Spontaneous Access to DNA Target Sites in Folded Chromatin Fibers

Abstract

DNA wrapped in nucleosomes is sterically occluded from many protein complexes that must act on it; how such complexes gain access to nucleosomal DNA is not known. In vitro studies on isolated nucleosomes show that they undergo spontaneous partial unwrapping conformational transitions, which make the wrapped nucleosomal DNA transiently accessible. Thus, site exposure might provide a general mechanism allowing access of protein complexes to nucleosomal DNA. However, existing quantitative analyses of site exposure focused on single nucleosomes, while the presence of neighbor nucleosomes and concomitant chromatin folding might significantly influence site exposure. In this work, we carried out quantitative studies on the accessibility of nucleosomal DNA in homogeneous nucleosome arrays. Two striking findings emerged. Organization into chromatin fibers changes the accessibility of nucleosomal DNA only modestly, from approximately 3-fold decreases to approximately 8-fold increases in accessibility. This means that nucleosome arrays are intrinsically dynamic and accessible even when they are visibly condensed. In contrast, chromatin folding decreases the accessibility of linker DNA by as much as approximately 50-fold. Thus, nucleosome positioning dramatically influences the accessibility of target sites located inside nucleosomes, while chromatin folding dramatically regulates access to target sites in linker DNA.

The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

“…Most important are the E-o-o-S fragments showing the presence of two nucleosomes. Because they are flanked with both EcoRI and SspI sites, the short 548-bp DNA fragment clearly contains two nucleosomes the restriction enzyme when the DNA is positioned on core histones (26).…”

“…The histones used here came from chicken red blood cells, which are transcriptionally inactive and whose histones are minimally acetylated (33). Recent in vitro analysis of the accessibility of restriction enzymes to chromatin would suggest, however, that acetylation alone would have only a modest effect (26).…”

“…5), two positioned nucleosomes must have been constantly present. As shown by Poirier et al (26), nucleosomes are resistant to cleavage with certain restriction enzymes contained within their sequences. Thus, plasmids cut by BamHI will not be sampled in our experiments because only intact circular plasmids are able to provide ampicillin or kanamycin resistance to E. coli (unpublished data).…”

“…1 B, presumably because in those tests 5× less BamHI and a 4.5× shorter time of incubation with the restriction enzymes were used. During prolonged incubation at 37°C, nucleosome "breathing" allows access to restriction enzymes (26). For mutation analysis of nucleo- Figure 5.…”

“…It is thus important to determine whether AID can deaminate cytosines in nucleosomal DNA. To answer this question, we have created a plasmid, pKMP2, with two 147-bp nucleosome positioning sequences, MP2 (26), placed between a T7 promoter and the start of an ampicillin resistance (Amp r ) gene (Fig. 1 A).…”