Selective Acetylation Reveals Distinct Roles of Histones H3 and H4 in Nucleosome Dynamics - a FRET Study

Histone tails and their posttranslational modifications play a crucial role in controlling genetic activity through alterations of nucleosome structure. Whether or not histone tails regulate DNA accessibility independently of each other or in a concerted fashion is currently under debate. Here we studied the structure-defining properties of selective histone acetylation and point mutations in the H4 tail in a combined bulk FRET - single molecule FRET assay. Nucleosome unwrapping was monitored by FRET between the linker ends of the DNA, while FRET experiments at an internal DNA site in the H2A/H2B binding region reported on nucleosome disassembly.By analysis of nucleosome unwrapping, structural heterogeneity during salt-induced disassembly and dimer exchange between nucleosomes we show that histones H3 and H4 assume significantly different roles in controlling nucleosome architecture. H4-acetylation opposes destabilization by H3-acetylation and reduces linker DNA unwrapping and dimer exchange at higher ionic strength, whereas its influence on nucleosome structure at physiological salt is minute. We found no increase in unwrapping when H3 and H4 were acetylated simultaneously, which challenges the idea of cooperativeness between tails that was observed for truncated H3 and H4. Our data suggest that the effect of lysine acetylation is not cumulative in nature but shows strong histone specificity. The specific role of the H4 tail was finally probed by comparing the effect of point mutations or acetylation of selective lysine residues at positions 5,8,12 and 16.Regardless of the state of acetylation nucleosomes disassemble via an intermediate state, which is suppressed at higher nucleosome concentration, confirming our proposed model of step-wise disassembly.