title: Investigating Histone Acetylation Stoichiometry and Turnover Rate.
abstract: Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrometry (MS) to comprehensively investigate histone acetylation dynamics. Turnover rates of histone acetylation are determined by measuring the kinetics of labeling from (13)C-labeled precursors of acetyl-CoA, which incorporates (13)C-carbon onto histones via the acetyltransferase reaction. Overall histone acetylation states are assessed from complete protease digestion to single amino acids, which is followed by MS analysis. Determination of site-specific acetylation stoichiometry is achieved by chemically acetylating endogenous histones with isotopic acetic anhydride, followed by trypsin digestion and LC-MS analysis. Combining metabolic labeling with stoichiometric analysis permits determination of both acetylation level and acetylation dynamics. When comparing genetic, diet, or environmental perturbations, these methods permit both a global and site-specific evaluation of how histone acetylation is dynamically regulated.
authors.affiliation: School of Medicine and Public Health, Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI, United States.
authors.forename: J
authors.initials: J
authors.lastname: Fan
authors.affiliation: School of Medicine and Public Health, Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI, United States.
authors.forename: J
authors.initials: J
authors.lastname: Baeza
authors.affiliation: School of Medicine and Public Health, Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI, United States; Morgridge Institute for Research, Madison, WI, United States. Electronic address: jmdenu@wisc.edu.
authors.forename: J M
authors.initials: JM
authors.lastname: Denu
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D006657:Histones; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases
keywords: Acetylation; Epigenetics; Histone; Kinetics; Mass spectrometry; Metabolic labeling; Stoichiometry