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

Linker histones (LH) have been shown to preferentially bind to AT-rich DNA, and specifically to A-tracts, contiguous stretches of adenines. Recently, using spFRET (single pair Forster/Fluorescence Resonance Energy Transfer), we showed that the globular domain (gH) of Xenopus laevis H1.0b LH orients towards A-tracts present on the linker-DNA (L-DNA) in LH:mononucleosome complexes. We investigated 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 containing three Widom 601 core sequences separated by about 40 bp linkers and A-tracts inserted on either the outer or the inner L-DNAs flanking the 1st and the 3rd 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, surprisingly, the extent of compaction was similar for trinucleosomes with A-tracts either on the two outer or on the two inner L-DNAs. Modeling constrained by the FRET efficiency suggests that the trinucleosomes adopt a zig-zagged conformation with the 1st and 3rd nucleosomes stacked on top of each other. Even though we expect that the gH of neighbouring (1st and 3rd) LHs are oriented towards the A-tracts, our models suggest that they are not sufficiently close to dimerize and affect compaction. Thus, despite differences in A-tract placements, the LH compacts trinucleosomes similarly. HighlightsO_LIDoes the A-tract-mediated orientation of the LH gH affect chromatin structure? C_LIO_LITrinucleosomes with A-tracts on the outer or inner L-DNAs of nucleosomes 1 and 3 were reconstituted C_LIO_LIspFRET-based modeling shows LH compacts trinucleosomes to a zig-zag conformation C_LIO_LIThe A-tract placement does not affect compaction of the LH-bound trinucleosomes. C_LI