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MBD2 is the only member of a family of methyl-CpGbinding proteins that has been reported to be both a transcriptional repressor and a DNA demethylase (dMTase). To understand the apparently contradictory function of MBD2/dMTase, we studied the effects of dMTase overexpression on the activity of various in vitro methylated promoters transiently transfected into HEK293 cells. We found that forced expression of a MBD2/dMTase expression vector (His-dMTase) differentially activated two methylated reporters, pSV40-CAT (the SV40 enhancerless promoter adjacent to the chloramphenicol acetyltransferase (CAT) reporter gene) and pGL2T؉I4xTBRE (a region of the p21 promoter next to the luciferase reporter gene), in a time-and dose-dependent manner. His-dMTase increased pSV40-CAT expression by 3-10-fold after 96 h, while pGL2T؉I4xTBRE expression was increased by 2-3-fold after only 48 h and did not further increase at 96 h. Gene activation was not universal because no effect was seen with the p19-ARF promoter. We then assessed whether activation might be due to demethylation within the promoter region. Using bisulfite mapping, we found that exogenous expression of His-dMTase induced demethylation at 8 of the 10 CpG sites within the SV40 promoter. The observation that His-dMTase increases the demethylase activity in the cells was also confirmed using an in vitro CpG demethylase assay with a mC32pG oligonucleotide substrate and purified Q-Sepharose fractions from HEK293 cells transfected with His-dMTase or empty pcDNA3.1His vector. We propose that a single protein possessing both repressor and demethylase functions has evolved to coordinate a program that requires suppression of some methylated genes and activation of others.The epigenome consists of an additional component that is part of the covalent structure of the genome, a coating of methyl groups. In vertebrates, 80% of cytosine residues within the dinucleotide sequence CpG are modified by methylation in a pattern that is tissue-specific and that is formed during development and maintained in somatic cells (1). It has been well established that the DNA methylation pattern is maintained exclusively by DNA methyltransferase activities, but we have recently proposed that DNA demethylase activities might also participate in the process (2-4) and that the methylation pattern is a steady state balance of reversible methylationdemethylation reactions (5, 6). We have shown that histone acetylation promotes active demethylation of ectopically methylated genes (3) and that inhibitors of histone acetylation inhibit demethylation (4).It is well documented that the state of activity of a gene, the chromatin structure, and DNA methylation are correlated (7) such that areas of the genome that are methylated are usually less expressed. One molecular mechanism that explains this relationship has recently been elucidated. Repressor complexes are recruited to methylated DNA via the binding of methylCpG binding domain proteins (MBDs).1 These complexes contain proteins that have histone deacet...

We demonstrate that expression of v-Ha-ras in mouse embryonal P19 cells results in genome-wide demethylation. Analysis of the pattern of methylation of specific genes reveals that different types of genes are demethylated in the ras transfectants: skeletal muscle specific genes, a gene specifically expressed in the adrenal cortex (c21), ubiquitous genes, and exogenously introduced sequences. Transient transfection and in vitro demethylation assays reveal that the ras transfectants express high levels of a general DNA demethylation activity. This demonstrates that the general DNA demethylation activity in mouse embryonal cells is controlled by an important cellular signal transducer and that DNA demethylase is a potential downstream effector of Ras.

The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. DNA-binding proteins that regulate gene expression play an important biological role and are potentially attractive therapeutic targets. However, the study of their role in different physiological and pathological processes has been hindered by the lack of specific inhibitors. These proteins are especially appealing as drug targets because their ligand is a DNA sequence that can be identified by standard molecular biology techniques and can be synthesized and modified by well established chemistries (1). In addition, recent observations suggest that double-stranded DNA-based oligonucleotides bearing transcription factor recognition sites can be delivered into cells in culture and in vivo and exhibit pharmacological effects (2, 3). A major limitation of this approach is that oligonucleotide antagonists that are identical to the transcription factors cognate site act as stochiometric competitors. Therefore, very high intracellular concentrations are required to effectively engage all the transcription factor available in the cell at all times. An ideal DNA-binding protein antagonist should exhibit higher affinity to the protein than the cognate sequence and bear a slow off rate. In this report we have tested the hypothesis that DNA-based inhibitors of DNA-binding proteins that address these requirements could be developed, using the DNA methyltransferase enzyme (DNA MeTase) 1 as a model DNA-binding protein.Basic oncogenic pathways such as the Ras-Jun signaling pathway have been shown to up-regulate DNA MeTase mRNA (4 -6) and the hyperactivation of DNA MeTase observed in many cancer cells (7,8) occurs in parallel with the development of the aberrant patterns of DNA methylation that these cells exhibit (9, 10). A number of studies suggest that the hyperactivation of DNA MeTase plays a causal role in oncogenesis. For example, the intraperitoneal injection of antisense oligonucleotide to DNA MeTase mRNA into LAF/1 mice bearing tumors derived from the syngeneic tumor cell line Y1 (11) inhibits tumor growth; and in vivo reduction of DNA MeTase levels by either 5-azaCdR treatment or by bearing one mutated allele of DNA MeTase reduces the frequency of appearance of intestinal adenomas in the Min mouse bearing a mutation in the adenomatosis polyposis coli gene (12).Uncovering the biological role of DNA MeTase in vivo requires the a...

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