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Identification of RING Finger Protein 4 (RNF4) As a Modulator of DNA

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Identification of RING Finger Protein 4 (RNF4) As a Modulator of DNA Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen Xiaoyi V. Hua, Tânia M. A. Rodriguesb, Haiyan Taoc, Robert K. Bakerb, Loren Miragliac, Anthony P. Orthc, Gary E. Lyonsb,1, Peter G. Schultza,1, and Xu Wuc,1 aDepartment of Chemistry, Scripps Research Institute, La Jolla, CA 92037; bDepartment of Anatomy, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53706; and cInstitute of the Novartis Research Foundation, San Diego, CA 92121 Contributed by Peter G. Schultz, June 29, 2010 (sent for review June 15, 2010) DNA methylation is an important epigenetic modification involved In plants, genetic and biochemical experiments suggest that in transcriptional regulation, nuclear organization, development, active DNA demethylation is mediated through a base excision aging, and disease. Although DNA methyltransferases have been repair (BER) pathway initiated by m5C-specific DNA glycosylases characterized, the mechanisms for DNA demethylation remain (16, 17). It is possible that active demethylation in mammals uses poorly understood. Using a cell-based reporter assay, we per- similar mechanisms. However, the mammalian counterpart of the formed a functional genomics screen to identify genes involved in m5C-specific DNA glycosylase has yet to be identified. Sponta- DNA demethylation. Here we show that RNF4 (RING finger protein neous or enzymatic m5C deamination would generate a G:T 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in mismatch in DNA duplex, and subsequent G:T mismatch repair maintaining genome stability, plays a key role in active DNA has, therefore, been proposed as a possible mechanism for active demethylation. RNF4 reactivates methylation-silenced reporters demethylation in mammalian cells (8, 18). and promotes global DNA demethylation. Rnf4 deficiency is embry- Functional genomics approaches have been used to identify onic lethal with higher levels of methylation in genomic DNA. factors that are involved in diverse biological processes (19–21). Mechanistic studies show that RNF4 interacts with and requires Here we use a cell-based functional genomics screen to reveal CELL BIOLOGY the base excision repair enzymes TDG and APE1 for active deme- a key role for RNF4 in active DNA demethylation. Rnf4 de- thylation. This activity appears to occur by enhancing the enzymatic ficiency is embryonic lethal, resulting in a higher content of m5Cin activities that repair DNA G:T mismatches generated from methyl- genomic DNA. Furthermore, RNF4 interacts with and requires cytosine deamination. Collectively, our study reveals a unique func- BER enzymes for demethylation. tion for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells. Results Genome-Wide Gain-of-Function Screen Identifies RNF4 as a Regulator epigenetics | DNA repair | base excision repair of DNA Demethylation. To identify genes that promote DNA demethylation, we carried out a gain-of-function genome-wide NA methylation plays important roles in transcriptional reg- screen using a cell-based assay in which the methylation-silenced Dulation, genomic imprinting, and mammalian development p16INK4a promoter drives the expression of a luciferase reporter. (1). Deregulation of this important epigenetic modification has The p16INK4a promoter is known to be aberrantly silenced by been implicated in a number of diseases, including cancer and DNA methylation in many cancers (22). The reporter construct developmental defects (2). Methylation of DNA occurs at the 5C was methylated in vitro, purified, and cotransfected into HEK293 position of the CpG dinucleotide and is mediated by DNA cells in 384-well format with an arrayed cDNA expression library methyltransferases (DNMTs) (3). The de novo methyltransferases composed of 9,624 mouse and 6,415 human full-length cDNAs DNMT3a and DNMT3b are mainly responsible for introducing from the Mammalian Gene Collection (MGC) (23). GADD45a cytosine methylation at previously unmethylated CpG sites, was used as a positive control based on its previously reported whereas the maintenance methyltransferase DNMT1 copies pre- demethylation activity and gave a 10-fold increase in luciferase existing methylation patterns into the newly synthesized DNA activity 48 h posttransfection (10). From the primary screen, 19 strand during DNA replication (4). genes were found to induce luciferase activity greater than 10-fold Dynamic DNA methylation is critical during development (1, 5). and were subsequently reconfirmed using an in vitro methylated Although maintenance and de novo methylation mediated by pFMR1-luc reporter (24) (full hit list in Table S1). Among them, DNMTs are relatively well understood (4), the mechanisms of DNA GADD45g has been reported to be involved in DNA methylation demethylation are still elusive (6). Passive demethylation can occur regulations (9, 10). Other genes with DNA repair activities have through the inhibition of DNMT1 activity, causing a loss in the also been identified (25). methylation pattern during DNA replication. Rapid genome-wide RNF4 (RING finger protein 4, or SNURF) was one of the most demethylation is observed during gametogenesis and postfertiliza- active hits in our screen with a >20-fold increase in reporter ac- tion, suggesting an active demethylation mechanism independent of tivity (Table S1). RNF4 has a conserved function in genome sta- DNA replication. Recent studies have identified several factors that bility and DNA repair in eukaryotes (26, 27). In addition, we found are involved in active DNA demethylation, including the activation- induced cytidine deaminase (AID), an enzyme catalyzing 5-methyl cytidine (m5C) deamination in single-stranded DNA to generate Author contributions: X.V.H., A.P.O., G.E.L., P.G.S., and X.W. designed research; X.V.H., – T.M.A.R., H.T., R.K.B., and L.M. performed research; X.V.H., G.E.L., P.G.S., and X.W. ana- thymine and a G:T mismatch (7 9), and GADD45a, a nuclear pro- lyzed data; and X.V.H., G.E.L., P.G.S., and X.W. wrote the paper. tein involved in the maintenance of genomic stability and DNA The authors declare no conflict of interest. repair (9–11). However, neither AID- nor Gadd45a-deficient mice 1To whom correspondence may be addressed. E-mail: [email protected], schultz@scripps. (12, 13) exhibit catastrophic developmental defects (14, 15), sug- edu, or [email protected]. gesting other factors might be involved in the regulation of active This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DNA demethylation. 1073/pnas.1009025107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1009025107 PNAS | August 24, 2010 | vol. 107 | no. 34 | 15087–15092 Downloaded by guest on September 28, 2021 that several methylation-silenced luciferase and GFP reporters Analysis of the methylation status of endogenous CpG islands in were also reactivated by RNF4 overexpression in different cell the p16INK4a promoter by bisulfite sequencing revealed that RNF4 lines (SV40-Luc, pOct4-Luc, and CMV-eGFP reporters; Fig. 1 A overexpression significantly reduces the methylation level of the INK4a and B), indicating that the activity of RNF4 is both reporter and p16 promoter in the HCT116 colorectal cancer cell line, with complete demethylation in >80% of clones analyzed (Fig. 1C). We next examined the methylation status of the p16INK4a promoters 2000 70000 U U SV40 40000 Oct4 A 1500 M using methylation-specific PCR (28). Overexpression of either 600 M 30000 GADD45a or RNF4 increases unmethylated-specific PCR prod- U U 400 20000 uct and decreases methylated-specific PCR product (Fig. 1D). RL RL 200 10000 Furthermore, removal of the repressive DNA methylation marks by RNF4 transfection led to partial reactivation of gene expres- 0 0 INK4a Ctrl GFP GADD45a RNF4 Ctrl GFP GADD45a RNF4 sion, as shown by 40% higher p16 mRNA levels (P < 0.001) measured by qRT-PCR (Fig. 1E). B Rnf4 Deficiency Is Embryonic Lethal, and Loss of RNF4 Results in an Increase in Global DNA Methylation. To address whether RNF4 plays an essential role in mammalian development, we took advantage of a recessive lethal gene-trap insertion mutation (29, 30) to generate Rnf4 knockout mice (Fig. S1A). Although het- Ctrl Gadd45a Rnf4 erozygous animals exhibited no obvious phenotype, homozygous null embryos were stunted and failed to develop to term (SI C Materials and Methods, Figs. S1–S3, and Table S2). The embryos died between E14 and E15, and exhibited ventricular septal de- fects and cardiac insufficiency, which probably accounted for − − PFG lethality. Genomic DNA from the Rnf4+/+, and Rnf4 / mouse A +/+ 50% 4FNR 78% *** D E 1.4 H 1.2 GFP GADD45a RNF4 APD 1.0 p16 U G 0.8 6/ B +/+ +/ - -/ - C p16 M p1 0.6 HMHM H M 0.4 100 p16 WT mRNA 0.2 0.0 80 * GFP RNF4 P CM 60 5 Fig. 1. RNF4 promotes DNA demethylation. (A) Luciferase reporter assays m transiently transfected with pCMV-sport6 (Ctrl) or the indicated genes. Lu- fd 40 ciferase expression was driven by the SV40 promoter in HEK293 cells, or the %o 20 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 0 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 p16INK4a promoter in HCT116 cells transiently transfected with the indicated genes. White and black circles represent unmethylated and Fig. 2. Rnf4 deficiency increases global methylation level. (A) Bisulfite se- methylated CpG, respectively. Images are representative result of three in- quencing of a maternal imprinted locus, Peg3 (paternal expressed gene 3), − − dependent experiments. (D) Methylation-specific PCR was performed using on genomic DNA from Rnf4+/+ or Rnf4 / MEFs. Representative results from bisulfate-treated genomic DNA as template with primers specificfor three independent experiments are shown. (B) Southern blot of DNA unmethylated CpG template (p16 U) and methylated CpG template (p16 M) methylation in minor satellite region.
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