Abstract
We demonstrate that core histones can affect the accessibility of a DNA element positioned outside of the classically defined nucleosome core region. The distance between a well positioned nucleosome and the binding site for the 5 S-specific transcription factor TFIIIA was systematically varied and the relative binding affinity for TFIIIA determined. We found that core histone-DNA interactions attenuate the affinity of TFIIIA for its cognate DNA element by a factor of 50-100-fold even when the critical binding region lies well outside of the classically defined nucleosome core region. These results have implications for the validity of parallels drawn between the accessibility of general nucleases to DNA sequences in chromatin and the activity of actual sequence-specific DNA binding factors.
Highlights
A major question is how transcription occurs within the context of nucleosomes
We examined the extent of influence of core histone-DNA interactions within a single nucleosome core on the binding of the transcription factor, TFIIIA
We show that the entire cognate element for TFIIIA must be moved well beyond the edge of the nucleosome core region to recover high affinity binding by this factor
Summary
Construction of Modified 5 S DNAs—The HpaII–XbaI fragment flanked by asymmetric BstXI linkers from pJHXI-BstXI, which contains the Xenopus borealis somatic 5 S RNA gene [22], was prepared and modified by standard three- and four-primer polymerase chain reaction methods. For the salt dialysis method, nonspecific carrier DNA (ϳ10 g), radiolabeled DNA (ϳ0.25 g), and purified core histones (mass ratio histone/DNA ϳ0.8) were mixed in a total volume of 200 l in 2.0 M NaCl. The sample was dialyzed for at least 1.5 h against several buffers containing 10 mM Tris-HCl, pH 8.0, 10 mM -mercaptoethanol, 1 mM EDTA, and decreasing NaCl concentrations of 1.2, 1.0, 0.8, and 0.6 M. Nucleosomes or (H3/H4) tetramer-DNA complexes were reconstituted as described and digested for 5 min at room temperature with 0.75, 0.4, 0.2, and 0.1 unit of micrococcal nuclease (Worthington). Nucleosome cores reconstituted onto HpaII-XbaI-labeled fragments were treated with hydroxyl radicals as described, and reactions were quenched with the addition of glycerol to a final concentration of 5% [22]. EDTA was omitted from all solutions [24]
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