MECHANISTIC STUDIES ON THE STIMULATION OF HDOT1L-MEDIATED H3K79 METHYLATION BY H2B-UB

Abstract

Ubiquitylation of histone H2B at lysine 120 (H2B-Ub), a post-translational modification (PTM) first discovered in 1980, plays a critical role in diverse nuclear processes including the regulation of transcription and DNA damage repair. In this thesis, we use a suite of protein chemistry methods to explore how H2B-Ub stimulates the activity of the protein lysine methyltransferase, hDot1L, leading to methylation of histone H3 on lysine 79 (H3K79me).

In chapter 2, a series of mutations is made on the catalytic domain of human Dot1L (hDot1L) to test a proposed auto-inhibition hypothesis. Though no evidence for auto-inhibition is found, the studies suggest that the structured core of hDot1L (i.e. amino acids 1 to 332) contains molecular elements required for H2B-Ub stimulation. In chapter 3, a highly optimized semisynthesis of H2BssUb* bearing a site-specifically installed photo-crosslinker is presented. By employing semisynthetic ‘designer’ chromatin containing H2BssUb*, we uncover a hitherto unknown interaction between a functional hotspot on ubiquitin and the N-terminus of histone H2A. We show that this interaction is required for normal stimulation of hDot1L activity. Moreover, our biochemical studies in chapter 4 show that H2B-Ub alters the binding of hDot1L to the nucleosome, indicating that this interaction leads to a repositioning of hDot1L on the nucleosomal surface, which likely places the active site of the enzyme proximal to H3K79. Biochemical studies in chapter 5 also support this hypothesis. Collectively, our data converge on a possible mechanism for hDot1L stimulation in which H2B-Ub physically ‘corrals’ the enzyme into a productive binding orientation.