Investigating the Residue Specificity of p300 in Human Histone Peptides

Within human cells, DNA is organized into chromatin, consisting of DNA, RNA, and associated proteins. The basic subunit of chromatin, a nucleosome, is an octamer of histone proteins wrapped in double stranded DNA. Post translational modifications (PTMs), i.e. the addition of chemical groups to a protein after it has been synthesized by the cell, have been shown to alter histone properties. Specifically, PTMs in the tails of histone proteins can affect the degree to which DNA is compacted, and thus its accessibility for transcription and gene expression. Acetylation in particular is associated with increased gene expression. The acetyltransferase p300 is known to acetylate lysine residues on many proteins, including histones. Here we focus on characterization of the amino acid sequence motifs that are acetylated by p300.

We constructed a library of histone H3 mutants to identify the impact that the amino acid sequences around a lysine residue have on histone acetylation patterns. The first forty-five amino acids of histone H3, containing seven distinct lysine residues that are acetylated, were used for library construction. The library is comprised of seven subsets, wherein three amino acids flanking each lysine were randomized, while the six other lysines were mutated to arginine. A previously described yeast surface display based platform (Waldman et al) was used to visualize protein expression, enzyme expression, and acetylation levels. The library was sorted using flow cytometry to identify subpopulations with distinct acetylation levels, controlling for protein and enzyme expression. Subsequently, we used next generation DNA sequencing to identify amino acid sequence determinants of histone acetylation by p300 and identified links to protein targets of disease relevance.