Chemical Approaches to Study the Effect of Histone Post-translational Modifications

Abstract

Genetic material is stored in a massive nucleoprotein complex called Chromatin. The basic unit of Chromatin is the Nucleosome Core Particle or NCP, made up of 147 bp DNA wound around a core of histone protein octamer comprising two of each histone H2A, H2B, H3 and H4. The positively charged tails of the histones extend outwards into the nucleosol and are subjected to various post-translational modifications (PTMs) by chromatin modifying enzymes. Histone PTMs are important regulators of a variety of critical cellular processes such as DNA damage repair, transcriptional regulation. Multiple human cancers, autoimmune and neurodegenerative diseases can occur from the misregulation and mistranslation of histone PTMs. Ubiquitylation and SUMOylation are two such examples of histone PTMs. Studies on the ubiquitylation of histone H2A in a nucleosomal context by the breast cancer predisposition genes BRCA1/BARD1 heterodimer, showed that missense mutations in the RING domain of BARD1 disrupts the ubiquitin ligase activity of the heterodimer as well as interferes with its ability to bind to nucleosomes. This in turn leads to loss of transcriptional control of genes that encode estrogen- metabolizing enzymes. These studies were facilitated by access to quantities of wild-type mononucleosomes produced in the Chatterjee lab at UW that was sufficient for carrying out biochemical assays and NMR experiments. The effect of the small ubiquitin-like modifier (SUMO) or SUMOylation is less well understood and requires access to uniformly and site-specifically modified NCPs. A successful protein-semisynthetic strategy developed by the Chatterjee lab utilized a chemical ligation auxiliary, 2-(aminooxy)ethanethiol in order to install SUMO at Lys 12 in histone H4 (suH4). This process generated sufficient quantities of suH4 that enabled investigations into its effect on chromatin and cross-talk with other histone PTMs such as acetylation and methylation, mediated by transcriptionally repressive complexes. The large-scale reconstitution of mononucleosomes and optimization of the semisynthesis of suH4 is described here.