Integrative chemical biology study on histone lysine crotonylation

Abstract

Protein posttranslational modifications (PTMs) play significant roles in regulating various biological processes, such as gene transcription, DNA replication and chromatin remodeling. Increasing evidence has implicated that PTMs on histone proteins would largely control the nucleosome structure and serve as docking sites to recruit a wide range of proteins (so called ‘readers’) to execute downstream chromatin-templated cellular events. Thus far, more than 20 types of PTMs have been identified on histones. Except for the several well-studied ones such as acetylation, phosphorylation, and methylation, we have limited knowledge for most of them on their regulatory mechanisms and biological significances. My research during the past four years mainly focuses on the investigation of functional roles of newly discovered histone modifications using integrative chemical biology approaches. Histone lysine crotonylation (Kcr) is a newly identified histone PTMs which is reported to specifically localize at active gene promoters and potential enhancers in mammalian genomes. Previously, our group identified that NAD-dependent deacetylase SIRT3 could act as decrotonylase in living cells. In Chapter 2, I demonstrated that SIRT3 could regulation the transcription level of its target gene via its decrotonylation activity. I observed dynamic changes of multiple Kcr marks during various physiological events, and demonstrated that H3K4cr is the crucial for normal cell cycle. In Chapter 3, I developed and characterized a new photo-reactive amino acid, photo-lysine, as a powerful chemical tool for the identification of protein-protein interactions, including those mediated by histone PTMs in vitro and in living cells. Furthermore, coupling the unnatural amino acid strategy, I incorporated crotonylated-photo-lysine into histone H3 and demonstrated the SIRT3 could perform decrotonylation toward H3K79cr. YEATS domain is a newly identified ‘reader’ of histone lysine crotonylation. Recent studies have shown that YEATS domain in involved in the initiation and maintenance of aggressive leukaemia. Our group previously designed and synthesized a series of potential inhibitors, and some of them showed promising potency and selectivity in vitro. In Chapter 4, I characterized one inhibitor in living cells and I showed that this designed inhibitor could readily pass cell membrane, target and inhibitor YEATS domains in cellular context. The study in this chapter provided not only chemical tool to study YEATS domain but also potential therapeutic strategies to treat leukaemia. Chapter 5 describes the experimental methods and related materials.