Date of Award
epigenetics, chromatin, histones, histone H2B, Dot1L, H3K79 methylation
Post-translational modification of histones plays an integral role in regulation of chromatin-templated processes through modulation of chromatin structure and function. One such modification, ubiquitylation of histone H2B on lysine 120 (uH2B), has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 by K79-specific methyltransferase, disruptor of telomeric silencing-like (Dot1L/KMT4). However, the specific function of uH2B in this crosstalk pathway was not understood, in part due to the challenges associated with isolating or generating homogeneously ubiquitylated H2B for use in biochemical studies. As both modifications are integral to transcriptional regulation and DNA damage repair, full elucidation of their functions is critical to understanding their roles in development and disease. In this thesis, a chemical strategy is presented for the preparation of native uH2B. Two traceless orthogonal expressed protein ligation (EPL) reactions were used for this purpose, one employing a photolytically removable ligation auxiliary, and the other, a cysteine-mediated ligation followed by a desulfurization to restore the native sequence. Reconstitution of semisynthetic uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed a direct role for uH2B in the stimulation of Dot1L-mediated methylation of H3K79. Although recruitment of Dot1L to the nucleosomal surface by uH2B could be excluded, comprehensive mechanistic analysis was precluded by systematic limitations in the ability to generate native uH2B in large-scale. To overcome this shortcoming, a highly optimized synthesis of ubiquitylated H2B bearing a Gly76Ala point mutation (uH2BG76A) was developed, yielding tens of milligrams of ubiquitylated protein. This mutant was indistinguishable from native uH2B by Dot1L, allowing for detailed studies of the resultant trans-histone crosstalk pathway. Kinetic and structure activity relationship analyses using uH2BG76A suggest a non-canonical role for ubiquitin in the enhancement of the chemical step of H3K79 methylation. This enhancement likely results from an allosteric change in the nucleosome and/or Dot1L following H2B ubiquitylation. Current work is aimed at further elucidation of the molecular mechanism of uH2B-mediated stimulation of Dot1L and the role of uH2B in other chromatin templated-processes.
McGinty, Robert Kendall, "Mechanistic Insights into the Stimulation of Dot1L-Mediated Methylation of Histone H3 by Semisynthetically Ubiquitylated Histone H2b" (2010). Student Theses and Dissertations. 75.