Roles of cell division and gene transcription in the methylation of CpG islands

De novo methylation of CpG islands within the promoters of eukaryotic genes is often associated with their transcriptional repression, yet the methyla tion of CpG islands located downstream of promoters does not block transcri ption. We investigated the kinetics of mRNA induction, demethylation, and r emethylation of the p16 promoter and second-exon CpG islands in T24 cells a fter 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment to explore the relationsh ip between CpG island methylation and gene transcription. The rates of reme thylation of both CpG islands were associated with time but not with the ra te of cell division, and remethylation of the p16 exon 2 CpG island occurre d at a higher rate than that of the p16 promoter. We also examined the rela tionship between the remethylation of coding sequence CpG islands and gene transcription, The kinetics of remethylation of the pld exon 2, PAX-6 exon 5, c-ABL exon 11, and MYF-3 exon 3 loci were examined following 5-Aza-CdR t reatment because these genes contain exonic CpG islands which are hypermeth ylated in T24 cells. Remethylation occurred most rapidly in the p16, PAX-6, and c-ABL genes, shown to be transcribed prior to drug treatment. These re gions also exhibited higher levels of remethylation in single-cell clones a nd subclones derived from 5-Aza-CdR-treated T24 cells. Our data suggest tha t de novo methylation is not restricted to the S phase of the cell cycle an d that transcription through CPG islands does not inhibit their remethylati on.