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

Post-translational modifications (PTMs) of histone proteins play a pivotal role in the regulation of chromatin condensation, with evidence increasingly pointing to the involvement of liquid–liquid phase separation (LLPS). Here, we report the significant impact of the acetylation site of the N-terminal histone H3 peptide on LLPS with nucleosomal-linker DNA. In our model system, which mimics a part of the structure of chromatin, non-acetylated H3 peptide and DNA undergo LLPS, driven primarily by electrostatic interactions. Acetylation of the H3 peptide significantly inhibits LLPS, with the inhibitory effects varying markedly depending on the acetylation site. Specifically, acetylation near the ends of the 20-residue H3 peptide (i.e., H3K4ac and H3K18ac) results in stronger inhibition of LLPS compared to acetylation near the center (i.e., H3K9ac and H3K14ac). Through experiments (circular dichroism and interaction assays using salts and 1,6-hexanediol) and molecular-dynamics (MD) simulations, it was revealed that differences in the DNA thermal stability, hydrophobic effects, and charge distribution of the H3 peptides/DNA complexes underlie this phenomenon. Our findings provide fundamental insights that link LLPS-mediated chromatin condensation to various biological phenomena depending on the acetylation sites on the histone proteins, thus shedding light on the mechanisms of gene regulation.