The unmethylated state of CpG islands in mouse fibroblasts depends on the poly(ADP-ribosyl)ation process.
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.
Zardo
G
G
Department of Biomedical Sciences and Technologies, University of L'Aquila, I-67100 L'Aquila, Italy.
Caiafa
P
P
D000818:Animals; D018899:CpG Islands; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics; D016350:Leucine Zippers / Q000235:genetics; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics; D016601:RNA-Binding Proteins; D015183:Restriction Mapping; D020931:ran GTP-Binding Protein
D009687:Nuclear Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; C091775:ran-binding protein 1; C070847:Htf9c protein, mouse; D011064:Poly Adenosine Diphosphate Ribose; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein