High-resolution biophysical analysis of the dynamics of nucleosome formation.

Akiko Hatakeyama,Malcolm Buckle,Claude Nogues,Brigitte Hartmann,Andrew Travers

doi:10.1038/srep27337

Abstract

We describe a biophysical approach that enables changes in the structure of DNA to be followed during nucleosome formation in in vitro reconstitution with either the canonical “Widom” sequence or a judiciously mutated sequence. The rapid non-perturbing photochemical analysis presented here provides ‘snapshots’ of the DNA configuration at any given moment in time during nucleosome formation under a very broad range of reaction conditions. Changes in DNA photochemical reactivity upon protein binding are interpreted as being mainly induced by alterations in individual base pair roll angles. The results strengthen the importance of the role of an initial (H3/H4)2 histone tetramer-DNA interaction and highlight the modulation of this early event by the DNA sequence. (H3/H4)2 binding precedes and dictates subsequent H2A/H2B-DNA interactions, which are less affected by the DNA sequence, leading to the final octameric nucleosome. Overall, our results provide a novel, exciting way to investigate those biophysical properties of DNA that constitute a crucial component in nucleosome formation and stabilization.

Highlights

We describe a biophysical approach that enables changes in the structure of DNA to be followed during nucleosome formation in in vitro reconstitution with either the canonical “Widom” sequence or a judiciously mutated sequence
There is much debate concerning the differential contribution of various factors such as sequence, remodellers and transcription factors to nucleosome positioning[1,2] in essence the central question concerns those molecular mechanisms that are involved in nucleosome formation, stabilisation and destabilisation
Our results provide the first dynamic analysis of nucleosome formation that indicates, in agreement with recent data on nucleosome unwrapping[18], that sequence dependent intrinsic properties of DNA strongly impact on nucleosome stability and, on the recruitment of (H3/H4)[2] that is the first stage of nucleosome formation in vitro

Summary

Introduction

We describe a biophysical approach that enables changes in the structure of DNA to be followed during nucleosome formation in in vitro reconstitution with either the canonical “Widom” sequence or a judiciously mutated sequence. The results strengthen the importance of the role of an initial (H3/H4)[2] histone tetramer-DNA interaction and highlight the modulation of this early event by the DNA sequence. DNA condensation may have been initiated by wrapping of DNA around prototype histones using positioning parameters inherent in the DNA sequence. Ground breaking studies to identify SELEX-generated DNA sequences that possessed advantageous parameters for nucleosome formation[7,8] lead to the suggestion of a positional code for nucleosome positioning and paved the way for crystallographic studies on reconstituted nucleosomes that provided remarkable insights in particular into the bound DNA shape[4,9,10] and DNA-histone interface[11]

Methods

Results

Conclusion