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The Language of Methylation in Genomics of Eukaryotes

P. Volpe

Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy; fax: (+39-06) 7259-4244; E-mail: volpe@bio.uniroma2.it

Received October 13, 2004
Background studies have shown that 6-methylaminopurine (m6A) and 5-methylcytosine (m5C), detected in DNA, are products of its post-synthetic modification. At variance with bacterial genomes exhibiting both, eukaryotic genomes essentially carry only m5C in m5CpG doublets. This served to establish that, although a slight extra-S phase asymmetric methylation occurs de novo on 5´-CpC-3´/3´-GpG-5´, 5´-CpT-3´/3´-GpA-5´, and 5´-CpA-3´/3´-GpT-5´ dinucleotide pairs, a heavy methylation during S involves Okazaki fragments and thus semiconservatively newly made chains to guarantee genetic maintenance of -CH3 patterns in symmetrically dimethylated 5´-m5CpG-3´/3´-Gpm5C-5´ dinucleotide pairs. On the other hand, whilst inverse correlation was observed between bulk DNA methylation, in S, and bulk RNA transcription, in G1 and G2, probes of methylated DNA helped to discover the presence of coding (exon) and uncoding (intron) sequences in the eukaryotic gene. These achievements led to the search for a language that genes regulated by methylation should have in common. Such a deciphering, initially providing restriction minimaps of hypermethylatable promoters and introns vs. hypomethylable exons, became feasible when bisulfite methodology allowed the direct sequencing of m5C. It emerged that, while in lymphocytes, where the transglutaminase gene (hTGc) is inactive, the promoter shows two fully methylated CpG-rich domains at 5´ and one fully unmethylated CpG-rich domain at 3´ (including the site +1 and a 5´-UTR), in HUVEC cells, where hTGc is active, in the first CpG-rich domain of its promoter four CpGs lack -CH3: a result suggesting new hypotheses on the mechanism of transcription, particularly in connection with radio-induced DNA demethylation.
KEY WORDS: maintenance and de novo post-synthetic modification, demethylation, Okazaki fragments, parental and daughter chains, eukaryotic gene structure, coding (exon) and uncoding (promoter and intron) regions, unique, repeated, foldback, viral and mitochondrial sequences, restriction minimaps, hTGc gene, m5C sequencing of bisulfite-converted sequences, CpG-rich domains, regulation of transcription