Effect of A-tract-mediated linker histone orientation on trinucleosome compaction

Abstract

AbstractLinker histones (LH) have been shown to preferentially bind to AT-rich DNA, particularly A-tracts, contiguous stretches of adenines. Using spFRET (single pair Förster/Fluorescence Resonance Energy Transfer), we recently found that the globular domain (gH) of Xenopus laevis H1.0b LH orients towards A-tracts on the linker-DNA (L-DNA) while binding on-dyad in LH:mononucleosome complexes. Here, we investigate the impact of this A-tract-mediated orientation of the gH on the compaction of higher-order structures by studying trinucleosomes as minimal models for chromatin. Two 600 bp DNA sequences were constructed, each containing three Widom 601 core sequences connected by about 40 bp L-DNA but differing in the positioning of A-tracts on either the outer or the inner L-DNAs flanking the first and the third Widom 601 sequences. The two inner L-DNAs were fluorescently labelled at their midpoints. Trinucleosomes were reconstituted using the doubly labelled 600 bp DNA, core histone octamers and the full-length H1.0b LH. SpFRET was performed for a range of NaCl concentrations. While the LH compacted the trinucleosomes, the extent of compaction was similar for the two DNA sequences. Modeling constrained by the measured FRET efficiency suggests that the trinucleosomes adopt a zig-zagged two-helical start arrangement with the first and third nucleosomes stacked on top of each other, but with the first and third LHs insufficiently close to interact and affect compaction. Thus, despite differences in the positioning of the A-tracts in the sequences studied, LH binding compacts the corresponding trinucleosomes similarly.HighlightsDoes the A-tract-mediated orientation of the gH affect chromatin structure?Trinucleosomes were reconstituted with A-tracts on the outer or inner linker DNAs.spFRET-based modeling shows LH compacts trinucleosomes to a zig-zag conformation.The A-tract placement does not affect compaction of the LH-bound trinucleosomes.