title: The proteomic investigation of chromatin functional domains reveals novel synergisms among distinct heterochromatin components.
abstract: Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status.
authors: Department of Experimental Oncology, European Institute of Oncology (IEO), Via Adamello 16, Milan, Italy.
authors: Monica
authors: M
authors: Soldi
authors: Tiziana
authors: T
authors: Bonaldi
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods; D004249:DNA Damage; D006367:HeLa Cells; D006570:Heterochromatin / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D000092468:Bromodomain Containing Proteins
chemical_list: D000251:Adenosine Triphosphatases; D002843:Chromatin; D006570:Heterochromatin; D006657:Histones; D008239:Lysine; D020543:Proteome; D014157:Transcription Factors; D014443:Tyrosine; C116750:BAZ1B protein, human; D000092468:Bromodomain Containing Proteins; C468783:H2AX protein, human; C086468:ISWI protein