Abstract
The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.
Highlights
Spaced at ϳ200-base pair intervals along the genomic DNA
We wished to determine whether the core histone tail domains contribute to the selection of sequence-dependent nucleosome translational positions and whether alteration of tail-DNA interactions would lead to mobilization and redistribution of nucleosome positions
We observed that the pattern of cross-links within nucleosomes assembled on a 215-bp DNA fragment containing a Xenopus 5S RNA gene was drastically altered upon proteolytic removal of the tail domains, suggesting that the tails contribute to choice of translational positions on this DNA [40]
Summary
Preparation of the Radiolabeled DNA Fragments—A 215-bp DNA fragment containing nucleotides Ϫ78 to ϩ137 of a Xenopus borealis somatic-type 5S RNA gene was obtained from plasmid pXP-10 [37] by digestion with EcoRI (New England Biolabs). Preparation of Tailless Nucleosomes Containing H2A-A12CAPB, H2A-A45C-APB, and H2B-S56C-APB—Five ml of nucleosomes (400 g) reconstituted with wild type H2A, H2AA12C-APB, H2A-A45C-APB, and H2B-S56C-APB were concentrated to 0.5 ml by spin filtration (Millipore, YM-50), treated with 0.04 ml of trypsin cross-linked agarose beads (Sigma) for 15 min at room temperature [42]. Restriction Enzyme Selection for Major Translational Positions—Five ml of nucleosomes containing H2A-A45CAPB reconstituted with the 215-bp 5S DNA fragment were concentrated to 1 ml using centrifugal-based filter (Millipore) as described above, treated with 50 units of BamHI (New England Biolabs) for 15 min at 37 °C. The major translational positions within the H2A-A45C-APB-containing 5S nucleosomes reconstituted onto the 238-bp DNA fragment were enriched by AluI digestion by the above protocol because AluI cleaves the DNA template at nucleotide Ϫ54 (Fig. 1). Determination of Restriction Enzyme Accessibility—The 5S nucleosomes were reconstituted, purified by sucrose gradient,
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