DNA Methyltransferase 1 Knock Down Induces Gene Expression by a Mechanism Independent of DNA Methylation and Histone Deacetylation

Snezana Milutinovic, Shelley E. Brown, Qianli Zhuang, Moshe Szyf
2004 Journal of Biological Chemistry  
DNA methyltransferase 1 (DNMT1) catalyzes the postreplication methylation of DNA and is responsible for maintaining the DNA methylation pattern during cell division. A long list of data supports a role for DNMT1 in cellular transformation and inhibitors of DNMT1 were shown to have antitumorigenic effects. It was long believed that DNMT1 promoted tumorigenesis by maintaining the hypermethylated and silenced state of tumor suppressor genes. We have previously shown that DNMT1 knock down by either
more » ... nock down by either antisense oligonucleotides directed at DNMT1 or expressed antisense activates a number of genes involved in stress response and cell cycle arrest by a DNA methylation-independent mechanism. In this report we demonstrate that antisense knock down of DNMT1 in human lung carcinoma A549 and embryonal kidney HEK293 cells induces gene expression by a mechanism that does not involve either of the known epigenomic mechanisms, DNA methylation, histone acetylation, or histone methylation. The mechanism of activation of the cell cycle inhibitor p21 and apoptosis inducer BIK by DNMT1 inhibition is independent of the mechanism of activation of the same genes by histone deacetylase inhibition. We determine whether DNMT1 knock down activates one of the nodal transcription activation pathways in the cell and demonstrate that DNMT1 activates Sp1 response elements. This activation of Sp1 response does not involve an increase in either Sp1 or Sp3 protein levels in the cell or the occupancy of the Sp1 elements with these proteins. The methylation-independent regulation of Sp1 elements by DNMT1 unravels a novel function for DNMT1 in gene regulation. DNA methylation was believed to be a mechanism for suppression of CG-rich Sp1-bearing promoters. Our data suggest a fundamentally different and surprising role for DNMT1 regulation of CG-rich genes by a mechanism independent of DNA methylation and histone acetylation. The implications of our data on the biological roles of DNMT1 and the therapeutic potential of DNMT1 inhibitors as anticancer agents are discussed. DNA modification by methylation of cytosines residing at the dinucleotide sequence CG plays an important role in epigenomic programming of gene expression (1). Not all CGs are methylated, and the pattern of distribution of methylated and unmethylated CGs is cell type-specific (2). DNA methylation in regulatory regions of genes plays a role in silencing genes either by directly inhibiting the interaction of transcription factors with their regulatory sequences (3, 4) or by attracting methylated DNA-binding proteins, which in turn recruit histone deacetylases and histone methyltransferases, resulting in an inactive chromatin structure (5, 6). DNA methylation is catalyzed by DNA methyltransferases DNMTs, 1 which transfer the methyl moiety from the methyl donor S-adenosylmethionine to 5th position on the cytosine ring (7) . DNMT1 is responsible for maintaining the DNA methylation pattern during embryonal development and cell division (8, 9) . DNMT1 deregulation was proposed to play a critical role in cellular transformation (10). Forced expression of DNMT1 was shown to transform NIH 3T3 cells (11), DNMT1Ϫ/Ϫ knockouts are resistant to colorectal tumorigenesis (12), and antisense knock down of DNMT1 reverses tumorigenesis in vitro (13, 14) and in vivo (15). The mechanisms through which DNMT1 causes cellular transformation and through which inhibition of DNMT1 reverses cellular transformation are unknown (16). The most obvious mechanism is that aberrant expression of DNMT1 causes methylation and silencing of tumor suppressor genes (17, 18) . This hypothesis is supported by numerous documentations of methylated tumor suppressor genes in tumors (19). In accordance with this hypothesis, knock down of DNMT1 by either antisense or siRNA results in demethylation and activation of tumor suppressor genes such as p16 (14, 20) . However, it was surprisingly previously shown that knock down of DNMT1 results in induction of the unmethylated tumor suppressor gene p21 by a mechanism that does not involve DNA methylation (21). More recently it was shown that DNMT1 interacts with histone deacetylases (HDACs) 1 (22) and 2 (23) as well as histone methyltransferase SUV39H1 (24), suggesting that DNMT1 silences gene expression by recruiting chromatin-modifying enzymes. It was also shown that ectopic expression of DNMT1 could suppress exogenous genes bearing E2F1 sites by recruiting Rb⅐E2F1⅐HDAC1 complex (25). However, it is not clear whether DNMT1 regulates endogenous genes by these mechanisms. We have previously shown that DNMT1 expression is regulated with the cell cycle (8, 26, 27) and that antisense knock down of DNMT1 results in an intra-S-phase arrest of DNA replication (28). This intra-S-phase arrest requires knock down of the DNMT1 protein rather than inhibition of DNA methyl-*
doi:10.1074/jbc.m312823200 pmid:15087453 fatcat:olczsnagbrhbfkxnijtltbwrga