Defining nucleosome features regulating the function of chromatin modifiers and remodelers

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All processes associated with the expression and maintenance of the genetic material are intimately linked to the dynamic structure of chromatin. The cell has developed an intricate network of factors not only to modulate chromatin structure but also to mark it as a signaling platform with different post-translational modifications. Chromatin modifiers and remodelers are critical regulators of most DNA-based processes, de facto controlling cell cycle progression and cell fate. These enzymes perform very precise modifications and structural alterations, which in turn are recognized by different effector modules/proteins. This is thought to be at the heart of the epigenetic phenomena, in which signals to chromatin mark different genomic loci and are read by effectors to translate a biological response and establish cellular memory during division/development. Accurate writing and reading of these epigenetic marks lead to changes in chromatin dynamics, in a targeted manner within the genome. Our work has characterized the structure and function of several multiprotein complexes that control chromatin modifications, remodeling and composition. The past decade has highlighted the need to use biochemistry to fully dissect the molecular mechanisms at play. Furthermore, we have repeatedly shown the need to use native-like molecular assemblies to recreate accurate specificity of the chromatin modifying/remodeling enzymes. We still lack complete structural and functional understanding of each complex' specific role/mechanism of action, as well as essential cross-talks with other factors/modifications implicated in gene regulation and genome maintenance. Our research program aims at filling this knowledge gap by using a wide array of different types of chromatin substrates to biochemically characterize the features that regulate the function of several important chromatin remodeling and modifying complexes. Our large collection of chromatin substrates includes mono-/di-nucleosomes with different linkers/spacings and post-translational modifications, mutated on specific regions or containing non-canonic histone variants. Each one of these features can have an impact on the binding and function of different chromatin modifying or remodeling complexes. In the short term, we will screen the binding and activities of four remodelers and three modifiers. Through these enzyme/substrate combinations we will obtain crucial new information on the specific features of chromatin that differentially regulate the function of these complexes. In the longer term, new interesting concepts uncovered through this biochemical approach will be tested in structural studies and functional assays in vivo. Overall, this research will provide a large amount of new molecular information that will greatly improve our understanding of key regulatory processes controlling expression and maintenance of the genome.