GADD45A does not promote DNA demethylation

Abstract

Although DNA methylation patterns in somatic cells are thought to be relatively stable, they undergo dramatic changes during embryonic development, gametogenesis, and during malignant transformation. The enzymology of DNA methyltransferases is well understood, but the mechanism that removes methylated cytosines from DNA (active DNA demethylation) has remained enigmatic. Recently, a role of the growth arrest and DNA damage inducible protein GADD45A in DNA demethylation has been reported [1]. We have investigated the function of GADD45A in DNA demethylation in more detail using gene reactivation and DNA methylation assays. Contrary to the previous report, we were unable to substantiate a functional role of GADD45A in DNA demethylation. The mechanism of active DNA demethylation in mammalian cells remains unknown.

Figure 1. GADD45A does not reactivate a methylation-silenced EGFP reporter gene.

HEK293 cells were transiently transfected with an unmethylated (pEGFP-N2) or CpG-methylated (Me-pEGFP-N2) plasmid and the GADD45A expression vector. Fluorescence microscopy indicates that expression of GADD45A does not promote reactivation of EGFP.

Figure 2. Overexpression of GADD45A does not promote demethylation of a methylated pOct4-EGFP plasmid.

A. Schematic diagram of the pOct4-EGFP plasmid (not to scale). The 2.4 kb Oct4 promoter sequences are upstream of the EGFP gene. DE is the distal element, PE is the proximal element, and P is the minimal promoter of Oct4. This plasmid was methylated in vitro with HpaII or HpaII + HhaI. Black circles indicate HpaII sites and black triangles indicate HhaI sites. B. Confirmation of the methylation status of the six HpaII sites (black circles in panel A) by sodium bisulfite sequencing. Closed circles are methylated sites and open circles are unmethylated sites. C. Southern blot assay of plasmids recovered from HEK293 cells after transient transfection of the methylated and unmethylated pOct4-EGFP plasmid with and without GADD45A overexpression. The plasmids were digested with HincII and HpaII after in vitro methylation of the plasmid with HpaII (left panel) or HpaII and HhaI (right panel) prior to transfection. Digestion of the recovered methylated plasmid with HpaII does not lead to cleavage indicating that demethylation did not occur in HEK293 cells that overexpress GADD45A. D. Confirmation of GADD45A expression in transfected HEK293 cells by Western blotting with anti-GADD45A antibody. Beta-tubulin was probed as a loading control.

Figure 3. Bisulfite sequencing of the methylated pOct4-GFP plasmid recovered from HEK293 cells transfected in the absence or presence of the GADD45A expression vector.

A. The plasmid was methylated with HpaII methylase only. B. The plasmid was methylated with HpaII and HhaI methylases. The recovered DNA was treated with sodium bisulfite and the pOct4-EGFP target sequences containing the six HpaII sites (see Figure 2A) were amplified from bisulfite-treated DNA. The PCR products were cloned and between 22 and 25 individual molecules were sequenced. Closed circles mark methylated HpaII sites and open circles mark unmethylated HpaII sites. Overexpression of GADD45A (right panels) did not induce demethylation of the plasmid.

Figure 4. GADD45A does not promote demethylation of the endogenous pOct4 promoter in NIH3T3 cells.

A. The transfection efficiency of NIH3T3 cells was analyzed with an EGFP expression vector and was calculated to be ∼60%. DIC, differential interference contrast image; GFP, green fluorescence. B. Schematic diagram of the mouse Oct4 promoter region. DE is the distal element, PE is the proximal element, and P is the minimal promoter of Oct4. Black triangles indicate HpaII sites and the open triangle shows a HhaI site. C and D. Bisulfite sequence analysis of all CpGs in the Oct4 upstream region. Closed circles are methylated CpGs and open circles are unmethylated CpGs. C, control vector transfected cells; D, GADD45A expression vector transfected cells.

Figure 5. Bisulfite sequencing of two endogenous methylated genes in HEK293 cells transfected in the absence or presence of a GADD45A expression vector.

The promoter sequences of the RASSF1A (A) and TIG1 (B) genes are highly methylated in HEK293 cells. Transfection with the control (pcDNA3.1) vector and the GADD45A cDNA expression vector shows no substantial difference in genomic methylation patterns. Closed circles mark methylated CpG dinucleotides and open circles are unmethylated CpG dinucleotides.

Figure 6. GADD45A does not promote demethylation of endogenous LINE1 sequences in human cells.

HEK293 cells were transfected with pcDNA3.1 control vector or the pcDNA3.1-GADD45A expression vector. Forty-eight hours after transfection, genomic DNA was isolated and treated with sodium bisulfite. A. LINE1 promoter sequences were amplified and the PCR products were digested with HinfI, which cleaves only the methylated PCR products. B. The intensity of each cleavage product (bands 1–5) obtained from control or GADD45A-transfected cells was quantitated by densitometry. The result from three independent experiments is displayed (mean +/− S.D.).