Mapping the Specificity of the p300 Acetyltransferase to H3 and H4 Histone Variants

In eukaryotic cells, DNA complexes with RNA and histone proteins to form chromatin. Conformationally free peptide chains on the N- and C-termini of these histone proteins can serve as substrates for post-translational modifications such as methylation, phosphorylation, and acetylation. These modifications act combinatorically to regulate chromatin structure and gene expression as an important part of the epigenetic code. Variants of these histone proteins, typically differing in their sequence by only a few amino acids, have been observed to display unique cell-cycle independent and tissue-specific behaviors. The origin and function of these histone variants is not well understood nor is their ability to serve as substrates for regulatory modification. An existing yeast-surface display system was modified to rapidly profile the interaction of the p300 acetyltransferase with a selective lysine knockout library of histone H3 and H4 variants. Following simultaneous expression in Saccharomyces cerevisiae of the acetyltransferase and substrate and subsequent endoplasmic reticulum sequestration, the histone variants were displayed on the surface of the cells. Cells displaying the enzyme expression proxy, a histone tail, and an acetylation at the available lysine site were sorted and profiled by next-generation sequencing to identify the variants and the lysine residues at which acetylation occurred. Site-specific H3 anti-acetylation antibodies were used in addition to a pan-acetyllysine antibody for validation purposes. Distinct populations of acetyllysine-positive cells were observed prior to sorting, hinting at the possibility of variant-dependent acetylation kinetics.