Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene

Citation
B. Zhu et al., Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene, P NAS US, 98(9), 2001, pp. 5031-5036
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN journal
0027-8424 → ACNP
Volume
98
Issue
9
Year of publication
2001
Pages
5031 - 5036
Database
ISI
SICI code
0027-8424(20010424)98:9<5031:OO5DGI>2.0.ZU;2-X
Abstract
We have shown that the DNA demethylation complex isolated from chicken embr yos has a G T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kid ney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus p romoter overexpress 5-MCDG, A 15- to 20-fold overexpression of 5-MCDG resul ts in the specific demethylation of a stably integrated ecdysone-retinoic a cid responsive enhancer-promoter linked to a p-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A tan analog ue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction . Demethylation occurs downstream of the hormone response elements. No geno me-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promot er-enhancer of the reporter gene. An increase in 5-MCDG activity does not i nfluence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy numbe r during selection of the clones with antibiotics. Immunoprecipitation comb ined with Western blot analysis showed that an antibody directed against 5- MCDG precipitates a complex containing the retinoid X receptor a. The assoc iation between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may targ et demethylation of the transgene in this system.