Chromatin Affinity Purification and Quantitative Mass Spectrometry Defining the Interactome of Histone Modification Patterns

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...

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Bibliographic Details
Published in:Molecular & cellular proteomics 2011-11, Vol.10 (11), p.M110.005371-M110.005371, Article M110.005371
Main Authors: Nikolov, Miroslav, Stützer, Alexandra, Mosch, Kerstin, Krasauskas, Andrius, Soeroes, Szabolcs, Stark, Holger, Urlaub, Henning, Fischle, Wolfgang
Format: Article
Language:English
Subjects:
Animals
Cell Line
Chromatin - chemistry
Chromatography, Affinity - methods
Histones - chemistry
Histones - metabolism
Humans
Immobilized Proteins - chemistry
Immobilized Proteins - metabolism
Isotope Labeling
Methylation
Mice
Peptide Fragments - chemistry
Protein Binding
Protein Interaction Mapping - methods
Protein Processing, Post-Translational
Proteolysis
Proteome - chemistry
Proteome - isolation & purification
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Tandem Mass Spectrometry
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Summary: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.
ISSN:1535-9476
1535-9484
DOI:10.1074/mcp.M110.005371