Journal of Molecular Biology
Volume 275, Issue 3, 23 January 1998, Pages 427-441
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Regular article
Positioning and stability of nucleosomes on MMTV 3′LTR sequences1

https://doi.org/10.1006/jmbi.1997.1464Get rights and content

Abstract

Uniquely positioned nucleosomes were mapped in vitro on mouse mammary tumor 3′ long terminal repeat (MMTV 3′LTR) DNA at base-pair resolution. Nucleosome A assembly was strongly favored over nucleosome B, and heating of each as a mononucleosome caused migration to the ends of the DNA fragment at a unique rate. Taken together with DNA sequence analysis, this suggests why MMTV 3′LTV nucleosome positions reported upstream of vector-derived sequences conflict and also how flanking genomic sequences could modulate the promoter in in vivo situations. Importantly, nucleosomes are shown to migrate for significant distances along DNA under physiologically relevant conditions, and the actual rates have been measured directly in solution. Exact positioning and shifting over greater than 60 bp has important consequences for transcription factor access to this MMTV promoter and for the role of nucleosomes in general.

Introduction

Chromatin structure is an important contributor to eukaryotic gene regulation due to its role in organizing DNA and its consequent effects on the access of transcription factors to promoter sequences Kornberg and Lorch 1995, Varga-Weisz and Becker 1995. The fundamental repeating unit of chromatin is the nucleosome, which consists of the core histone octamer surrounded by a 147 bp DNA superhelix, 20 to 80 bp of linker DNA, and the linker histone H1 Richmond et al 1984, van Holde 1989. Although capable of forming on any DNA sequence, nucleosomes have been shown to assemble preferentially on several different sequences (Simpson & Stafford, 1983), and when associated with promoter elements, they may take on a positive or negative role in transcription activation Ganter et al 1993, McPherson et al 1993. For example, a DNA site displayed on the surface of a positioned nucleosome may take on a conformation which is specially recognized by a protein factor that facilitates activation Imbalzano et al 1994, Li and Wrange 1995. More generally, nucleosomes partially mask DNA and therefore repress transcription. To overcome this negative effect, chromatin-remodeling factors apparently become specifically localized where required for derepression of genes Cote et al 1994, Tsukiyama et al 1994.

The paradigm for chromatin-dependent regulation and remodeling is the mouse mammary tumor virus 3′ long terminal repeat (Godowski, 1993). At the promoter within the 3′ LTR, nucleosome B within a phased array of nucleosomes becomes hypersensitive to DNase I cleavage upon glucocorticoid induction Peterson 1985, Richard-Foy and Hager 1987. At least four hormone receptor binding sites (HREs) are located in the region of nucleosome B, and these DNA elements appear to be responsible for a structural alteration of this nucleosome on glucocorticoid receptor (GR) binding, which allows the protein NF-I to bind to an otherwise occluded site Archer et al 1992, Truss et al 1995. Once bound, NF-I promotes the formation of the pre-initiation complex at the TATA-box located immediately downstream of nucleosome B (Miksicek et al., 1987). During this process, elements of nucleosome B structure are preserved though characteristic DNase I hypersensitive sites also appear Mymryk et al 1995, Truss et al 1995. The precise position of nucleosome B in the promoter is commonly believed to be important because GR binding affinity varies with HRE position in the nucleosome Li and Wrange 1993, Li and Wrange 1995, and because different relative positions of the GR to the histone octamer within the nucleosome could influence subsequent chromatin remodeling. However, despite the obvious importance of nucleosome positioning, investigations using both histone octamer assembled in vitro and at lower resolution using episome constructs in cell lines have yielded a contradictory variety of results whose usefulness remains to be determined Perlmann and Wrange 1988, Pina et al 1990a, Archer et al 1991, Bresnick et al 1992b, Roberts et al 1995, Fragoso et al 1995, Mymryk et al 1995, Truss et al 1995. We have applied a base-pair resolution mapping method (Flaus et al., 1996) in vitro to the two promoter-proximal nucleosomes A and B of the MMTV 3′LTR to study their assembly and shifting on the LTR sequence. The results suggest how the flanking sequences derived from vector DNA in previous cell-line studies may have perturbed the true array of MMTV LTR nucleosomes.

Furthermore, nucleosomes are often viewed as rigid structures under physiological conditions, although rigorous characterization of their position and mobility based on features such as DNA sequence is so far incomplete Thoma and Simpson 1985, Travers and Klug 317. We have investigated the behaviour of the two MMTV 3′LTR nucleosomes and show that they can move along DNA for significant distances under low ionic strength conditions. Importantly, nucleosomes A and B exhibit distinctly different stability which can be related to the DNA sequences that they contain, illustrating a novel potential role for underlying DNA sequence in modulating transcriptional activation.

Section snippets

Results

We have recently developed a true base-pair resolution mapping procedure, utilising bacterially expressed histone octamers with histone H4 modified to carry an EDTA-derived reagent at residue 47 (Flaus et al., 1996). When loaded with Fe3+ and provided with ascorbate as a reductant, the reagent catalyses a site-specific generation of hydroxy radicals from hydrogen peroxide. Because H4 residue 47 is located close to the inner surface of the nucleosomal DNA, the hydroxy radicals cut each DNA

Discussion

Although the MMTV 3′LTR nucleosomes are a paradigm for the role of chromatin in gene regulation, their behavior during gene induction by steroid hormone receptors is not well understood Godowski 1993, Beato et al 1995, Lewin 1994. Serious discrepancies remain between the many reports of nucleosome positions, even though the release of nucleosomal repression is known to depend on the organization of the promoter. We have investigated how DNA sequence influences nucleosome positioning and

DNA fragments

All DNA sequences except AB442tc are derived from MMTV isolate REPRO (EMBL J02274) except that base −204 is G, as in plasmid pM50 kindly provided by Dr G. Hager and used as a PCR template (Archer et al., 1991). Numbering is relative to the transcription initiation site (+1), labeled Watson (W) and Crick (C) for transcribed and non-transcribed strands, respectively. DNA fragments are named by the nucleosome region they include and their length (e.g. 242 bp nucleosome A-inclusive A242). Fragment

Acknowledgements

We thank K. Luger, A. Mäder, T. Rechsteiner and S. Tan for discussions, and together with R. Richmond, for assistance in purifying histones. We also thank Professor F. Thoma for his comments, Drs S. Halford, G. Hager, M. Truss and Professor M. Beato for materials, and Professor C. Weissman for the use of a phosphorimager. This work was supported by the Swiss National Fund for Science.

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