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Role of Protein Methyltransferases and Demethylases

其他書名	In Kaposi's Sarcoma-associated Herpesvirus Infection and Cancer
出版	University of California, Davis, 2015
ISBN	13218065669781321806564
URL	http://books.google.com.hk/books?id=tiTLjgEACAAJ&hl=&source=gbs_api

註釋Accumulating evidence suggests epigenetic alterations are as important as genetic mutations in cancer development. Similar to other epigenetic modifications such as DNA methylation and histone acetylation, histone methylation is a dynamic and reversible process that alters the chromatin structure through covalent modifications, resulting in gene transcription regulation and ultimately defining the cell's identity. In even more recent studies, protein methyltransferases and demethylases have been shown to target a growing list of non-histone protein substrates, providing a new layer of protein regulation and biological outcomes. Not surprisingly, aberrant control of these sets of enzymes can lead to multiple diseases including cancer and thus, makes them attractive targets for oncogenic viruses. Hence, a better understanding of how lysine/arginine methyltransferases and lysine demethylases function in viral and cancer settings will allow scientists to develop more effective therapeutics for oncogenic viral infections and cancers. For my doctoral research, my goal was to explore the molecular mechanisms of some of the lysine/arginine methyltransferases and lysine demethylases in the context of 1) epigenetic control via histone demethylation during the oncogenic Kaposi's sarcoma-associated herpes virus (KSHV) infection, 2) regulation of the retinoblastoma (pRb) tumor suppressor protein by arginine methylation in cell proliferation, and 3) protein lysine methylation of human epidermal growth factor receptor 2 (HER2). These studies are covered in Chapters 2, 3, and 4, respectively. In Chapter 2, I show that the KSHV latency associated nuclear antigen (LANA) protein associates with the epigenetic modifying protein and cellular lysine demethylase, KDM3A in vitro as well as in naturally infected B-cell lymphoma cells. Accordingly, this interaction is required for co-recruitment of LANA and KDM3A on a subset of viral lytic genes. Furthermore, I have found that co-recruitment of LANA and KDM3A leads to demethylation or removal of the repressive histone marks, histone H3 lysine 9 dimethyl (H3K9me2) marks on a subset of viral promoters. Lastly, from my knockdown studies, depletion of KDM3A significantly impaired gene expression of KSHV lytic genes, suggesting the functional importance of KDM3A for efficient transitioning from KSHV latency to lytic replication. In Chapter 3, I investigated a novel post-translation modification on the tumor suppressor retinoblastoma protein (pRb). I have demonstrated that pRb is asymmetrically di-methylated by the Protein Arginine Methyltransferase 4 (PRMT4) on arginine (R) residues R775, R787, and R798 in vitro and R787 in vivo. Importantly, methylation defective pRb mutant decreases phosphorylation on adjacent serine (S) and threonine (T) residues. In line with these observations, stable isogenic expression of methyl mimetic pRb mutant displays decreased binding to E2F-1 when compared to wild-type pRb expression in U2OS cells. Transcription activation studies using a luciferase reporter system show that methylation is important for pRb mediated E2F-1 repression. Collectively, my pRb studies suggests that arginine methylation may negatively regulate pRb tumor suppressor function in part by reducing pRb phosphorylation of neighboring sites and abrogating pRb-E2F-1 interaction during cell cycle control. In Chapter 4, I demonstrate that human epidermal growth factor receptor 2 (HER2) is a novel substrate of SET7/9 targeted lysine methylation. SET7/9 targeted HER2 lysine methylation occurs on the extracellular domain on lysine residues K200 and K228. In a reporter luciferase assay, transient overexpression of SET7/9 specifically activated the MAPK ERK1/2 pathway. In addition, my SET7/9 knockdown studies show that depletion of SET7/9 reduces phosphorylation levels of MAPK ERK1/2, a well-known substrate of HER2. These results suggest that SET7/9 may be important for MAPK ERK1/2 phosphorylation and activation, which may be dependent on HER2 lysine methylation.