Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements

Abstract

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...

DNA methyltransferase 1 expression and promoter methylation of E‐cadherin in mucoepidermoid carcinoma

“…4 Genomic methylation patterns may be established through cooperation among these three DNMTs. 39,40 Moreover, studies have indicated that DNMT expression is coordinated in many malignancies, 12 and DNMTs have different expression levels in variable tumors or in a tissue-specific manner. 12,13 Therefore, it is hypothesized that other DNMTs are required in silencing E-cadherin in MEC.…”

Dynamic and reversibility of heterochromatic gene silencing in human disease

“…Genetic and biochemical analyses have shown that double knockout of DNMT1 and DNMT3b diminishes most of the genomic methylation while single knock-out has limited effect on DNA methylation pattern [30]; that DNMT1 alone is insufficient to restore DNA methylation after 5-azaCdR treatment [31] and that DNMT1 can form complex with DNMT3A and 3B [32] (Fig. 1).…”

Identification of DNMT1 (DNA methyltransferase 1) hypomorphs in somatic knockouts suggests an essential role for DNMT1 in cell survival

“…Because the DNMT1 hypomorphic cell line had presumably lost the ability to interact with PCNA and therefore might not be able to fully methylate its target sequences during S phase, it might be anticipated that the cells would show an increased level of hemimethylation after depletion of WT DNMT1. We therefore measured the level of hemimethylation within three regions of the p16 locus by using an assay we have previously described that couples methylation-sensitive restriction enzyme digestion with bisulfite treatment and methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) analysis (20,21). The ability of the assay to detect hemimethylation at three sites [within the 5Ј region of the p16 gene (H1), at a CpG-poor site (H2), and at a CpG-rich site in intron 1 (H3)] was first validated by using T24 cells after 24-h treatment with 5-aza-CdR (20).…”