Date of Award
Doctor of Philosophy (PhD)
Histone post-translational modifications modulate chromatin structure and function either by directly altering the intrinsic physical properties of the chromatin fiber or by nucleating the recruitment and activity of a host of transacting nuclear factors. Histone ubiquitylation is one class of histone PTMs where the 76 amino acid protein, ubiquitin, is ligated to the ε-nitrogen of a lysine amino acid residue within the histone substrate. 36 unique ubiquitin sites across the 4 canonical histones have been annotated, with 7 of these sites associated with chromatin-templated processes. One such modification, the ubiquitylation of Histone H2B at lysine 120 (H2B-Ub) is enriched at the 5’ end of active genes and has been implicated in transcriptional elongation and chromatin conformation, interacting with over 90 trans-acting nuclear factors. Further, it is additionally responsible for the regulation of H3K4 and H3K79 methylations, through the direct stimulation of methyltransferase activity specific to the installation of these marks. Interested in the surface features on ubiquitin required for H2B-Ub stimulation of the human H3K79 methyltransferase, hDot1L, we developed a strategy for the site-specific chemical ligation of ubiquitin to preassembled mononucleosomes to greatly expedite structure activity studies of ubiquitin in a nucleosome context. Accordingly, we synthesized a library of H2Bligated ubiquitin alanine mutant nucleosomes and tested their ability to stimulate hDot1L-mediated H3K79 methylation. A functional hotspot on Ub that is required for the stimulation of hDot1L activity in vitro was identified. We additionally investigated the structural implication of this functional hotspot in the context of nucleosomes and nucleosomal array compaction. Both nucleosomal structural and H2B-Ub-induced impairment of chromatin fiber compaction was not affected by this functional surface. Lastly, this functional hotspot was further tested with the yeast H3 methyltransferases, yDot1 and ySet1C. Surprisingly, this functional surface was dispensable for yDot1-mediated H3K79 methylation both in vivo and in vitro. Further insights into yDot1-mediated H3K79 methylation was obtained through an alanine scan of the Ub surface in the context of the nucleosome. Interestingly, the hDot1L hotspot within ubiquitin was found to be important for the regulation of ySet1-mediated H3K4 methylation. Collectively, these data delineate the multi-functionality of H2B-Ub as a histone post-translational modification.
Holt, Matthew Thomas, "Identification of a Functional Hotspot on Ubiquitin Required for Stimulation of Methyltransferase Activity on Chromatin" (2014). Student Theses and Dissertations. 212.