Studying Reversible Histone Acylations in Nucleosome Contexts
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PROJECT SUMMARY/In eukaryotes, chromatin is organized into nucleosomes through wrapping double stranded DNAaround the histone core. The histone core is composed of eight subunits, two each of histones H2A,H2B, H3, and H4. Histones are rich in lysine residues that undergo several types of acylation includingacetylation and novel acylations such as propionylation, butyrylation, crotonylation, malonylation,succinylation, and glutarylation. Sirtuins are a group of histone deacylases that potentially catalyze theremoval of both acetylation and novel acylations from chromatin. Previous studies have focused onusing acetyl/acyl-peptide substrates for sirtuin studies. However, acetyl/acyl-peptides don''t representthe native sirtuin substrates in cells and therefore critical information such as what roles thenucleosome scaffold plays in sirtuin catalyzed nucleosomal deacetylation/deacylation cannot beextracted by using acetyl/acyl-peptide substrates. To characterize fundamental epigenetic roles ofsirtuins in regulating chromatin acylations, we have developed strategies for swift preparation of anumber of homogenous acetyl/acyl-nucleosomes and used them as substrates for sirtuin studies. Ourpreliminary data indicates that Sirt1 shows substrate sequence selectivity when catalyzing nucleosomaldeacetylation but the nucleosome scaffold inhibits Sirt1 activity. However, Sirt6 displays uniquesubstrate sequence selectivity and the nucleosome scaffold is required for its activation. Encouraged byour exciting preliminary study, we will continue our endeavor of understanding reversible nucleosomalacetylation/acylation by pursuing three specific aims: 1) Study Sirt1 with acetyl/acyl-nucleosomesubstrates to understand the inhibitory role of the nucleosome scaffold towards Sirt1 activity andregulation of Sirt1 by protein and small molecule factors such as AROS, c-Jun, and resveratrol; 2)Study Sirt6 with acetyl/acyl-nucleosome substrates to understand the activating role of the nucleosomescaffold towards Sirt6 activity, substrate sequence selectivity of Sirt6, and potential indirect regulation ofH3K56 acetylation by Sirt6 ; 3) Build methods to synthesize succinyl-nucleosomes for screening Sirt5-targeted nucleosomal deacetylation sites and understanding potentially negative impacts of lysinesuccinylation on the nucleosome assembly.
