Multistep Chromatin Assembly on Supercoiled Plasmid DNA by Nucleosome Assembly Protein-1 and ATP-utilizing Chromatin Assembly and Remodeling Factor

Takeya Nakagawa,Michael Bulger,Masami Muramatsu,Takashi Ito

doi:10.1074/jbc.m101331200

Abstract

We examine in vitro nucleosome assembly by nucleosome assembly protein-1 (NAP-1) and ATP-utilizing chromatin assembly and remodeling factor (ACF). In contrast to previous studies that used relaxed, circular plasmids as templates, we have found that negatively supercoiled templates reveal the distinct roles of NAP-1 and ACF in histone deposition and the formation of an ordered nucleosomal array. NAP-1 can efficiently deposit histones onto supercoiled plasmids. Furthermore, NAP-1 exhibits a greater affinity for histones H2A-H2B than does naked DNA, but in the presence of H3-H4, H2A-H2B are transferred from NAP-1 to the plasmid templates. These observations underscore the importance of a high affinity between H2A-H2B and NAP-1 for ordered transfer of core histones onto DNA. In addition, recombinant ACF composed of imitation switch and Acf1 can extend closely packed nucleosomes, which suggests that recombinant ACF can mobilize nucleosomes. In the assembly reaction with a supercoiled template, ACF need not be added simultaneously with NAP-1. Regularly spaced nucleosomes are generated even when recombinant ACF is added after core histones are transferred completely onto the DNA. Atomic force microscopy, however, suggests that NAP-1 alone fails to accomplish the formation of fine nucleosomal core particles, which are only formed in the presence of ACF. These results suggest a model for the ordered deposition of histones and the arrangement of nucleosomes during chromatin assembly in vivo.

Highlights

The assembly of genomic DNA and histones into chromatin is a fundamental process of eukaryotes that affects a broad range of biological phenomena, including DNA replication, DNA repair, gene expression, and progression through the cell cycle
Among H3 and H4 chaperones, Drosophila anti-silencing function-1 and chromatin assembly factor-1 were shown to be associated with newly synthesized, acetylated core histones [17, 18]. These core histone chaperones can mediate the deposition of histones onto DNA via an ATP-independent process that yields chromatin consisting of randomly distributed nucleosomes
We demonstrated that the assembly of nucleosomal arrays is a multistep process consisting of ordered core histone transfer by nucleosome assembly protein-1 (NAP-1) and subsequent nucleosome maturation by ACF

Summary

Introduction

The assembly of genomic DNA and histones into chromatin is a fundamental process of eukaryotes that affects a broad range of biological phenomena, including DNA replication, DNA repair, gene expression, and progression through the cell cycle (for reviews, see Refs. 1–7). Among H3 and H4 chaperones, Drosophila anti-silencing function-1 and chromatin assembly factor-1 were shown to be associated with newly synthesized, acetylated core histones [17, 18] To varying extents, these core histone chaperones can mediate the deposition of histones onto DNA via an ATP-independent process that yields chromatin consisting of randomly distributed nucleosomes. A similar distribution of nucleosomes can be accomplished by the method of salt gradient dialysis or by using polyanions, such as polyglutamate or RNA, which can interact with the histones and prevent their nonspecific aggregation with DNA This random deposition process, is generally inefficient and does not yield periodic arrays of nucleosomes that resemble those seen in native chromatin This chromatin assembly reaction, which can be carried out in a purified reconstituted system, requires ACF, core histones, DNA, ATP, and NAP-1

Methods

Results

Conclusion