No abstract

CpG islands are distinguishable from the bulk of vertebrate DNA for being unmethylated and CpG-rich. Since CpG doublets are the specific target of eukaryotic DNA methyltransferases, CpG-rich sequences might be expected to be good methyl-accepting substrates in vitro, despite their unmethylated in vivo condition. This was tested using a partially purified DNA-methyltransferase from human placenta and several cloned CpG-rich or CpG-depleted sequences. The efficiency of methylation was found to be proportional to the CpG content for CpG-depleted regions, which are representative of the bulk genome. However, methylation was much less efficient for CpG frequencies higher than 1 in 12 nucleotides, reaching only 60% of the expected level. That suggests that the close CpG spacing typical of CpG-islands somehow inhibits mammalian DNA methyltransferase. The implications of these findings on the in vivo pattern of DNA methylation are discussed.

The CpG-rich promoter of the retinoblastoma tumor suppressor gene (Rb-1) is normally unmethylated. However, aberrant methylation of CpG dinucleotides within the Rb-1 promoter has been depicted in certain tumors, which determines transcriptional inactivity of the gene and absence of the pRb retinoblastoma protein. Here we have concentrated on an E2F-binding site in the Rb-1 promoter. We show that the E2F site is required for cell-cycle regulated Rb-1 transcription in non-transformed cells. The function of the E2F site is associated with its ability to interact with several activating factors of the E2F family. In contrast, in vitro methylation of two tandemly arranged CpGs in the E2F recognition site prevents binding by E2F factors, and determines instead the recruitment of the general repressor methylcytosine-binding protein 2 (MeCP2). These results suggest that the interaction of MeCP2 with the methylated version of the E2F site may represent a step towards Rb-1 promoter inactivity in tumor cells.

In HeLa cells, under conditions where normal semiconservative synthesis is suppressed by hydroxyurea, the excision repair process after irradiation by UV results in a small amount of incorporation of nucleotides into nonreplicated DNA. By labelling the cytosine moieties of these repair patches, and measuring the ratio between cytosine and 5-methylcytosine, we have found that the level of methylation of cytosine in repair patches five hours after UV-irradiation of the cells is about half of that observed in normal semiconservatively synthesized DNA.

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 beta 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 pre-existing 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-m5dC).poly(dG-m5dC), which is highly inhibitory for eukaryotic enzymes and almost ineffective on prokaryotic enzymes.