Histone Phosphorylation Combined with Acetylation Dramatically Increase Nucleosome Accessibility

Abstract

Histone post translational modifications (PTMs) regulate DNA transcription, replication and repair. A number of histone PTMs have been identified within the DNA entry-exit region of the nucleosome including phosphorylation of H3 at tyrosine 41 (H3Y41ph) and threonine 45 (H3T45ph). Each of these modifications are implicated to occur with H3K56ac, which is known to increase DNA unwrapping and accessibility. However, the influence of H3Y41ph and H3T45ph on nucleosome dynamics and stability with and without H3K56ac remains undetermined. Micrococcal nuclease digestion, small angle x-ray scattering and fluorescence resonance energy transfer (FRET) measurements reveal that each phosphorylation mimics H3Y41E and H3T45E, and the exact modification, H3Y41ph that was prepared by sequential native ligation, increase nucleosome unwrapping and enhance DNA accessibility to protein binding by three fold. Combinations of either phosphorylation site with H3K56ac increased DNA accessibility multiplicatively, while the combination of the phosphorylation sites did not increase accessibility beyond single phosphorylations. These results show that H3Y41 and H3T45 phosphorylation increase nucleosome unwrapping and accessibility similarly to H3K56 acetylation, and that combinations of PTMs can function multiplicatively to increase DNA accessibility by more than an order of magnitude.