Site-specific effects of acetylation within histone H3 tail peptide on liquid-liquid phase separation with DNA

Post-translational modifications (PTMs) of histone proteins are recognized as critical factors governing the regulation of chromatin condensation through liquid-liquid phase separation (LLPS). Here, we report the impact of acetylation sites within the N-terminal histone H3 peptide on LLPS with nucleosomal linker DNA. In our model system mimicking partial chromatin structures, non-acetylated H3 peptide and DNA undergo LLPS driven primarily by electrostatic interactions. We found that enzymatic acetylation of the H3 peptide significantly inhibits LLPS, and further experiments using synthetic H3 peptides with different acetylation sites revealed that inhibitory effects of acetylation vary markedly depending on the modification site; acetylation near the ends of the 20-residue H3 peptide (i.e., H3K4ac and H3K18ac) inhibits LLPS more than that near the center (i.e., H3K9ac and H3K14ac). The degree of this inhibitory effect correlates with the ability to thermally stabilize double-stranded DNA, and 1,6-hexanediol assays suggest that not only electrostatic interactions but also hydrophobic interactions play an important role in droplet formation when H3 peptide is acetylated. Our findings provide fundamental insights that link LLPS-mediated chromatin condensation to various biological phenomena dependent on acetylation sites within histone proteins.