Role of de Novo DNA Methyltransferases and Methyl CpG-binding Proteins in Gene Silencing in a Rat Hepatoma*

The expression of metallothionein-I (MT-I), a known antioxidant, was suppressed in a transplanted rat hepatoma because of promoter methylation and was induced by heavy metals only after demethylation by 5-azacytidine (5-AzaC). Treatment of the tumor-bearing rats with 5-AzaC resulted in significant regression of the hepatoma. When the inhibitor-treated tumor was allowed to grow in a new host, MT-I promoter was remethylated, which suggested de novo methylation. The activities of both de novo (3-fold) and maintenance DNA methyltransferases (DNMT) (5-fold) were higher in the hepatoma than in the host liver. The mRNA levels of the de novomethyltransferases DNMT3a and DNMT3b were 3- and 6-fold higher, respectively, in the tumor implicating transcriptional up-regulation of these two genes in this tissue. Immunohistochemical analysis showed exclusive localization of DNMT3a in the nuclei of both the liver and hepatoma, whereas DNMT3b was detected in the nuclei as well as the cytoplasm. Immunoblot assay showed that the levels of DNMT1, DNMT3a, and DNMT3b proteins in the hepatoma were 5-, 10-, and 4-fold higher, respectively, than in the liver. The mRNA level of the major methyl CpG-binding protein (MeCP2) was 8-fold higher in the tumor compared with the liver. Immunohistochemical studies showed that MeCP2 is localized exclusively in the nuclei of both tissues. A chromatin immunoprecipitation assay demonstrated that MeCP2 was associated with the MT-I promoter in the hepatoma implicating its involvement in repressing the methylated promoter. Analysis of the DNA isolated from the liver and hepatoma by RLGS-M (restriction landmark genomic scanning with methylation-sensitive enzyme) (NotI) showed that many genes in addition to MT-I were methylated in the hepatoma. These data demonstrate suppression of theMT-I gene and probably other genes in a solid tumor by promoter methylation and have provided potential molecular mechanisms for the altered methylation profile of the genes in this tumor.

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