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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-08, Vol.107 (34), p.15087-15092
• Main Authors: Hu, Xiaoyi V., Rodrigues, Tânia M. A., Tao, Haiyan, Baker, Robert K., Miraglia, Loren, Orth, Anthony P., Lyons, Gary E., Schultz, Peter G., Wu, Xu
• Format: Article
• Language: English
• Subjects: Aging; Animals; Base excision repair; Base Sequence; Biological Sciences; Cell Line; Deamination; Demethylation; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA methylation; DNA Methylation - genetics; DNA Methylation - physiology; DNA methyltransferase; DNA mismatch repair; DNA Mismatch Repair - genetics; DNA Mismatch Repair - physiology; DNA repair; Embryos; Enzymatic activity; Enzyme kinetics; Enzymes; Epigenetics; Female; Gene regulation; Genes, Lethal; Genes, p16; Genes, Reporter; Genetic screening; Genomics; HEK293 cells; Humans; Mammalian cells; Methylation; Mice; Mice, Knockout; Nuclear Proteins - deficiency; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Plasmids; Pregnancy; Promoter Regions, Genetic; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RING finger proteins; Stem cells; Transcription; Transcription Factors - deficiency; Transcription Factors - genetics; Transcription Factors - metabolism; Ubiquitin
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1009025107

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.