Abstract
Chromatin structure and gene expression are dynamically regulated by posttranslational modifications of histones. Recent advance in mass spectrometry has identified novel types of lysine acylations, such as butyrylation and malonylation, whose functions and regulations are likely different from those of acetylation. Sirtuins, nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases, catalyze various deacylations. However, it is poorly understood how distinct sirtuins regulate the histone acylation states of nucleosomes that have many lysine residues. Here, we provide mass spectrometry-based quantitative information about the acyl group- and site-selectivity of all human sirtuins on acylated nucleosomes. The acyl group- and site-selectivity of each sirtuin is unique to its subtype. Sirt5 exclusively removes negatively-charged acyl groups, while Sirt1/2/3/6/7 preferentially remove hydrophobic acyl groups; Sirt1 and Sirt3 selectively remove acetyl group more than butyryl group, whereas Sirt2 and Sirt6 showed the opposite selectivity. Investigating site-selectivity for active sirtuins revealed acylated lysines on H4 tails to be poor substrates and acylated H3K18 to be a good substrate. Furthermore, we found Sirt7 to be a robust deacylase of H3K36/37, and its activity reliant on nucleosome-binding at its C-terminal basic region. All together, our quantitative dataset provides a useful resource in understanding chromatin regulations by histone acylations.
Highlights
The structure of chromatin, a polymer of nucleosomes, is dynamically regulated by post-translational modifications (PTMs) of the histones in nucleosomes
We envisioned that acetylated or malonylated nucleosomes could be used as substrates for histone deacetylases (HDACs)-promoted deacylation to compare the site- and acyl group-selectivity of HDAC isoforms
We have provided LC–MS/MS-based quantitative information about site- and acyl group-selectivity of all the human sirtuins against acetylated, butyrylated, and malonylated nucleosomal substrates and found that site- and acyl-group selectivity of each sirtuin are different from each other
Summary
The structure of chromatin, a polymer of nucleosomes, is dynamically regulated by post-translational modifications (PTMs) of the histones in nucleosomes. HDACs can be classified into two major groups: zinc-dependent HDACs (HDAC1–11) and nicotinamide adenine dinucleotide (NAD+)-dependent ones[3] The latter, called sirtuins, are known to regulate important physiologic functions, such as metabolism, cell cycle, genome maintenance, and aging processes[4,5,6,7,8]. Our biochemical analyses using recombinant nucleosomes that are acetylated or malonylated by artificial catalyst systems suggest that histone malonylation has a greater effect on inter-nucleosome interactions than acetylation does[20,21], while the functions of acidic acyl groups on histones in living cells remain to be elucidated. We report on characterization of the acyl group- and site-selectivity of seven human sirtuins using acylated nucleosomes as substrates, by quantitative mass spectrometry-based analysis
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