, and should allow reconstitution of the other electrophoretic forms of nucleosomes which we have described previously.

Highlights

Approach-In the present study the role of histone Hl in nucleosome heterogeneity and structure has been studied using a reconstitution procedure. This approach depends on selectively removing histone Hl and non-histone proteins from enzymatically fragmented chromatin under conditions which hopefully prevent nucleosome sliding and interpolynucleosomal core histone exchange
Sliding would be expected to lead to misalignment of the ends of DNA molecules in nucleosome components and a reduction in the sharpness of the 160-base pair nuclease digestion barrier upon histone Hl reassociation and redigestion. In accord with this view, we find a weaker 160-base pair nuclease digestion barrier in enzymatically fragmented chromatin which has been depleted of histone Hl and non-histone protein by prolonged exposure to 0.6 M Na+ ion as a prelude to histone Hl reassociation and redigestion
The results of the present report show that histone Hl reassociation occurs with fidelity when Dowex 5OW-X2stripped chromatin is used as the acceptor

Summary

Introduction

The simplest fundamental particle of chromatin structure, termed the nucleosome core, has been subject to extensive investigation (reviewed in Ref. 2) This component is known to be composed of an octamer of the four smaller histones along with about 140 base pairs of DNA. Besides understanding the structure of this fundamental particle, it is important to consider how this unit is modeled into polynucleosomal arrays Our approach toward this problem has been to develop and utilize two-dimensional electrophoretic methods to fractionate nucleosome components. The precursor nucleosome classes, MI1 to MV, differ from MI in that they contain histone Hl or non-histone proteins (or both) [5, 6] These particles have longer DNA lengths and possess barriers toward nuclease digestion of National Institutes of Health and by a grant from The National. Accessory proteins are believed to be the chemical determinants of heterogeneity in nucleosome spacing, since arrays of different electrophoretic forms of mononucleosomes (or their immediate precursors) exist in chromatin with different DNA repeat lengths [5]

Objectives

Results

Conclusion