DNA methylation in mammals plays a crucial role in development, X chromosome inactivation, epigenetic silencing, aging, carcinogenesis and certain diseases. Although methylation at CpG islands is known to occur in several genes that leads to suppression of their expression, the molecular mechanism of silencing the genes by this process and its potential role in neoplastic transformation have not been completely elucidated. We showed that (a) unlike the parental tissues (thymus and liver), mouse lymphosarcoma cells P1798 and rat hepatoma 3924A are incapable of inducing metallothionein-I and II (MT-I and II) by heavy metals as a result of hypermethylation of their promoters and (b) P1798 cells contain a unique mentyl C-binding protein, designated MeCP-L that is distince from the well characterized MeCP2 and the partially characterized MeCP1. The long term goal of this proposal is to elucidate the molecular mechanisms by which DNA methylation suppresses gene expression in the cancer cells, its functional and potential clinical implications. The present study will (a) purify the unique MeCP- L from the nuclear extracts of the tumor cells and characterize it biochemically, clone the cDNA for the factor, express it in suitable expression system, study the mechanism by which this MeCP-L inhibits the MT-I promoter activity by an in vitro transcription assay (b) identify the polypeptides that specifically associate with MeCP-L to form a functional complex and identify the repressor domain of this protein in vitro (c) explore the role of MeCP-L in silencing the methylated genes in vivo by expressing MeCP-L in drosophila SL2 cells (that contains insignificant amounts of MeCPs) and methylated MT-I promoter reporter construct, and also using the antisense MeCP-L for stable transfection of the P1798 cells, (d) elucidate the molecular mechanism by which DNA methylation of different regions of the MT-I gene represses its expression in vivo using transient transfection assay, (e) study the effect of MT overexpression in P1798 cells on the growth and transformation potential of these cells,(f) investigate whether silencing of MT genes is a unique characteristic of tumors of lymphoid and hepatic orgin, and test the possibility that glucocorticoid receptor (GR) promoter methylation is repsonsible for the resistance of some lymphoid cancer cells to glucocorticoid, a potent drug used in the treatment of the cancers of lymphatic orgin. It is hoped that this study could provide the impetus to explore ways to reactivate these repressed genes that may result in the arrest of specific neoplastic growth.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES010874-03
Application #
6518208
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Reinlib, Leslie J
Project Start
2000-04-10
Project End
2005-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
3
Fiscal Year
2002
Total Cost
$328,500
Indirect Cost
Name
Ohio State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210
Majumder, Sarmila; Kutay, Huban; Datta, Jharna et al. (2006) Epigenetic regulation of metallothionein-i gene expression: differential regulation of methylated and unmethylated promoters by DNA methyltransferases and methyl CpG binding proteins. J Cell Biochem 97:1300-16
Majumder, Sarmila; Ghoshal, Kalpana; Datta, Jharna et al. (2006) Role of DNA methyltransferases in regulation of human ribosomal RNA gene transcription. J Biol Chem 281:22062-72
Bai, Shoumei; Ghoshal, Kalpana; Jacob, Samson T (2006) Identification of T-cadherin as a novel target of DNA methyltransferase 3B and its role in the suppression of nerve growth factor-mediated neurite outgrowth in PC12 cells. J Biol Chem 281:13604-11
Motiwala, Tasneem; Jacob, Samson T (2006) Role of protein tyrosine phosphatases in cancer. Prog Nucleic Acid Res Mol Biol 81:297-329
Ghoshal, Kalpana; Datta, Jharna; Majumder, Sarmila et al. (2005) 5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal. Mol Cell Biol 25:4727-41
Datta, Jhrana; Majumder, Sarmila; Bai, Shoumei et al. (2005) Physical and functional interaction of DNA methyltransferase 3A with Mbd3 and Brg1 in mouse lymphosarcoma cells. Cancer Res 65:10891-900
Bai, Shoumei; Ghoshal, Kalpana; Datta, Jharna et al. (2005) DNA methyltransferase 3b regulates nerve growth factor-induced differentiation of PC12 cells by recruiting histone deacetylase 2. Mol Cell Biol 25:751-66
Jacob, Samson T; Motiwala, Tasneem (2005) Epigenetic regulation of protein tyrosine phosphatases: potential molecular targets for cancer therapy. Cancer Gene Ther 12:665-72
Motiwala, Tasneem; Kutay, Huban; Ghoshal, Kalpana et al. (2004) Protein tyrosine phosphatase receptor-type O (PTPRO) exhibits characteristics of a candidate tumor suppressor in human lung cancer. Proc Natl Acad Sci U S A 101:13844-9
Ghoshal, Kalpana; Majumder, Sarmila; Datta, Jharna et al. (2004) Role of human ribosomal RNA (rRNA) promoter methylation and of methyl-CpG-binding protein MBD2 in the suppression of rRNA gene expression. J Biol Chem 279:6783-93

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