Date of Award

5-2013

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Advisor(s)

Ramesh Raina

Second Advisor

Michael S. Cosgrove

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Biology

Abstract

Cells employ elaborate mechanisms to introduce structural and chemical variation into chromatin in the form of covalent post-translational modifications. Covalent modifications of histones contribute to the dynamic states of chromatin structure that govern nearly all of DNA-coupled processes such as transcription, replication, and repair. The mechanism by which covalent modifications of histones contribute to these activities remains a central question to understanding genome regulation and its dysfunction in human disease. Although there is extensive literature documenting the identification of many of the enzymes that place histone modifications, far less is known about how the enzymes are targeted and how their enzymatic activities are regulated. The long-term goal of the work in this thesis seeks to understand the mechanisms underlying the accessibility of genes in chromatin. In particular, this study focuses on identifying the underlying molecular mechanisms involved in the regulation of one such element of variation in chromatin, the methylation of lysine four on histone H3.

Histone H3 lysine 4 methylation (H3K4me) is an evolutionarily conserved epigenetic mark that is correlated with transcriptional activation in eukaryotes. H3K4 methylation has been shown to be important for a number of biological processes including gene expression and DNA replication. This mark is mainly catalyzed by a group of enzymes that contain an evolutionarily conserved SET domain. The evolutionarily conserved SET domain was originally named for its presence in three Drosophila melanogaster proteins: the position effect variegation modifier SU(VAR)3-9, the polycomb group protein E(z), and the trithorax group protein (TRX) . In vertebrates, the Mixed Lineage Leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3K4 methyltransferases. The catalytic activity of MLL1 is regulated by a conserved group of proteins that include the Tryptophan-Aspartate-repeat protein-5 (WDR5), the retinoblastoma-binding protein-5 (RbBP5) and the absent small homeotic-2-like protein (Ash2L).

The focus of this investigation is the RbBP5 component of the MLL1 core complex. The RbBP5 subunit of the MLL1 core complex has been shown to be required for enzymatic activity and disruption of RbBP5 is frequently observed in patients with malignant primary brain tumors. To gain insight into the functional role of RbBP5 in the regulation of the enzymatic activity of the MLL1 core complex, mutations targeting individual residues in a highly conserved stretch of amino acid residues in RbBP5 were generated. Biochemical and biophysical analyses of the variant proteins were utilized to assess the functional role of each amino acid on the intrinsic properties of RbBP5 alone or when assembled within the context of the entire complex. These studies identify several conserved aromatic residues and one acidic residue in RbBP5 required for interaction with MLL1 and the overall dimethyltransferase activity of the complex. The residues identified constitute a previously uncharacterized MLL1 interaction motif located in RbBP5. Therefore, this study provides important insights into how the RbBP5 subunit of the MLL1 core complex contributes to its overall enzymatic activity. Understanding the role that RbBP5 plays in facilitating proper H3K4 methylation may provide insight into how misregulation of RbBP5 can lead to brain tumor genesis.

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