Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen

Abstract

DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells.

Fig. 1.

RNF4 promotes DNA demethylation. (A) Luciferase reporter assays transiently transfected with pCMV-sport6 (Ctrl) or the indicated genes. Luciferase expression was driven by the SV40 promoter in HEK293 cells, or the Oct4 promoter in P19 cells. Reporter plasmids were unmethylated (U) or in vitro methylated (M). Error bars indicate SEM (n = 6). (B) GFP reporter assay in HEK293 cells transiently transfected with the indicated genes. pEGFP-N1 vector was in vitro methylated. (Scale bar: 20 μm.) (C) Bisulfite sequence analysis of the p16^INK4a promoter in HCT116 cells transiently transfected with the indicated genes. White and black circles represent unmethylated and methylated CpG, respectively. Images are representative result of three independent experiments. (D) Methylation-specific PCR was performed using bisulfate-treated genomic DNA as template with primers specific for unmethylated CpG template (p16 U) and methylated CpG template (p16 M) in HCT116 cells. Untreated genomic DNA was used as a loading control (p16 WT). (E) p16 mRNA was determined by qRT-PCR in HCT116 cells. GAPDH mRNA was used for normalization. P = 0.001; error bar indicate SEM (n = 3).

Fig. 2.

Rnf4 deficiency increases global methylation level. (A) Bisulfite sequencing of a maternal imprinted locus, Peg3 (paternal expressed gene 3), on genomic DNA from Rnf4^+/+ or Rnf4^−/− MEFs. Representative results from three independent experiments are shown. (B) Southern blot of DNA methylation in minor satellite region. Genomic DNA was digested with the methylation-sensitive enzyme HpaII (H) or methylation-insensitive enzyme MspI (M), blotted, and probed by minor satellite pMR-150 probe. (C) m⁵C content on CCGG sequences in genomic DNA is determined using the end-labeling assay followed by TLC separation. The intensity of the spot corresponding to dm⁵CMP is quantified by scintillation counting. Representative results from three independent experiments are shown. P < 0.05; error bars indicate SEM (n = 3).

Fig. 3.

RNF4 interacts and requires BER pathway enzymes for active demethylation. (A) RNF4 overexpression induces demethylation of pGL3 plasmid. Methylated or unmethylated pGL3 plasmids were used as negative or positive controls, respectively (lanes 1 and 2). Plasmids recovered from cell culture were digested with HpaII, and the products were analyzed by Southern blot. Arrow, methylated plasmid; Arrowhead, unmethylated fragment. (B) RNF4 interacts with TDG and APE1 in cells. Endogenous TDG and APE1 were coimmunoprecipitated from Flag-tagged RNF4-transfected HEK293 cells. (C) Reverse Co-IP detected RNF4 association with myc-tagged TDG and APE1. (D) TDG and APE1 can synergize with RNF4 for active demethylation. Methylated pGL3 plasmid was recovered from cells cotransfected with suboptimal amounts of RNF4 (0.5 μg) and the designated genes (0.5 μg), digested with HpaII, and analyzed by Southern blot. Arrow, methylated plasmid; arrowhead, unmethylated fragment. (E and F) TDG and APE1 are required for RNF4-induced active demethylation. shRNAs targeting TDG and APE1 were used to knock down the expression of the corresponding protein (Western blot, Lower). Active demethylation by RNF4 is analyzed by Southern blot using recovered plasmids from cells cotransfected with corresponding shRNAs (Upper). Arrow, methylated plasmid; arrowhead, unmethylated fragment.

Fig. 4.

RNF4 promotes DNA G:T mismatch repair. (A) The time-dependent generation of DNA nicking was assayed by incubation of immunoprecipitated TDG and APE1 with double-stranded 60-mer substrate (50 nM) containing a single G:T mismatch (39). Average numbers of three independent assays were graphed. (B) Relative response ratio for G:T mismatch firefly luciferase reading vs. Renilla luciferase reading (Materials and Methods). P = 0.01; error bars indicate SEM (n = 6). The number of AP sites per 10⁵-bp genomic DNA was assayed using aldehyde reactive probe and colorimetric detection (Materials and Methods). P < 0.0001; error bars indicate SEM (n = 6). HEK293 genomic DNA (C) and RNF4 MEFs genomic DNA (D) were assayed.