ESTABLISHMENT AND OPTIMIZATION OF THE CHROP APPROACH, COMBINING CHIP AND MS-BASED PROTEOMICS, FOR THE CHARACTERIZATION OF THE CHROMATOME AT DISTINCT FUNCTIONAL DOMAINS

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 the subsequent mass spectrometry analysis and thus remains a very attractive unachieved goal. I contributed in this direction developing and optimizing a proteomic strategy that combines chromatin immunoprecipitation 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. This chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, thus suggesting new regulatory pathways, such as an 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.