The Obesity-AssociatedFTOGene Encodes a 2-Oxoglutarate-Dependent Nucleic Acid Demethylase

Abstract

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)-and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)-and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the Copyright 2007 by the American Association for the Advancement of Science; all rights reserved. ||To whom correspondence should be addressed. E-mail: chris.ponting@dpag.ox.ac.uk (C.P.P.); frances.ashcroft@dpag.ox.ac.uk (F.M.A.); so104@medschl.cam.ac.uk (S.O.); christopher.schofield@chem.ox.ac.uk (C.J.S.). * These authors contributed equally to this work. † These authors contributed equally to this work. ‡ These authors contributed equally to this work. § These authors contributed equally to this work. Recent studies have revealed a strong association between common variants in the first intron of FTO and obesity in both children and adults, with ~16% of studied populations 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 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 (2OG) oxygenases [for a review of these enzymes, see (6)] (Fig. 1). The predicted DSBH fold of FTO contains four conserved residues characteristic of Fe(II) and 2OG binding sites (7,8), and its sequence is highly conserved in organisms ranging from mammals to green algae ( Fig. 1 and fig. S1). 2OG oxygenases are involved in diverse processes, including DNA repair, fatty acid metabolism, and posttranslational modifications, for example, proline hydroxylation and histone lysine demethylation [reviewed in (6, 9)]. They require nonheme iron [Fe(II)] as a cofactor, use oxygen and, almost always, 2OG as cosubstrates, and produce succinate and carbon dioxide as by-products.To determine whether FTO is a 2OG oxygenase, we expressed the murine Fto gene in Escherichia coli and purified N-terminally hexa-His tagged Fto (10). Some 2OG oxygenases catalyze 2OG turnover without a "prime" substrate provided that a reducing agent, typically ascorbate, is present (uncoupled turnover We next considered the identity of the prime FTO substrate. Among 2OG oxygenases with known substrates, the FTO sequence is most similar to that of the E. coli enzyme AlkB (11) and its eukaryotic hom...

Human obesity as a heritable disorder of the central control of energy balance

“…Working together with Tomas Lindahl from the Cancer Research UK labs at South Mimms, UK, and later with Chris Schofield, Chris Ponting, and Fran Ashcroft from Oxford, who had independently spotted the homology, we established unequivocally that FTO was a member of this family of enzymes and was capable of demethylating specific forms of nucleic acid. 48 We also showed that FTO was particularly highly expressed in hypothalamic nuclei concerned with energy balance and, in at least one of those nuclei (the arcuate), that feeding and fasting influence its expression. Although the link between nucleic acid demethylase activity of FTO and its role in obesity is still entirely obscure, the fact that FTO is highly expressed in the hypothalamus and is nutritionally regulated there suggests that it may also play a role in the 'hypothalamic setpoint' of appetite and satiety.…”

BAC transgenic zebrafish reveal hypothalamic enhancer activity around obesity associated SNP rs9939609 within the human FTO gene

“…Transgenic larvae with this BAC inserted in their genomes had weak ubiquitous expression that appeared strongest in the eye and in muscle fibers. This is in line with in situ hybridization analyses showing that mouse Fto has low‐level ubiquitous expression (Gerken et al ., 2007; Smemo et al ., 2014). The BAC transgene also confirmed an earlier result with a very similar BAC construct, showing that replacing the first exon of Fto with a lacZ cassette recapitulated the endogenous Fto expression pattern (Smemo et al ., 2014).…”

FTO levels affect RNA modification and the transcriptome

“…Modification of brain RNA in wild-type and Fto-knockout mice As shown by Gerken et al, 12 3-methyl-uracil and 3-methyl-thymine in single-stranded RNA and DNA, respectively, are the preferred in vitro substrates of mouse and human Fto/FTO. Given that RNA Total RNA from whole brains of wild-type and Fto À/ À mice was prepared and separated into two fractions: large RNAs (4200 bases) and small (o 200 bases) RNAs.…”