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

Sarmila Majumder,Kalpana Ghoshal,Jharna Datta,Shoumei Bai,Xiaocheng Dong,Ning Quan,Christoph Plass,Samson T Jacob

doi:10.1074/jbc.m111662200

Sarmila Majumder, Kalpana Ghoshal + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m111662200

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Abstract

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 novo methyltransferases 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 the MT-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.

Highlights

Treatment of the tumor-bearing decade, the silencing of tumor suppressor genes by promoter rats with 5-AzaC resulted in significant regression of the methylation in tumorigenesis emerged as an important alterhepatoma
Besides the well established tumor suppressor genes, the list of hypermethylated genes in tumors that are not documented as tumor suppressors is growing in number. The list of these important genes includes p15INK4a [4], ER [5], GSTPi [6], TIMP3 [7], SOC [8], DAPK1 [9], and p73 [10]. These genes are silenced in clusive localization of DNMT3a in the nuclei of both the different tumors ranging from acute myeloid lymphoma to liver and hepatoma, whereas DNMT3b was detected in breast cancer
The expression of MT-III is restricted to glutaminergic neurons of the brain [19], and that of MT-IV is confined to squamous epithelial cells of the skin, tongue, and intestinal lining [20]

Summary

Introduction

Analysis of the DNA isolated from MT-II, MT-III, and MT-IV [18] Of these isoforms, MT-I and 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. DNA methylation was considered a under stress [23], in response to viral infection [24] and in mechanism to control precisely the expression of a small por- Cu,Zn-superoxide dismutase null mice [25]. By virtue of their heavy metal binding capacity, MTs participate in maintaining

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