DNA methylation in mammals is required for embryonic development, X chromosome inactivation and imprinting. Previous studies have shown that methylation patterns become abnormal in malignant cells and may contribute to tumorigenesis by improper de novo methylation and silencing of the promoters for growth-regulatory genes. RNA and protein levels of the DNA methyltransferase DNMT1 have been shown to be elevated in tumors, however murine stem cells lacking Dnmt1 are still able to de novo methylate viral DNA. The recent cloning of a new family of DNA methyltransferases (Dnmt3a and Dnmt3b) in mouse which methylate hemimethylated and unmethylated templates with equal efficiencies make them candidates for the long sought de novo methyltransferases. We have investigated the expression of human DNMT1, 3a and 3b and found widespread, coordinate expression of all three transcripts in most normal tissues. Chromosomal mapping placed DNMT3a on chromosome 2p23 and DNMT3b on chromosome 20q11.2. Significant overexpression of DNMT3b was seen in tumors while DNMT1 and DNMT3a were only modestly over-expressed and with lower frequency. Lastly, several novel alternatively spliced forms of DNMT3b, which may have altered enzymatic activity, were found to be expressed in a tissue-specific manner.

Zebularine is a stable DNA demethylating agent and the first drug in its class able to reactivate an epigenetically silenced gene by oral administration.

We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.The mammalian DNA methyltransferases (DNA methyltransferase 1 [Dnmt1], Dnmt3a, and Dnmt3b) establish and maintain genomic methylation patterns which are of critical importance in various biological processes, including development, genomic imprinting, silencing of parasitic sequence elements, and tumorigenesis (3,14,17,31). The individual role of each of the DNA methyltransferases in establishing and maintaining these patterns is still unclear and has been confounded by their overlapping activities with respect to their abilities to methylate unmethylated and hemimethylated DNA in the test tube (21, 30). Embryonic stem (ES) cells deficient in one or more of these enzymes can be used in one of several approaches to elucidate the roles of the individual enzymes in living cells. Earlier studies using cells deficient in the Dnmt1 enzyme showed considerable decreases in the level of genomic DNA methylation at CpG-rich repetitive elements and imprinted genes (17,25,27). Recent studies using cells deficient in both the Dnmt3a and -3b enzymes showed that CpG-rich retroviral and intracisternal A particle (IAP) elements became slightly demethylated, and Igf-2 and Xist became extensively demethylated, in the absence of these enzymes, implying that Dnmt1 by itself had sequence specificity in maintaining the methylation of these sequences (20).These previous studies all focused on the methylation of CpG-rich sequences in knockout cells. However, most methylation in mammalian cells is found in non-CpG-rich regions of DNA (5), and the roles of the various enzymes in establishing and maintaining these methylation patterns have not been investigated. We have therefore used a genome-scanning approach to investigate the patterns of methylation in the various knockout cells in CpG-poor and CpG-rich regions to determine the roles of the enzymes in carrying out the bulk of methylation in mouse ES cells.We found that methylatio...

Almost 1-2% of the human genome is located within 500 bp of either side of a transcription initiation site, whereas a far larger proportion (Ϸ25%) is potentially transcribable by elongating RNA polymerases. This observation raises the question of how the genome is packaged into chromatin to allow start sites to be recognized by the regulatory machinery at the same time as transcription initiation, but not elongation, is blocked in the 25% of intragenic DNA. We developed a chromatin scanning technique called ChAP, coupling the chromatin immunoprecipitation assay with arbitrarily primed PCR, which allows for the rapid and unbiased comparison of histone modification patterns within the eukaryotic nucleus. Methylated lysine 4 (K4) and acetylated K9͞14 of histone H3 were both highly localized to the 5 regions of transcriptionally active human genes but were greatly decreased downstream of the start sites. Our results suggest that the large transcribed regions of human genes are maintained in a deacetylated conformation in regions read by elongating polymerase. Common models depicting widespread histone acetylation and K4 methylation throughout the transcribed unit do not therefore apply to the majority of human genes.