Analysis of Promoter Methylation and Its Role in Silencing Metallothionein I Gene Expression in Tumor Cells

Ghoshal, Kalpana; Majumder, Sarmila; Jacob, Samson T.

doi:10.1016/s0076-6879(02)53070-6

Cited by 23 publications

(23 citation statements)

References 12 publications

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“…Amplification of MT-I promoter from mock-digested and HpaII-digested DNA but not from MspI-digested DNA (Fig. 3C, lanes 1-3) confirmed our earlier observation that the MT-I promoter is methylated in P1798 cells (25,34). Amplification of the MT-I promoter from DNA pulled down by Dnmt3a but not by preimmune serum showed specific association of Dnmt3a Figure 3.…”

Section: Resultssupporting

confidence: 86%

“…In vivo association of Dnmt3a with Mbd3 and Brg1 in the mouse lymphosarcoma cells (P1798) led us to explore the functional significance of their interaction in modulating gene expression. Because the MT-I gene is silent in P1798 cells due to extensive promoter methylation (25,34), we investigated whether these three proteins involved in epigenetic regulation of genes associate with the promoter by chromatin immunoprecipitation assay. The formaldehyde cross-linked chromatin from P1798 cells was immunoprecipitated with antibodies against Dnmt3a, Mbd3, or Brg1.…”

Section: Resultsmentioning

confidence: 99%

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RETRACTED: Physical and Functional Interaction of DNA Methyltransferase 3A with Mbd3 and Brg1 in Mouse Lymphosarcoma Cells

Datta¹,

Majumder²,

Bai³

et al. 2005

Cancer Research

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Dnmt3a and Dnmt3b are de novo DNA methyltransferases that also act as transcriptional repressors independent of methyltransferase activity. To elucidate the underlying mechanism of transcriptional repression, Dnmt3a was purified from mouse lymphosarcoma cells (P1798) by extensive fractionation on five different chromatographic matrices followed by glycerol density gradient centrifugation. Liquid chromatography electrospray tandem mass spectrometry analysis of Dnmt3a-associated polypeptides identified the methyl CpG binding protein Mbd3, histone deacetylase 1(Hdac1), and components of Brg1 complex (Brg1, Baf155, and Baf57) in the purified preparation. Association of Dnmt3a with Mbd3 and Brg1 was confirmed by coimmunoprecipitation and coimmunolocalization studies. Glutathione S-transferase pulldown assay showed that the NH 2 -terminal ATRX homology domain of Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and with both bromo and ATPase domains of Brg1. Chromatin immunoprecipitation assay revealed that all three proteins are associated with transcriptionally silent methylated metallothionein (MT-I) promoter in the mouse lymphosarcoma cells. To understand the functional significance of their association with the promoter, their role on the MT-I promoter activity was analyzed by transient transfection assay. The results showed that Mbd3 and Dnmt3a specifically inhibited the methylated promoter, and the catalytic activity of Dnmt3a was dispensable for the suppression. In contrast, the wild-type but not the ATPase-inactive mutant of Brg1 suppressed MT-I promoter irrespective of its methylation status, implicating involvement of ATP-dependent chromatin remodeling in the process. Coexpression of two of the three interacting proteins at a time augmented their repressor function. This study shows physical and functional interaction of Dnmt3a with components of nucleosome remodeling machinery. (Cancer Res 2005; 65(23): 10891-900)

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

RETRACTED: Physical and Functional Interaction of DNA Methyltransferase 3A with Mbd3 and Brg1 in Mouse Lymphosarcoma Cells

Datta¹,

Majumder²,

Bai³

et al. 2005

Cancer Research

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“…Our laboratory has been studying the roles of different factors involved in the epigenetic process, particularly DNA methyltransferases (Dnmts), histone deacetylases (Hdacs), and methyl-CpG binding proteins (MBDs), on gene expression (23,24,35). Methylation of DNA at position 5 of cytosine within CpG dinucleotides is the most abundant covalent modification in the eukaryotic genome (27,31).…”

mentioning

confidence: 99%

DNA Methyltransferase 3b Regulates Nerve Growth Factor-Induced Differentiation of PC12 Cells by Recruiting Histone Deacetylase 2

Bai¹,

Ghoshal²,

Datta³

et al. 2005

Molecular and Cellular Biology

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“…Genomic DNA from liver and HCCs were subjected to bisulfite conversion as described (26)(27)(28) followed by amplification of the promoter region of each MT isoform using strandspecific primers (see Supplementary Data). MT-2A PCR product was also subjected to sequencing using dideoxy termination kit (26).…”

Section: Methodsmentioning

confidence: 99%

Metallothionein Expression Is Suppressed in Primary Human Hepatocellular Carcinomas and Is Mediated through Inactivation of CCAAT/Enhancer Binding Protein α by Phosphatidylinositol 3-Kinase Signaling Cascade

et al. 2007

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Reactive oxygen species (ROS) resulting from chronic inflammation cause liver injury leading to transformation of regenerating hepatocytes. Metallothioneins (MT), induced at high levels by oxidative stress, are potent scavengers of ROS. Here, we report that the levels of MT-1 and MT-2A are drastically reduced in primary human hepatocellular carcinomas (HCCs) and in diethylnitrosamine-induced liver tumors in mice, which is primarily due to transcriptional repression. Expression of the transcription factor, MTF-1, essential for MT expression, and its target gene Zn-T1 that encodes the zinc transporter-1 was not significantly altered in HCCs. Inhibitors of both phosphatidylinositol 3-kinase (PI3K) and its downstream target AKT increased expression of MT genes in HCC cells but not in liver epithelial cells. Suppression of MT-1 and MT-2A by ectopic expression of the constitutively active PI3K or AKT and their up-regulation by dominant-negative PI3K or AKT mutant confirmed negative regulation of MT expression by PI3K/AKT signaling pathway. Further, treatment of cells with a specific inhibitor of glycogen synthase kinase-3 (GSK-3), a downstream effector of PI3K/AKT, inhibited MT expression specifically in HCC cells. Short interfering RNAmediated depletion of CCAAT/enhancer binding protein A (C/EBPA), a target of GSK-3, impeded MT expression, which could not be reversed by PI3K inhibitors. DNA binding activity of C/EBPA and its phosphorylation at T222 and T226 by GSK-3 are required for MT expression. MTF-1 and C/EBPA act in concert to increase MT-2A expression, which probably explains the high level of MT expression in the liver. This study shows the role of PI3K/AKT signaling pathway and C/EBPA in regulation of MT expression in hepatocarcinogenesis.

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Analysis of Promoter Methylation and Its Role in Silencing Metallothionein I Gene Expression in Tumor Cells

Cited by 23 publications

References 12 publications

RETRACTED: Physical and Functional Interaction of DNA Methyltransferase 3A with Mbd3 and Brg1 in Mouse Lymphosarcoma Cells

RETRACTED: Physical and Functional Interaction of DNA Methyltransferase 3A with Mbd3 and Brg1 in Mouse Lymphosarcoma Cells

DNA Methyltransferase 3b Regulates Nerve Growth Factor-Induced Differentiation of PC12 Cells by Recruiting Histone Deacetylase 2

Metallothionein Expression Is Suppressed in Primary Human Hepatocellular Carcinomas and Is Mediated through Inactivation of CCAAT/Enhancer Binding Protein α by Phosphatidylinositol 3-Kinase Signaling Cascade

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