title: Substrate Specificity Profiling of Histone-Modifying Enzymes by Peptide Microarray.
abstract: The dynamic addition and removal of covalent posttranslational modifications (PTMs) on histone proteins serves as a major mechanism regulating chromatin-templated biological processes in eukaryotic genomes. Histone PTMs and their combinations function by directly altering the physical structure of chromatin and as rheostats for effector protein interactions. In this chapter, we detail microarray-based methods for analyzing the substrate specificity of lysine methyltransferase and demethylase enzymes on immobilized synthetic histone peptides. Consistent with the "histone code" hypothesis, we reveal a strong influence of adjacent and, surprisingly, distant histone PTMs on the ability of histone-modifying enzymes to methylate or demethylate their substrates. This platform will greatly facilitate future investigations into histone substrate specificity and mechanisms of PTM signaling that regulate the catalytic properties of histone-modifying enzymes.
authors.affiliation: Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI, United States.
authors.forename: E M
authors.initials: EM
authors.lastname: Cornett
authors.affiliation: Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI, United States.
authors.forename: B M
authors.initials: BM
authors.lastname: Dickson
authors.affiliation: Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI, United States.
authors.forename: R M
authors.initials: RM
authors.lastname: Vaughan
authors.affiliation: University of Michigan, Ann Arbor, MI, United States.
authors.forename: S
authors.initials: S
authors.lastname: Krishnan
authors.affiliation: University of Michigan, Ann Arbor, MI, United States.
authors.forename: R C
authors.initials: RC
authors.lastname: Trievel
authors.affiliation: University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
authors.forename: B D
authors.initials: BD
authors.lastname: Strahl
authors.affiliation: Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI, United States. Electronic address: scott.rothbart@vai.org.
authors.forename: S B
authors.initials: SB
authors.lastname: Rothbart
mesh_terms: D000818:Animals; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods; D042421:Histone Code; D056466:Histone Demethylases / Q000378:metabolism*; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism*; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
chemical_list: D006657:Histones; D010455:Peptides; D056466:Histone Demethylases; D011495:Histone-Lysine N-Methyltransferase
keywords: Chromatin; Demethylases; Epigenetics; Erasers; Histone code; Histones; Methyltransferases; Peptide microarray; Posttranslational modifications; Writers