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The language of methylation in genomics of eukaryotes
1Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy. volpe@bio.uniroma2.it
Abstract:
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.
Insights
DNA methylation patterns, specifically 5-methylcytosine (m5C), are crucial for eukaryotic gene regulation. Differential methylation in the transglutaminase gene promoter correlates with its activity, suggesting new transcription mechanisms and radio-induced demethylation insights.
Area of Science:
- Epigenetics and Molecular Biology
- Genomics and Gene Regulation
- Biochemistry of DNA Modification
Background:
- Eukaryotic genomes primarily feature 5-methylcytosine (m5C) in m5CpG doublets, unlike bacterial genomes with both m6A and m5C.
- DNA methylation, particularly during S phase, is vital for maintaining genetic patterns, especially in symmetrically dimethylated 5'-m5CpG-3'/3'-Gpm5C-5' dinucleotide pairs.
- An inverse correlation exists between bulk DNA methylation in S phase and bulk RNA transcription in G1 and G2 phases.
Purpose of the Study:
- To investigate the role of DNA methylation in eukaryotic gene regulation.
- To explore the relationship between DNA methylation patterns and gene transcription.
- To hypothesize new mechanisms of transcription, particularly concerning radio-induced DNA demethylation.
Main Methods:
- Analysis of DNA methylation patterns in eukaryotic genomes.
- Utilizing bisulfite methodology for direct sequencing of 5-methylcytosine (m5C).
- Comparative analysis of DNA methylation in the transglutaminase gene (hTGc) promoter in different cell types (lymphocytes vs. HUVEC cells).
Main Results:
- Eukaryotic genes contain coding (exon) and uncoding (intron) sequences, discovered through probes of methylated DNA.
- In lymphocytes (inactive hTGc), the promoter exhibits two methylated and one unmethylated CpG-rich domain.
- In HUVEC cells (active hTGc), the first CpG-rich domain of the promoter shows four unmethylated CpGs, suggesting altered methylation patterns influence transcription.
Conclusions:
- DNA methylation patterns in gene promoters are directly linked to gene activity.
- Differential methylation within CpG-rich domains of the hTGc promoter may regulate its transcription.
- These findings propose novel hypotheses regarding transcription mechanisms and the impact of DNA demethylation, potentially induced by radiation.
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