Nucleic Acids Research, Vol 27, Issue 11 2332-2338, Copyright © 1999 by Oxford University Press
GZ Qu and M Ehrlich
In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid
usually greatly inhibits gene expression in mammalian cells. However, we
found that in vitro methylation of all CpGs in episomal or non-episomal
plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving
expression of an antibiotic-resistance gene decreased the formation of
antibiotic-resistant colonies by only approximately 30-45% upon stable
transfection of HeLa cells. In contrast, when expression of the
antibiotic-resistance gene was driven by the Rous sarcoma virus long
terminal repeat or the herpes simplex virus thymidine kinase promoter, this
methylation decreased the yield of antibiotic-resistant HeLa transfectant
colonies approximately 100-fold. The low sensitivity of the SV40 Pr/E to
silencing by in vitro methylation was probably due to demethylation upon
stable transfection. This demethylation may be targeted to the promoter and
extend into the gene. By genomic sequencing, we showed that four out of six
of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for
demethylation in the HeLa transfectants. High frequency demethylation at
Sp1 sites was unexpected for a non-embryonal cell line and suggests that
DNA demethylation targeted to certain aberrantly methylated regions may
function as a repair system for epigenetic mistakes.
ARTICLES
Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells
Department of Biochemistry, Hayward Genetics Center, and Tulane Cancer Center, Tulane Medical School, New Orleans, LA 70122, USA.
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