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

Abstract

We have shown that the DNA demethylation complex isolated from chicken embryos has a G(.)T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a beta-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A (an analogue 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 genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor alpha. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.

Figure 1

Silenced methylated reporter genes can be reactivated with 5-azacytidine. (Top) β-Galactosidase activity from cells stably transfected with the β-galactosidase gene under the control of an nonmethylated promoter-enhancer. Ponasterone A (10 μM) was added to the cultures and induction of β-galactosidase was tested 48 h later. (Middle) Same cell type stably transfected with a β-galactosidase construct containing the methylated promoter-enhancer. (Bottom) Pretreatment of above cells (silent reporter gene) with 2.5 μM 5-azacytidine for 5 days and then further a treatment with 10 μM ponasterone A for 48 h.

Figure 2

Overexpression of 5-MCDG in human embryonal kidney cells allows the induction of the reporter gene β-galactosidase by ponasterone A from a methylated promoter-enhancer. (Top) The empty vector was stably transfected in the cell line containing the silent methylated reporter gene. (Middle) 5-MCDG was stably transfected into the cell line containing the silent reporter gene. (Bottom) Inactive 5-MCDG mutant was transfected as above. When the reporter gene was demethylated, it could be induced by ponasterone A (10 μM for 48 h).

Figure 3

Relative specific activity of β-galactosidase from cells not treated or treated with ponasterone A. These cell lines were permanently transfected with the nonmethylated or the demethylated reporter gene. In the latter, the cell line was further stably transfected with an empty vector or a vector containing 5-MCDG or a mutant of 5-MCDG. Cells treated with 10 μM ponasterone A were tested for β-galactosidase activity 48 h later. ONPG, ortho-nitrophenyl-β-D-galactopyranoside.

Figure 4

Western blot of nuclear extract with anti-5-MCDG (G⋅T mismatch glycosylase) antibodies. (Upper) Lanes: 1, nuclear extract from parent cells (293 M+); 2, nuclear extract from parent cells transfected with the inactive mutant of 5-MCDG; 3, nuclear extract from cells transfected with the empty vector; 4, nuclear extract from cells transfected with 5-MCDG cDNA. (Lower) Total protein loading with Ponceau staining before immunoreaction.

Figure 5

Activity of 5-MCDG from nuclear extracts. Average values (triplicates) obtained by cutting out the specific band (27-mer) from the gel and measuring the radioactivity. (Inset) Separation of the reaction product (27-mer) on a sequencing gel (20% polyacrylamide). Lanes: 1, parent cell line; 2, cell line transfected with the empty vector; 3, cell line transfected with the inactive mutant of 5-MCDG; 4, cell line transfected with the 5-MCDG; 5, blank.

Figure 6

Southern blot of genomic DNA from clones of stably transfected kidney cells digested as follows. Lanes: D, BglII and AflII; M, BglII, AflII, and MspI; H, BglII, AflII, and HpaII. Vc2 and Vc3 are clones stably transfected with the empty vector. HGc2, HGc5, and HGc7 are clones stably transfected with 5-MCDG. (These last three clones stain positive for β-galactosidase activity after ponasterone A treatment). When the promoter-enhancer is demethylated, digestion with HpaII gives DNA fragments of 180–300 bp.

Figure 7

State of methylation of the promoter-enhancer region (600 bp) of the reporter gene in cell lines containing the empty vector (Top), the vector and 5-MCDG (Middle), and the vector and an inactive mutant of 5-MCDG (Bottom). For one cell line (HGC2 from Fig. 6), the bisulfite genomic sequencing of individual clones is shown. Solid circles are methylated CpGs, and vertical bars without circle are nonmethylated CpGs. TSS, transcription start site.

Figure 8

(Upper) Copy number determination of the integrated lacZ gene. Lanes: 1, parent cell line 293 M+; 2, mutant 5-MCDG; 3, cell line with empty vector; 4, cell line with 5-MCDG. Total genomic DNA was digested with EcoRI. EcoRI cuts only once in the integrated construct with a second cut generating the 6-kb fragment in surrounding genomic DNA. In Southern blots, the coding region of lacZ was used as a labeled probe. (Lower) Under the antibiotic selection of transformants, there is no amplification of the reporter gene. Slot blots of total genomic DNA (cleaved with EcoRI) where the coding region of lacZ was used as labeled probe. Rows: A, cell line with 5-MCDG; B, cell line with the empty vector; C, cell line with the mutant 5-MCDG; D, parent cell line 293 M+. 293 M+ is the parent cell line and is transfected with the mutant of 5-MCDG, the empty vector, or 5-MCDG.

Figure 9

Antibodies against 5-MCDG precipitate a complex containing RXRα. (A) Immunoprecipitation of a labeled retinoic acid binding protein with anti-5-MCDG. Bars: 1, nuclear extract of cells transfected with the empty vector; 2, with the inactive mutant 5-MCDG; 3, with the active 5-MCDG. Data are the mean ± SD (n = 3). (B) Western blot of the immunoprecipitates of A with RXRα antibodies.