Abstract
In vivo and in vitro experiments carried out on L929 mouse fibroblasts suggested that the poly(ADP-ribosyl) ation process acts somehow as a protecting agent against full methylation of CpG dinucleotides in genomic DNA. Since CpG islands, which are found almost exclusively at the 5'-end of housekeeping genes, are rich in CpG dinucleotides, which are the target of mammalian DNA methyltransferase, we examined the possibility that the poly(ADP-ribosyl)ation reaction is involved in maintaining the unmethylated state of these DNA sequences. Experiments were conducted by two different strategies, using either methylation-dependent restriction enzymes on purified genomic DNA or a sequence-dependent restriction enzyme on an aliquot of the same DNA, previously modified by a bisulfite reaction. With the methylation-dependent restriction enzymes, it was observed that the "HpaII tiny fragments" greatly decreased when the cells were preincubated with 3-aminobenzamide, a well known inhibitor of poly(ADP-ribose) polymerase. The other experimental approach allowed us to prove that, as a consequence of the inhibition of the poly(ADP-ribosyl)ation process, an anomalous methylation pattern could be evidenced in the CpG island of the promoter fragment of the Htf9 gene, amplified from DNA obtained from fibroblasts preincubated with 3-aminobenzamide. These data confirm the hypothesis that, at least for the Htf9 promoter region, an active poly(ADP-ribosyl)ation protects the unmethylated state of the CpG island.
Highlights
During pre-implantation development, most DNA sequences undergo extensive demethylation
The aim of this paper was to verify if, in cells preincubated with 3-aminobenzamide, the DNA methyltransferase becomes able to modify the unmethylated state of CpG islands, which normally remain untouched by the action of DNA methyltransferase even though they are located in the promoter region of housekeeping genes [7,8,9], which are permanently accessible to the transcription factors
The methylation state of CpG islands was investigated by digesting, with methylation-dependent restriction enzymes, the genomic DNAs purified from nuclei in which the DNA methyl-accepting ability was saturated in the presence of 16 M S-AdoMet, exploiting endogenous DNA methyltransferase activity
Summary
During pre-implantation development, most DNA sequences undergo extensive demethylation. Our results, obtained using two different experimental approaches, allowed us to observe that the block of the poly(ADP-ribosyl)ation process modifies the methylation pattern of CpG islands in general and in particular introduces an anomalous methylation pattern in the Htf promoter region [10] when it is purified from cells treated with 3-aminobenzamide. These data confirm the hypothesis that, at least for the Htf promoter region, an active poly(ADP-ribosyl)ation protects its unmethylated state
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