FIGURE 2.

RNF4 does not induce active DNA demethylation. A, MeGFP vector was co-transfected in 293T cells with RNF4 or factors involved in active DNA demethylation, including Tet2, TDG, and AID. Representative images show the expression of GFP. B, the number of GFP-positive cells per field in A was quantified, and the value of vector control was set as 1. Results from three independent experiments ± S.D. are shown. C, RNF4 does not affect 5caC level. Immunostaining with 5caC antibody detects accumulation of 5caC in Tet2-expressing 293T cells. 5caC level is not affected by either knockdown or overexpression of RNF4. In contrast, expression of TDG completely removes 5caC, and the activity of TDG is not impaired by RNF4 knockdown. siCT, scrambled siRNA control. D, RNF4 knockdown is confirmed by Western blot analysis. Densitometry quantification of RNF4 signal relative to tubulin is provided. E, purified RNF4 and TDG proteins were incubated with 5caC-containing oligonucleotide (arrow) for in vitro glycosylase assay. The lower bands (arrowhead) represent oligonucleotide nicked by TDG, which is not affected by the addition of RNF4. F, MeGFP co-expressed with empty vector or RNF4 was extracted from cells for bisulfite sequencing. The CMV promoter remains predominantly methylated (filled circles) in both control and RNF4 cells.