Abstract
Eukaryotic genomes are packaged into linker-oligonucleosome assemblies, providing compaction of genomic DNA and contributing to gene regulation and genome integrity. To define minimal requirements for initial steps in the transition of compact, closed chromatin to a transcriptionally active, open state, we developed a model in vitro system containing a single, unique, "target" nucleosome in the center of a 25-nucleosome array and evaluated the accessibility of the linker DNA adjacent to this target nucleosome. We found that condensation of H1-lacking chromatin results in ∼60-fold reduction in linker DNA accessibility and that mimics of acetylation within all four core histone tail domains of the target nucleosome synergize to increase accessibility ∼3-fold. Notably, stoichiometric binding of histone H1 caused >2 orders of magnitude reduction in accessibility that was marginally diminished by histone acetylation mimics. Remarkably, a nucleosome-free region (NFR) in place of the target nucleosome completely abrogated H1-dependent restriction of linker accessibility in the immediate vicinity of the NFR. Our results suggest that linker DNA is as inaccessible as DNA within the nucleosome core in fully condensed, H1-containing chromatin. They further imply that an unrecognized function of NFRs in gene promoter regions is to locally abrogate the severe restriction of linker DNA accessibility imposed by H1s.
Highlights
Eukaryotic genomes are packaged into linker– oligonucleosome assemblies, providing compaction of genomic DNA and contributing to gene regulation and genome integrity
Our analysis revealed that DraIII sites in the TNuc linker DNA were about 60-fold less accessible in the condensed chromatin compared with the naked DNA template (Table 1), commensurate with previous measurements for 601 arrays [16]
Given that accessibility of the linker DNA is already ϳ60-fold inhibited in such buffers in the absence of H1, these results suggest that the restriction of linker DNA accessibility approaches that found within the nucleosome core region
Summary
To investigate the effects of chromatin remodeling events that initiate opening closed chromatin, we generated 25-nucleo-. Quantitative determinations of the effect of linker histones on DNA accessibility in chromatin are lacking, hampered by the difficulty in precisely controlling the stoichiometry of H1 binding, as slight excesses of H1 can cause aggregation of chromatin samples, whereas undersaturation can lead to drastically different results compared with saturated conditions. We found that the effect of the NFR is largely localized to the vicinity of the TNuc as the remainder of the array exhibits roughly equivalent, inhibited rates of digestion with EcoRV in the presence of H1, regardless of whether the central position was a WT nucleosome or an NFR (Fig. 5B) These results indicate that H1 imposes a dominant constraint on DNA accessibility in native chromatin and suggests that the NFR, which lacks a canonical H1-binding site, locally counteracts H1-mediated condensation. These results are consistent with wholegenome mapping studies, which show that H1 is widely distributed across genomes except for in the immediate vicinity of NFRs of active genes (54 –57)
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