The impact of 5CG-3′ methylation on the activity of different eukaryotic promoters: A comparative study (original) (raw)
The inhibiting or inactivating effects of position-specific promoter methylation in different viral or human cellular promoters Ad2 E2AL, SV40, LTR-MMTV, HSV-tk, TNFa) have been compared by in vitro 5'-CCGG-3' methylation by M-&a11 or the M-&I DNA-methyltransferase, respectively. In most promoters, 5'-CG-3' methylation reduces activity to a few percent of that of mock-methylated controls. The number of 5'-CG-3' dinucleotides in a promoter does not strictly correlate with the extent of methylation inhibition. The LTR-MMTV promoter, which lacks 5'-CG-3' dinucleotides, is not affected by methylation. The late E2A promoter of Ad2 DNA cannot be inactivated by 5'-CCGG-3' methylation when the construct carries the strong cytomegalovirus enhancer devoid of this sequence. In contrast, 5'-CG-3' methylation shuts this promoter off even in the presence of this enhancer.
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Proceedings of the National Academy of Sciences of the United States of America, 1991
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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.
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