title: Chromatin affinity purification and quantitative mass spectrometry defining the interactome of histone modification patterns.
abstract: DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated.
authors.affiliation: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Nikolov
authors.forename: Alexandra
authors.initials: A
authors.lastname: Stützer
authors.forename: Kerstin
authors.initials: K
authors.lastname: Mosch
authors.forename: Andrius
authors.initials: A
authors.lastname: Krasauskas
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Soeroes
authors.forename: Holger
authors.initials: H
authors.lastname: Stark
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Fischle
mesh_terms: D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D008745:Methylation; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D002843:Chromatin; D006657:Histones; D055767:Immobilized Proteins; D010446:Peptide Fragments; D020543:Proteome; D011994:Recombinant Proteins