Accelerating Chromatin Biochemistry: A DNA-Barcoded Nucleosomal Array Library

Abstract

Post-translational modifications of histones (PTMs) can affect chromatin structure, ultimately leading to changes in gene expression. Methylation at lysine 9 of histone 3 (H3K9) is associated with transcriptional repression of genes. One chromatin-modifying enzyme that is responsible for methylation at H3K9 is histonemethyltrasnferase G9a. Interestingly, G9a has two domains: a SET domain, which catalyzes the methylation, and an ankyrin repeat domain, which binds H3K9me1/2. G9a is one of many chromatin-modifying enzymes that contains domains within the same protein for both making and recognizing a PTM. It is hypothesized that G9a operates under the mechanism of spreading, in which product binding on the ankyrin repeats could help propagate the methyl mark by positioning G9a to the SET domain of a neighboring nucleosome. Although G9a's ankyrin repeat domain has been long recognized, it is still not known whether G9a operates under the mechanism of spreading, as it is difficult to obtain suitable substrates for assays to study how pre-installed marks on neighboring nucleosomes affect enzymatic activity. This thesis presents the development of a chemical biology tool, a DNA-barcoded nucleosome array library, that will be used to probe the dynamics of PTM spreading for G9a. Specifically, native chemical ligation (NCL), along with solid phase peptide synthesis (SPPS) and recombinant protein production, was employed to synthesize designer histones. Modified histones can be reconstituted into mononucleosomes, which can later be ligated to nucleosome arrays and included in the DNA-barcoded nucleosome array library. Furthermore, results from the histone methyltransferase assay show two G9a inhibitors: sinefungin and H3 peptide (residues 1-14) with a norleucine mutation at K9. These inhibitors can later be used in a spreading assay with G9a. This DNA-barcoded array library is a powerful tool in epigenetics research, as it offers the opportunity for greater understanding of the mechanisms involved in PTM deposition, recognition, and removal.