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-r ibosyl)-ation on genomic DNA methylation, and in particular on CpG islands, can also be seen on foreign DNA transfected in cells where inhibition of t he 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 S V40 early promoter was transfected in L929 mouse fibroblast cells. The bisu lfite reaction, which is capable of immortalizing the methylation state of cytosine on DNA, was performed before amplification of the plasmid DNA frag ment, then used for sequence analysis. Our results have shown that I) when transfected in control cells, the plasmid maintains its characteristic unme thylated pattern, whereas this pattern is lost when the plasmid is transfec ted in cells treated with S-aminobenzamide; and 2) the presence of new meth yl groups on plasmid DNA is paralleled by a decrease of CAT reporter gene e xpression. These data confirm that Poly(ADP-ribosyl)ation is a process tigh tly involved in protecting genomic DNA from full methylation and suggest th e use of 3-aminobenzamide as a possible experimental strategy to mime other conditions of DNA hypermethylation in cells.