Study of a novel evolutionarily conserved pattern of histone acetylation

The eukaryotic genome is packaged into a highly ordered chromatin structure. Even though chromatin structure is important for maintaining genomic integrity, it is a barrier to numerous DNA-based processes such as DNA replication, transcription and DNA repair. Histones contain a bewildering diversity of covalent modifications that are mostly but not exclusively concentrated within their amino-terminal tails. Histone modifications play a central role in regulating chromatin structure and function. However, determining the stoichiometry of site-specific modifications, identifying patterns of modifications and establishing their physiological roles remain formidable challenges.    In this study, we exploited mass spectrometry to determine the stoichiometry of acetylation at specific histone lysine residues. In general, histone lysine residues lacking acetylation are derivatized to render the resulting peptides chemically equivalent but distinguishable by mass from their acetylated counterparts. However, this method is insufficient to study peptides that contain more than one acetylatable lysine, such as those derived from the N-terminal tail of histones H3 and H4. Tryptic digestion of such peptides generates ‘positional isomers’, isomers that have the same mass but bearing acetyl groups located at different positions. Accurate quantification of site-specific acetylation in those peptides is, therefore, a major analytical challenge.   In the second chapter, we describe a novel method for quantifying site-specific acetylation of co-eluting isomeric and isobaric peptides that combines high-resolution LC-MS/MS data with a novel bioinformatics algorithm, Iso-PeptidAce. Using tandem mass spectra (MS/MS) of synthetic peptides, fragmentation products diagnostic of each positional isomer were identified and were used to deconvolute spectra that arise from mixtures of positional isomers and quantify the abundance of each isomer. We then tested the applicability of Iso-PeptidAce to quantify time-dependent increases in histone acetylation of K562 erythroleukaemia cells treated with histone deacetylase (HDAC) inhibitors. Using our method, we also found that histones H3 and H4 associated with CAF-1, a chromatin assembly factor, have a high stoichiometry of acetylation on multiple residues of H3 and H4, compared with total histones.   In Chapter 3, we applied Iso-PeptidAce to determine the stoichiometry of acetylation in fission yeast histone deacetylase mutants. Consistent with previous reports implicating Clr3 and Sir2 in heterochromatin function, we observed that cells lacking these HDACs showed an increase in H3-K14 acetylation only on those peptides where it co-exists with di/trimethylated H3-K9, a mark of heterochromatin.   In chapter 4, we describe the discovery of very high levels of acetylation on two lysine residues. We found that a high stoichiometry of acetylation at H3-K14 and H3-K23, and low stoichiometry of acetylation at H3-K9 and H3-K18, is an evolutionarily conserved global pattern of H3 acetylation. Using fission yeast (S. pombe) strains harboring histone mutations H3-K14R and/or H3-K23R that prevent acetylation, we demonstrate that H3-K14 and H3-K23 have separable functions. Surprisingly, we found that the phenotypes observed in H3-K14R mutant cells are largely due to mutation of the lysine residue, rather than loss of acetylation. Using S. pombe strains that lack histone acetyltransferases (HATs) we identified the acetyltransferases that contribute to H3-K14ac and H3-K23ac in vivo.   Very few studies have aimed at specifically determining the acetylation stoichiometries of histones. Our results suggest that, on average, over the entire genome, every second or third nucleosome contains an H3 molecule with K14 and/or K23 acetylation. This leads us to surmise that genome-wide acetylation of histone H3 may have an important role in chromosome function. It is imperative to understand the functional significance of this acetylation pattern given that epigenetic therapy is actively pursued as a strategy to treat many diseases.

Keyphrases: Histones, Schizosaccharomyces pombe, Mass spectrometry