PROJECT SUMMARY/ABSTRACT In eukaryotes, chromatin is organized into nucleosomes through wrapping double stranded DNA around 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 including acetylation and novel acylations such as propionylation, butyrylation, crotonylation, malonylation, succinylation, and glutarylation. Sirtuins are a group of histone deacylases that potentially catalyze the removal of both acetylation and novel acylations from chromatin. Previous studies have focused on using acetyl/acyl-peptide substrates for sirtuin studies. However, acetyl/acyl-peptides don't represent the native sirtuin substrates in cells and therefore critical information such as what roles the nucleosome scaffold plays in sirtuin catalyzed nucleosomal deacetylation/deacylation cannot be extracted by using acetyl/acyl-peptide substrates. To characterize fundamental epigenetic roles of sirtuins in regulating chromatin acylations, we have developed strategies for swift preparation of a number of homogenous acetyl/acyl-nucleosomes and used them as substrates for sirtuin studies. Our preliminary data indicates that Sirt1 shows substrate sequence selectivity when catalyzing nucleosomal deacetylation but the nucleosome scaffold inhibits Sirt1 activity. However, Sirt6 displays unique substrate sequence selectivity and the nucleosome scaffold is required for its activation. Encouraged by our exciting preliminary study, we will continue our endeavor of understanding reversible nucleosomal acetylation/acylation by pursuing three specific aims: 1) Study Sirt1 with acetyl/acyl-nucleosome substrates to understand the inhibitory role of the nucleosome scaffold towards Sirt1 activity and regulation 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 nucleosome scaffold towards Sirt6 activity, substrate sequence selectivity of Sirt6, and potential indirect regulation of H3K56 acetylation by Sirt6 ; 3) Build methods to synthesize succinyl-nucleosomes for screening Sirt5- targeted nucleosomal deacetylation sites and understanding potentially negative impacts of lysine succinylation on the nucleosome assembly.