Study on the cellular function of histone succinylation and development of genetically encoded yeats domain inhibitor

Abstract

Protein post-translational modifications (PTMs) play significant roles in expanding the structural and functional diversity of the proteome, and they are involved in nearly all biological processes. Histones, which interact with deoxyribonucleic acid (DNA) and pack DNA into chromatin, are extensively post-translationally modified. Histone PTMs play fundamental roles in regulating nucleosome and chromatin dynamics, thereby implicated in various DNA-templated biological events, such as DNA replication, DNA transcription, and DNA damage repair. The dysregulation of histone PTMs is involved in many human diseases, such as cancer, autoimmune disease and diabete. To date, more than 20 different types of PTMs have been identified in histones; however, only a few of them have been extensively studied. Therefore, it is necessary to investigate the regulatory mechanisms as well as the biological functions of histone PTMs, especially these newly identified ones. During the past four years, I mainly focused on two newly identified PTMs: histone lysine succinylation and crotonylation. In Chapter 2, an genetic mimic approach was used to investigate the biological functions of two histone lysine succinylation marks, namely, H2BK34succ and H4K77succ, in yeast. To mimic the negatively charged succinylation, yeast strains whose histones bear a lysine-to-glutamic acid (K-to-E) mutation at the modification site were generated. It was observed that yeast cells bearing a K-to-E mutation on histones display unique and significant defects in nucleosome stability and chromatin structure. To gain further insight into the biological functions of histone succinylation, in Chapter 3, a chemical proteomics approach was used to comprehensively profile the histone succinylation “binder” proteins in yeast. Two histone succinylation marks, namely, H2AK13succ and H3K27succ, which are located at histone tails, were selected. CDC1 stood out as a potential histone succinylation “binder.” To better characterize this potential “binder,” different strategies were used to purify this protein from yeast. However, all attempts ultimately failed. To obtain homogenous histone proteins with site-specific succinylation at the stoichiometry level via the genetic code expansion (GCE) approach, in Chapter 4, a directed evolution platform for pyrrollysine aminoacyl-tRNA synthetase (PylRS) was established. Then, directed evolution of PylRS toward two lysine succinylation precursors, namely, photocaged succinyllysine and tBu-caged succinyllysine, whose genetic incorporation and deprotection will afford succinyllysine, was conducted. However, no active PylRS mutants were identified from the evolution. Our group has previously developed a serial of peptide-based competitive inhibitors of the YEATS domain, the “reader” of histone crotonylation, by targeting π-π-π stacking. The inhibition is achieved by replacing the crotonyl group with an expanded π system. We reasoned that the π system can be directly incorporated into proteins in cells by GCE. To this end, in Chapter 5, a 2-furancarbonyl lysine-specific PylRS was identified by directed evolution, and a genetically encoded YEATS domain inhibitor was developed. It was further demonstrated that this inhibitor disrupts the chromatin and genomic localization of YEATS-domain-containing proteins.