CHROP APPROACH DISSECTS THE DYNAMIC PROFILING OF CHROMATOME AT ENHANCERS OF INFLAMMATORY GENES

Inflammatory stimuli drive a fine rearrangement of cell-specific chromatin determinants at cis-regulatory regions of inflammatory genes. Although a few determinants are known (i.e. H3K4me1 and Pu.1), a global picture of the enhancers’ molecular signature and how specific factors dynamically synergize during the inflammatory response remain incomplete. The aim of my thesis is the global characterization of the enhancers’ determinants and their dynamic profiling during inflammatory response. I addressed this issue by employing the chromatin proteomics approach (ChroP), which combines chromatin immunoprecipitation and mass spectrometry-based proteomics, to dissect histone post-translational modifications (modificome) and chromatin-binding proteins (interactome) associated with a specific chromatin region. We used H3K4me1 and Pu.1 antibodies to specifically enrich enhancers from macrophage-derived RAW264.7 cells. Native chromatin and formaldehyde-fixed chromatin from SILAC-labeled cells were used for the hPTMs profiling and the identification of chromatin binding proteins, respectively. Furthermore, a triple-SILAC setup was used in time-course experiments to profile the gene transcriptional activation triggered by lipopolysaccharide. Our findings suggest that enhancers have in basal condition an overall higher-ordered structure that is maintained during the inflammation; while a subset of proteins displays a dynamic behavior, i.e. the PBAF complex and Dnmt1, that seem to synergize with specific hPTMs to set an environment permissive to transcription. Interestingly, a number of factors are newly recruited at enhancers, suggesting a role in fine-tuning the appropriate gene expression: among them, Junb and Stat1 are well characterized to be involved in inflammatory response, while others (i.e. DDIT3 and Ifi204) are novel, very promising and under further investigation.