HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

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...