Inhibition of poly(ADP-ribosyl)ation introduces an anomalous methylation pattern in transfected foreign DNA.
The aim of this paper is to verify whether the control played by poly(ADP-ribosyl)ation on genomic DNA methylation, and in particular on CpG islands, can also be seen on foreign DNA transfected in cells where inhibition of the poly(ADP-ribosyl)ation process was obtained by treating them with 2 mM 3-aminobenzamide for 24 h. The CpG island-like pVHCk plasmid containing the bacterial chloramphenicol acyltransferase (CAT) gene under the control of SV40 early promoter was transfected in L929 mouse fibroblast cells. The bisulfite reaction, which is capable of immortalizing the methylation state of cytosine on DNA, was performed before amplification of the plasmid DNA fragment, then used for sequence analysis. Our results have shown that 1) when transfected in control cells, the plasmid maintains its characteristic unmethylated pattern, whereas this pattern is lost when the plasmid is transfected in cells treated with 3-aminobenzamide; and 2) the presence of new methyl groups on plasmid DNA is paralleled by a decrease of CAT reporter gene expression. These data confirm that poly(ADP-ribosyl)ation is a process tightly involved in protecting genomic DNA from full methylation and suggest the use of 3-aminobenzamide as a possible experimental strategy to mime other conditions of DNA hypermethylation in cells.
Zardo
G
G
Department of Biomedical Sciences and Technologies, University of L'Aquila, Rome, Italy.
Marenzi
S
S
Perilli
M
M
Caiafa
P
P
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D015226:Dinucleoside Phosphates / Q000378:metabolism; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D015500:Chloramphenicol O-Acetyltransferase; D015226:Dinucleoside Phosphates; D011064:Poly Adenosine Diphosphate Ribose; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases; D011994:Recombinant Proteins; C015772:CpG dinucleotide