The Obesity-Associated FTO Gene Encodes a 2-Oxoglutarate-Dependent Nucleic Acid Demethylase Thomas Gerken, et al. Science 318, 1469 (2007); DOI: 10.1126/science.1151710 The following resources related to this article are available online at www.sciencemag.org (this information is current as of January 9, 2008 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/cgi/content/full/318/5855/1469 Supporting Online Material can be found at: http://www.sciencemag.org/cgi/content/full/1151710/DC1 A list of selected additional articles on the Science Web sites related to this article can be found at: This article cites 28 articles, 10 of which can be accessed for free: http://www.sciencemag.org/cgi/content/full/318/5855/1469#otherarticles This article appears in the following subject collections: on January 9, 2008 Medicine, Diseases http://www.sciencemag.org/cgi/collection/medicine Information about obtaining reprints of this article or about obtaining permission to reproduce this article in whole or in part can be found at: http://www.sciencemag.org/about/permissions.dtl www.sciencemag.org Downloaded from Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2007 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS. REPORTS Fe(II)and2OGbindingsites(7, 8), and its se- The Obesity-Associated FTO Gene quence is highly conserved in organisms ranging from mammals to green algae (Fig. 1 and fig. S1). Encodes a 2-Oxoglutarate–Dependent 2OG oxygenases are involved in diverse processes, including DNA repair, fatty acid metabolism, and posttranslational modifications, for example, pro- Nucleic Acid Demethylase line hydroxylation and histone lysine demethyla- tion [reviewed in (6, 9)]. They require nonheme 1 2 3 1† Thomas Gerken, Christophe A. Girard, * Yi-Chun Loraine Tung, * Celia J. Webby, iron [Fe(II)] as a cofactor, use oxygen and, almost 3† 1,4† 3† 1† Vladimir Saudek, Kirsty S. Hewitson, Giles S. H. Yeo, Michael A. McDonough, always, 2OG as cosubstrates, and produce suc- Sharon Cunliffe,4† Luke A. McNeill,1,4† Juris Galvanovskis,5† Patrik Rorsman,5 Peter Robins,6 3 3 3 3 3 cinate and carbon dioxide as by-products. Xavier Prieur, Anthony P. Coll, Marcella Ma, Zorica Jovanovic, I. Sadaf Farooqi, To determine whether FTO is a 2OG oxy- 6 7 6 8‡ Barbara Sedgwick, Inês Barroso, Tomas Lindahl, Chris P. Ponting, §|| genase, we expressed the murine Fto gene in 2‡ ’ 3 1‡ Frances M. Ashcroft, §|| Stephen O Rahilly, §|| Christopher J. Schofield §|| Escherichia coli and purified N-terminally hexa- His tagged Fto (10). Some 2OG oxygenases cat- FTO Variants in the (fat mass and obesity associated) gene are associated with increased body mass alyze 2OG turnover without a “prime” substrate index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with provided that a reducing agent, typically ascorbate, – Fe(II)- and 2-oxoglutarate dependent oxygenases. We find that recombinant murine Fto catalyzes is present (uncoupled turnover). 2OG uncoupled the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with turnover assays with Fto, monitoring conversion concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a of [1-14C]-2OG into [14C]-carbon dioxide (10), potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. revealed that Fto catalyzed 2OG decarboxylation, Fto Studies of wild-type mice indicate that messenger RNA (mRNA) is most abundant in the brain, a reaction that was stimulated by ascorbate and particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the FeSO4 (fig. S2A). 2OG turnover was inhibited arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward by known 2OG oxygenase inhibitors (fig. S2B) determining the physiologically relevant FTO substrate and how nucleic acid methylation status is and by the absence of Fe(II) and ascorbate. linked to increased fat mass. We next considered the identity of the prime FTO substrate. Among 2OG oxygenases with on January 9, 2008 ecent studies have revealed a strong asso- (2OG) oxygenases [for a review of these enzymes, known substrates, the FTO sequence is most ciation between common variants in the see (6)] (Fig. 1). The predicted DSBH fold of FTO similar to that of the E. coli enzyme AlkB (11) Rfirst intron of FTO and obesity in both contains four conserved residues characteristic of and its eukaryotic homologs, members of the children and adults, with ~16% of studied pop- ulations homozygous for the risk alleles (1–4). As adults, these individuals weigh ~3 kg more than those homozygous for the low risk alleles as a result of a specific increase in fat mass (2). FTO mRNA is expressed in a wide range of www.sciencemag.org human tissues (2). The Fto gene was first cloned after identification of a fused-toe mutant mouse whose phenotype results from a 1.6-Mb deletion of six genes, including Fto (5). Sequence analysis predicts that FTO protein contains a double-stranded beta-helix (DSBH) fold homologous to those of Fe(II) and 2-oxoglutarate Downloaded from 1Chemistry Research Laboratory and Oxford Centre for Integrative Systems Biology, University of Oxford, 12 Mansfield Road, Oxford, Oxon OX1 3TA, UK. 2Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford, Oxon OX1 3PT, UK. 3University of Cambridge, Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK. 4ReOx Ltd., Magdalen Centre, The Oxford Science Park, Oxford, Oxon OX4 4GA, UK. 5Oxford Centre Fig. 1. Multiple sequence alignment of FTO from human (Homo sapiens;Hs),mouse(Mus musculus; for Diabetes, Endocrinology and Metabolism, Churchill Mm) and green algae (Ostreococcus tauri; Ot) with Escherichia coli (Ec) and Shewanella woodyi (Sw) Hospital, Oxford, Oxon OX3 7LJ, UK. 6Cancer Research UK AlkB and human ABH2 and ABH3. A comparison of Ot FTO with the nonredundant protein sequence London Research Institute, Clare Hall Laboratories, South 28 7 database at the National Center for Biotechnology Information using PSI-BLAST ( )revealed Mimms, Hertfordshire EN6 3LD, UK. Metabolic Disease E Group, Wellcome Trust Sanger Institute, Hinxton, Cam- significant similarity ( < 0.005) between human FTO, bacterial AlkB, and its human homologs, within bridge, UK. 8MRC Functional Genetics Unit, Department of two iterations. Conserved residues highlighted in red are histidine and carboxylate (Asp or Glu) Fe(II) Physiology, Anatomy and Genetics, University of Oxford, binding residues of 2OG oxygenases (His-228/231, Asp-230/233, and His-304/307 in murine Fto/human South Parks Road, Oxford, Oxon OX1 3QX, UK. FTO, respectively), as well as an arginine (Arg-313/316), which binds the 2OG C-5 carboxylate in a 2OG *These authors contributed equally to this work. oxygenase subfamily (7, 8). The cylinders and arrows represent a helices and b strands, respectively, †These authors contributed equally to this work. assigned as in a crystal structure of ABH3 (29). Secondary structure in yellow represents the N-terminal ‡These authors contributed equally to this work. region, blue strands represent an assigned (29) substrate binding lid for ABH3, green strands labeled §These authors contributed equally to this work. with roman numerals identify the eight b strands that form the conserved double-stranded beta-helix of ||To whom correspondence should be addressed. E-mail: [email protected] (C.P.P.); frances.ashcroft@ the 2OG oxygenases. GenInfo numbers: human FTO: 122937263; mouse Fto: 6753916; green algae dpag.ox.ac.uk (F.M.A.); [email protected] (S.O.); FTO: 116060758; human ABH2: 48717226; human ABH3: 21040275; E. coli AlkB: 113638; S. woodyi [email protected] (C.J.S.) AlkB: 118070714. www.sciencemag.org SCIENCE VOL 318 30 NOVEMBER 2007 1469 REPORTS ABH (AlkB homolog) family (Fig. 1). AlkB is We screened potential Fto substrates, in- were also inhibitors in the 2OG uncoupled a DNA repair enzyme that repairs cytotoxic cluding a synthetic single-stranded 1-methyl turnover assays (Fig. 2C). 1-methyladenine (1-meA) and 3-methylcytosine adenine (1-meA) methylated oligonucleotide, Using a liquid chromatography–mass spec- (3-meC) lesions by methyl group hydroxylation, Lys-9 methylated histone H3, hypoxia-inducible trometry assay that directly monitors DNA followed by a retro-aldol reaction. Among the factor-1a (HIF-1a) subunit fragments, IkBa, demethylation [without the need for radiolabeled various human ABHs, only two, ABH2 and coenzyme A derivatives, and other known (co-)substrates or coupled assays (10)], we dem- ABH3, have been shown to exhibit DNA de- substrates of human 2OG oxygenases (10). onstrated that Fto catalyzes Fe(II)- and 2OG- methylation activity analogous to that of AlkB Only the 1-meA methylated oligonucleotide dependent DNA demethylation. This activity was (12, 13). ABH 2 and ABH3 are ubiquitously stimulated turnover of 2OG above control levels stimulated by ascorbate, as observed for other 2OG expressed, and their expression is not known to (Fig. 2A). This activity was inhibited by N- oxygenases (6) (Fig. 2B). Significantly reduced be altered by physiological stimuli. oxalylglycine, fumarate, and succinate, which turnover was observed when the reaction was on January 9, 2008 Fig. 2. Fto is a 2-oxoglutarate–dependent DNA demethylase in vitro. (A) The oxygen control reaction was carried out in an atmosphere of <1% O2; Demethylation of 1-meA is dependent on Fto in 2OG decarboxylation assays; (C) Inhibition of Fto-catalyzed 1-meA demethylation in 2OG decarboxylation (B) Cofactor/cosubstrate dependence of FTO activity on a 3-meT substrate assays.