Histones are the major scaffolding proteins which organize and compact nuclear DMAin chromatin. These proteins undergo numerous types of chemical modifications that govern a variety of processes in the nucleus. Methylation of the amino acid lysine occurs in several histones and has been linked to the regulation of gene expression as well as in DNA repair processes and cell division. Histone lysine methyltransferases (HKMTs) are enzymes that catalyze the methylation of specific lysines within histones. HKMTs have been shown to function as both positive and negative regulators of gene expression, depending on the specific lysine that they methylate in histones. Recently, some HKMTs have been shown to methylate transcription factors (DNA binding proteins that activate gene expression), suggesting that these enzymes have broader roles in controlling transcription than previously believed. The importance of HKMTs in regulating gene expression is underscored by direct relationships between these enzymes and numerous forms of cancer. Deletions, mutations, or over-expression of several HKMTs have been linked malignancies such as leukemias and prostate, breast, lung, colorectal, and hepatic cancers. In order to gain insight into the functions of HKMTs in transcriptional regulation and how disruption of these enzymes can lead to cancer, we propose several experiments to characterize their protein substrate specificities and elucidate the chemical mechanism through which they methylate specific lysine residues in nuclear proteins. To achieve these goals, we will use a combination of biochemistry and structural biology in which we will determine the molecular structures of these enzymes bound to their protein substrates. Taken together, this research will provide not only novel insights into the functions of HKMTs in the nucleus, but will also provide avenues for the development of specific inhibitors against these enzymes in the treatment of cancer.
Aim1 : Determine the chemical mechanism of lysine methylation of HKMTs via biochemical and structural studies of a plant HKMT homolog known as Rubisco large subunit methyltransferase (LSMT).
Aim2 : Determine the substrate specificity of human SET7/9, a histone and transcription factor methyltransferase, through structural and biochemical techniques.
Aim3 : Determine the structure and substrate specificity of human SETS, an HKMT that is pivotal in regulating gene silencing and proper chromosome separation during cell division.
|Krishnan, Swathi; Collazo, Evys; Ortiz-Tello, Patricia A et al. (2012) Purification and assay protocols for obtaining highly active Jumonji C demethylases. Anal Biochem 420:48-53|
|Horowitz, Scott; Yesselman, Joseph D; Al-Hashimi, Hashim M et al. (2011) Direct evidence for methyl group coordination by carbon-oxygen hydrogen bonds in the lysine methyltransferase SET7/9. J Biol Chem 286:18658-63|
|Krishnan, Swathi; Horowitz, Scott; Trievel, Raymond C (2011) Structure and function of histone H3 lysine 9 methyltransferases and demethylases. Chembiochem 12:254-63|
|Bulfer, Stacie L; McQuade, Thomas J; Larsen, Martha J et al. (2011) Application of a high-throughput fluorescent acetyltransferase assay to identify inhibitors of homocitrate synthase. Anal Biochem 410:133-40|
|Takahashi, Yoh-hei; Westfield, Gerwin H; Oleskie, Austin N et al. (2011) Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human. Proc Natl Acad Sci U S A 108:20526-31|
|Del Rizzo, Paul A; Trievel, Raymond C (2011) Substrate and product specificities of SET domain methyltransferases. Epigenetics 6:1059-67|
|Magnani, Roberta; Dirk, Lynnette M A; Trievel, Raymond C et al. (2010) Calmodulin methyltransferase is an evolutionarily conserved enzyme that trimethylates Lys-115 in calmodulin. Nat Commun 1:43|
|Bulfer, Stacie L; Scott, Erin M; Pillus, Lorraine et al. (2010) Structural basis for L-lysine feedback inhibition of homocitrate synthase. J Biol Chem 285:10446-53|
|Del Rizzo, Paul A; Couture, Jean-François; Dirk, Lynnette M A et al. (2010) SET7/9 catalytic mutants reveal the role of active site water molecules in lysine multiple methylation. J Biol Chem 285:31849-58|
|Trievel, Raymond C; Shilatifard, Ali (2009) WDR5, a complexed protein. Nat Struct Mol Biol 16:678-80|
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