Nucleosome positioning on chicken and human globin gene promoters in vitro. Novel mapping techniques

We have developed two new techniques to assess the positions adopted by core histone octamers when reconstituted onto DNA. These, together with a previously described technique, were applied to mapping binding sites on plasmid DNAs containing either the human zeta-globin or chicken beta-globin gene promoters. Each of the approaches enabled the sites occupied by histone octamers to be measured at high resolution and, in qualitative terms, revealed the same pattern of multiple, overlapping sites. Monomer extension, one of the novel techniques, can be used to reveal binding sites over extensive stretches of a single reconstitute (approximately 1000 bp). We found the distribution of histone octamer binding sites to be largely independent of the conditions employed for reconstitution, the topology of the DNA substrate and prolonged incubation under various post-reconstitution conditions. These properties, and features of the binding site maps that we derived, suggest that histone octamer positioning on these DNAs is predominantly a characteristic of the DNA sequence itself and, by implication, that nucleosome-nucleosome interactions and the formation of nucleosome arrays are of minor influence. Some of the techniques provide quantitative information concerning the relative binding strengths of the core histone octamer for different positioning sequences. In this context, it is notable that the majority of potential binding sites compete very poorly for the histone octamer, demonstrating that under the conditions pertinent to our analysis, the range of binding strengths exhibited by the octamer for particular DNA sequences is extensive, and greater than that observed when competitive binding has been studied by methods that do not reflect precise positioning.