Clonal inheritance of the pattern of DNA methylation in mouse cells (original) (raw)
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J Mol Biol, 1997
The mechanisms that establish and maintain methylation patterns in the mammalian genome are very poorly understood, even though perturbations of methylation patterns lead to a loss of genomic imprinting, ectopic X chromosome inactivation, and death of mammalian embryos. A family of sequence-speci®c DNA methyltransferases has been proposed to be responsible for the wave of de novo methylation that occurs in the early embryo, although no such enzyme has been identi®ed. A universal mechanism-based probe for DNA (cytosine-5)-methyltransferases was used to screen tissues and cell types known to be active in de novo methylation for new species of DNA methyltransferase. All identi®able de novo methyltransferase activity was found to reside in Dnmt1. As this enzyme is the predominant de novo methyltransferase at all developmental stages inspected, it does not ®t the de®nition of maintenance methyltransferase or hemimethylase. Recent genetic data indicate that de novo methylation of retroviral DNA in embryonic stem cells is likely to involve one or more additional DNA methyltransferases. Such enzymes were not detected and are either present in very small amounts or are very different from Dnmt1. A new method was developed and used to determine the sequence speci®city of intact Dnmt1 in whole-cell lysates. Speci®city was found to be con®ned to the sequence 5 H -CpG-3 H ; there was little dependence on sequence context or density of CpG dinucleotides. These data suggest that any sequence-speci®c de novo methylation mediated by Dnmt1 is either under the control of regulatory factors that interact with Dnmt1, or is cued by alternative secondary structures in DNA.
DNA (cytosine-5)-methyltransferases in mouse cells and tissues. studies with a mechanism-based probe
Journal of Molecular Biology, 1997
The mechanisms that establish and maintain methylation patterns in the mammalian genome are very poorly understood, even though perturbations of methylation patterns lead to a loss of genomic imprinting, ectopic X chromosome inactivation, and death of mammalian embryos. A family of sequence-speci®c DNA methyltransferases has been proposed to be responsible for the wave of de novo methylation that occurs in the early embryo, although no such enzyme has been identi®ed. A universal mechanism-based probe for DNA (cytosine-5)-methyltransferases was used to screen tissues and cell types known to be active in de novo methylation for new species of DNA methyltransferase. All identi®able de novo methyltransferase activity was found to reside in Dnmt1. As this enzyme is the predominant de novo methyltransferase at all developmental stages inspected, it does not ®t the de®nition of maintenance methyltransferase or hemimethylase. Recent genetic data indicate that de novo methylation of retroviral DNA in embryonic stem cells is likely to involve one or more additional DNA methyltransferases. Such enzymes were not detected and are either present in very small amounts or are very different from Dnmt1. A new method was developed and used to determine the sequence speci®city of intact Dnmt1 in whole-cell lysates. Speci®city was found to be con®ned to the sequence 5 H -CpG-3 H ; there was little dependence on sequence context or density of CpG dinucleotides. These data suggest that any sequence-speci®c de novo methylation mediated by Dnmt1 is either under the control of regulatory factors that interact with Dnmt1, or is cued by alternative secondary structures in DNA.
Journal of Molecular Biology, 1997
The mechanisms that establish and maintain methylation patterns in the mammalian genome are very poorly understood, even though perturbations of methylation patterns lead to a loss of genomic imprinting, ectopic X chromosome inactivation, and death of mammalian embryos. A family of sequence-speci®c DNA methyltransferases has been proposed to be responsible for the wave of de novo methylation that occurs in the early embryo, although no such enzyme has been identi®ed. A universal mechanism-based probe for DNA (cytosine-5)-methyltransferases was used to screen tissues and cell types known to be active in de novo methylation for new species of DNA methyltransferase. All identi®able de novo methyltransferase activity was found to reside in Dnmt1. As this enzyme is the predominant de novo methyltransferase at all developmental stages inspected, it does not ®t the de®nition of maintenance methyltransferase or hemimethylase. Recent genetic data indicate that de novo methylation of retroviral DNA in embryonic stem cells is likely to involve one or more additional DNA methyltransferases. Such enzymes were not detected and are either present in very small amounts or are very different from Dnmt1. A new method was developed and used to determine the sequence speci®city of intact Dnmt1 in whole-cell lysates. Speci®city was found to be con®ned to the sequence 5 H -CpG-3 H ; there was little dependence on sequence context or density of CpG dinucleotides. These data suggest that any sequence-speci®c de novo methylation mediated by Dnmt1 is either under the control of regulatory factors that interact with Dnmt1, or is cued by alternative secondary structures in DNA.
Influence of Pre-existing Methylation on the de Novo Activity of Eukaryotic DNA Methyltransferase †
Biochemistry, 1998
Aberrant de novo methylation of CpG island DNA sequences has been observed in cultured cell lines or upon malignant transformation, but the mechanisms underlying this phenomenon are poorly understood. Using eukaryotic DNA (cytosine-5)-methyltransferase (of both human and murine origin), we have studied the in vitro methylation pattern of three CpG islands. Such sequences are intrinsically poor substrates of the enzyme, yet are efficiently methylated when a small amount of 5-methylcytosine is randomly introduced by the M.SssI prokaryotic DNA (cytosine-5)-methyltransferase prior to in vitro methylation by the eukaryotic enzyme. A stimulation was also found with several other double-stranded DNA substrates, either natural or of synthetic origin, such as poly(dG-dC)‚poly(dG-dC). An A+T-rich plasmid, pHb 1S, showed an initial stimulation, followed by a severe inhibition of the activity of DNA (cytosine-5)-methyltransferase. Methylation of poly(dI-dC)‚poly(dI-dC) was instead inhibited by preexisting 5-methylcytosines. The extent of stimulation observed with poly(dG-dC)‚poly(dG-dC) depends on both the number and the distribution of the 5-methylcytosine residues, which probably must not be too closely spaced for the stimulatory effect to be exerted. The activity of the M.SssI prokaryotic DNA methyltransferase was not stimulated, but was inhibited by pre-methylation on either poly(dG-dC)‚poly-(dG-dC) or poly(dI-dC)‚poly(dI-dC). The prokaryotic and eukaryotic DNA methyltransferases also differed in sensitivity to poly(dG-m 5 dC)‚poly(dG-m 5 dC), which is highly inhibitory for eukaryotic enzymes and almost ineffective on prokaryotic enzymes.
DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase
Nucleic Acids Research, 1985
Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (>65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivO.
Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases
Nucleic Acids Research, 2001
We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K m AdoMet , for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 µM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K m DNA values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 µM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 µM when poly(dG-dC)poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT ≥ CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.
Biomedicine & Pharmacotherapy, 1992
Methylation of C residues at specific sites in DNA of higher eukaryotes appears to play a role in regulating gene expression during development and differentiation. Silencing of genes by methylation can in part explain genomic imprinting and alterations in patterns of methylation during the early stages of carcinogenesis may account for some changes in gene expression in tumor cells [l]. Although the distribution of 5mC in mammalian DNA is tissue-and species-specific, little is known about MTases role in establishing specific patterns of methylation. Mammalian DNA (mDNA)MTase is most active in methylating C residues in hemi-methylated CpG sites and prefers substrates with high GC content and multiple CpGs-15 residues apart [21. To more fully characterize the specificity of mDNA MTase, we analyzed its activity with a variety of defined DNA substrates and compared it with a bacterial MTase, Sssi, which is reported to act on hemi-methylated and unmethylated CpG sites with equal efficiency [3]. Using defined unmethylated single(ss)-and double stranded(DNAs as substfates, we find that: 1) the initial rate of methylation of both ss-and ds-DNA-by Sssl and mDNA MTase is influenced by sequence, often in opposite ways; 2) depending on sequence, Sssf may be more active on ds-than ss-DNA. Activity of mDNA Mtase with ss-DNA is highly variable and dependant on sequence but is always low with unmethylated ds-DNA. Using SmC-substituted substrates, we find that hemi-methylation either has no effect or inhibits activity of Sssl but always activates mDNA MTase. mDNA MTase is most active with hemi-methylated ds-DNA and ss-DNA with 5mC near the 5'-end. These results demonstrate that although both MTases catalyze the same reaction, methylation of C residues in CpG dinucleotides. they have quite different specificities. This should be considered when these enzymes are used to quantitate hypomethylation at CpG sites in DNA resulting from exposure of mammalian cells or tissues to conditions or agents that affect DNA methylation. Finally, our findings suggest that 5mC residues in specific sequences in ss-DNA may serve to activate de novo methylation in mammalian cells allowing alteration of existing methylation patterns.