Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by post-translational modifications is rather limited. In this study, we report on the generation of site-specifically mono-and diacetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification mass spectrometry and show that site-specific acetylation results in overlapping but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD +dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together, our data suggest that site-specific acetylation of H1.2 plays a role in modulating protein−protein interactions.

Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by posttranslational modifications (PTMs) is rather limited. In this study, we report on the generation of site-specifically mono- and di-acetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification-mass spectrometry (AP-MS) and show that site-specific acetylation results in overlapping, but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD+-dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together our data suggests that site-specific acetylation of H1.2 plays a role in modulating protein-protein interactions.