The Role of SAS-I in Mediating Crosstalk Between Histone Modifications

Abstract

Histone post translational modifications (HPTM) such as methylation, acetylation, phosphorylation, and ubiquitination play a role in regulating many cell processes including cell cycling, transcription, and DNA damage repair. Regulation of such processes is achieved through the ability of HPTMs to 1) recruit activating and repressive complexes via 'effector' proteins and 2) alter chromatin structure through changing DNA, inter-, and intranucleosomal interactions. Many marks, when studied alone, can correlate with an increase and/or a decrease in global gene expression.However, recent studies suggest that one HPTM rarely affects gene expression without 'crosstalk' with one or several other HPTMs.The focus of this dissertation is to elucidate the mechanisms of targeting and regulation of Dot1 via histone crosstalk. Dot1 is a nonprocessive histone methyltransferase (HMT) responsible for H3K79 methylation. Dot1 requires H2BK123 ubiquitination in order to di-and trimethylate H3K79. However, how each H3K79 methylation state is regulated and what roles each state plays in specific cellular processes is unknown. Evidence provided in this dissertation illustrates that H3K79 trimethylation by Dot1 is dependent on H4K16 acetylation by the histone acetyltransferase (HAT) complex, SAS-I. In vitro HMT assays suggest that H4K16 acetylation-dependent trimethylation of H3K79 is achieved through changes in internuclesomal interactions, subsequent chromatin decondensation, and possible allosteric stimulation of Dot1 by H4K16ac. Upon loss of H4K16 acetylation, a significant loss in H3K79me3 is exhibited at genes bodies, while a loss of all three H3K79 methylation states is exhibited at subtelomeric regions. These results suggest that H3K79/H4K16 crosstalk may play a specific role in transcriptional regulation. IV Data provided in this dissertation also shows that Dot1 is targeted to chromatin via various HPTMs that have been linked to conserved pathways involved in transcriptional regulation. Results shown here indicate that Dot1 binds to unmodified H4 tail and modified H3 peptides, including H3K4me, H3R2me, and H3K14/18ac. In addition, loss of H3K4 and H3R2 methylation is shown to affect Dot1 activity.Communication between H3K79 and the modifications discussed in this dissertation ultimately alters the degree to which H3K79 is methylated. Elucidating the mechanisms involved in regulating Dot1 will provide addition therapeutic targets for patients suffering from leukemia caused by the mistargeting of Dot1 and consequent dysregulatin of genes involved in hematopoietic development. Chromatin OrganizationChromatin is the organization of the eukaryotic genome into a condensed form due the function of many proteins and RNAs. In humans, approximately 2.0 meters of DNA is compacted into 24 chromosomes that fit into a nucleus that is 6 µm in diameter.The fundamental unit of the highly ordered chromatic fiber is the nucleosome, which consists of approximately146 base pairs of DNA wrapped around an octamer of core histones that c...