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Doctor of Philosophy (PhD)




Michael S. Cosgrove


Crystallography, Epigenetics, Isothermal Titration Calorimetry, Mixed Lineage Leukemia

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Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are associated with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 associates with a sub-complex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this dissertation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoprecipitate WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic human SET1 family Win motif sequences (MLL1-4, SETd1a and SETd1b) inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. We show that the MLL3 Win motif peptide is the best inhibitor of the H3K4 dimethylation activity of MLL1 core complex. To understand the structural basis for these differences, we determined three-dimensional structures of WDR5 bound to six different naturally occurring Win motif sequences (MLL1-4, SETd1a and SETd1b). The structural analysis reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. Based on the structure-function analysis, we deduce structural rules to facilitate the design of two additional Win motif -based inhibitors (Ac-10-mer and six-residue Win motif peptides) that bind WDR5 with <10 nM affinities. To understand the structural basis for this low nanomolar affinity, we determined X-ray three-dimensional structures of the Ac-10-mer Win motif peptide bound to WDR5. The structures suggest that the presence of additional intramolecular hydrogen bonds might contribute to the increased affinities for WDR5 possibly through the stabilization of the bound 310-helical conformation. We extend this structure-function analysis further to identify other peptidomimetics by characterizing peptides identified in a randomized phage display screen, which are also highly specific inhibitors of MLL1 core complex. Crystal structures of these peptidomimetics reveal novel protein structural features that contribute to increased affinity. We also present preliminary evidence suggesting that the MLL3 Win motif based peptide that has a cell penetrating sequence is readily taken up by mammalian cells. This MLL3 Win motif-based peptide (MLL3-FITC-TAT) is localized to euchromatin regions of cell nuclei, induces nuclear defects and inhibits global levels of H3K4 trimethylation. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.


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