Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D)1,2,3. Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes4,5,6. However, no direct connection between the obesity-associated variants and FTO expression or function has been made7,8,9. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Dina, C. et al. Variation in FTO contributes to childhood obesity and severe adult obesity. Nature Genet. 39, 724–726 (2007)
Frayling, T. M. et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science 316, 889–894 (2007)
Scuteri, A. et al. Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits. PLoS Genet. 3, e115 (2007)
Church, C. et al. Overexpression of Fto leads to increased food intake and results in obesity. Nature Genet. 42, 1086–1092 (2010)
Fischer, J. et al. Inactivation of the Fto gene protects from obesity. Nature 458, 894–898 (2009)
Gao, X. et al. The fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice. PLoS ONE 5, e14005 (2010)
Grunnet, L. G. et al. Regulation and function of FTO mRNA expression in human skeletal muscle and subcutaneous adipose tissue. Diabetes 58, 2402–2408 (2009)
Klöting, N. et al. Inverse relationship between obesity and FTO gene expression in visceral adipose tissue in humans. Diabetologia 51, 641–647 (2008)
Wåhlén, K., Sjolin, E. & Hoffstedt, J. The common rs9939609 gene variant of the fat mass- and obesity-associated gene FTO is related to fat cell lipolysis. J. Lipid Res. 49, 607–611 (2008)
McMurray, F. et al. Adult onset global loss of the fto gene alters body composition and metabolism in the mouse. PLoS Genet. 9, e1003166 (2013)
Jin, F. et al. A high-resolution map of the three-dimensional chromatin interactions in human cells. Nature 503, 290–294 (2013)
Houweling, A. C. et al. Gene and cluster-specific expression of the Iroquois family members during mouse development. Mech. Dev. 107, 169–174 (2001)
van Tuyl, M. et al. Iroquois genes influence proximo-distal morphogenesis during rat lung development. Am. J. Physiol. Lung Cell. Mol. Physiol. 290, L777–L789 (2006)
Gerken, T. et al. The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase. Science 318, 1469–1472 (2007)
Qi, L. et al. Fat mass-and obesity-associated (FTO) gene variant is associated with obesity: longitudinal analyses in two cohort studies and functional test. Diabetes 57, 3145–3151 (2008)
Stratigopoulos, G. et al. Regulation of Fto/Ftm gene expression in mice and humans. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1185–R1196 (2008)
Ragvin, A. et al. Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX, SOX4, and IRX3. Proc. Natl Acad. Sci. USA 107, 775–780 (2010)
Visel, A. et al. VISTA Enhancer Browser—a database of tissue-specific human enhancers. Nucleic Acids Res. 35 (Database issue). D88–D92 (2007)
Bosse, A. et al. Identification of the vertebrate Iroquois homeobox gene family with overlapping expression during early development of the nervous system. Mech. Dev. 69, 169–181 (1997)
Dixon, J. R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012)
Gamazon, E. R. et al. Enrichment of cis-regulatory gene expression SNPs and methylation quantitative trait loci among bipolar disorder susceptibility variants. Mol. Psychiatry 18, 340–346 (2012)
Speliotes, E. K. et al. Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index. Nature Genet. 42, 937–948 (2010)
Kong, D. et al. GABAergic RIP-Cre neurons in the arcuate nucleus selectively regulate energy expenditure. Cell 151, 645–657 (2012)
Shi, Y. C. et al. Arcuate NPY controls sympathetic output and BAT function via a relay of tyrosine hydroxylase neurons in the PVN. Cell Metab. 17, 236–248 (2013)
Mori, H. et al. Critical role for hypothalamic mTOR activity in energy balance. Cell Metab. 9, 362–374 (2009)
Dankel, S. N. et al. Switch from stress response to homeobox transcription factors in adipose tissue after profound fat loss. PLoS ONE 5, e11033 (2010)
Hagège, H. et al. Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nature Protocols 2, 1722–1733 (2007)
Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Meth. Mol. Biol. 132, 365–386 (2000)
Dekker, J. et al. Capturing chromosome conformation. Science 295, 1306–1311 (2002)
Noordermeer, D. et al. The dynamic architecture of Hox gene clusters. Science 334, 222–225 (2011)
Splinter, E. et al. Determining long-range chromatin interactions for selected genomic sites using 4C-seq technology: from fixation to computation. Methods 58, 221–230 (2012)
Stadhouders, R. et al. Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions. Nature Protocols 8, 509–524 (2013)
Denholtz, M. et al. Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and polycomb proteins in genome organization. Cell Stem Cell 13, 602–616 (2013)
Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009)
Kent, W. J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002)
Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009)
ENCODE. A user's guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol. 9, e1001046 (2011)
Zhou, X. & Wang, T. Using the Wash U Epigenome Browser to examine genome-wide sequencing data. Curr. Protoc. Bioinformatics 40, 10.10.1–10.10.14 (2012)
Li, G. et al. Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cell 148, 84–98 (2012)
Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–50 (2005)
Smemo, S. et al. Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease. Hum. Mol. Genet. 21, 3255–3263 (2012)
Wilkinson, D. G. & Nieto, M. A. Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol. 225, 361–373 (1993)
Elbein, S. C. et al. Genetic risk factors for type 2 diabetes: a trans-regulatory genetic architecture? Am. J. Hum. Genet. 91, 466–477 (2012)
Zhang, S. S. et al. Iroquois homeobox gene 3 establishes fast conduction in the cardiac His-Purkinje network. Proc. Natl Acad. Sci. USA 108, 13576–13581 (2011)
Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001)
Li, Z. J. et al. Kif7 regulates Gli2 through Sufu-dependent and -independent functions during skin development and tumorigenesis. Development 139, 4152–4161 (2012)
Eppig, J. T. et al. The Mouse Genome Database (MGD): comprehensive resource for genetics and genomics of the laboratory mouse. Nucleic Acids Res. 40 (Database issue). D881–D886 (2012)
Smith, C. L. & Eppig, J. T. The mammalian phenotype ontology: enabling robust annotation and comparative analysis. Biol. Med. 1 (3), 390–399 (2009)
Smith, R. N. et al. InterMine: a flexible data warehouse system for the integration and analysis of heterogeneous biological data. Bioinformatics 28, 3163–3165 (2012)
Dimitrieva, S. & Bucher, P. UCNEbase–a database of ultraconserved non-coding elements and genomic regulatory blocks Nucleic Acids Res. 41 (Database issue). D101–D109 (2013)
The authors thank F. Gage, C. Marchetto, B. Ren and F. Jin for their generosity in sharing reagents and data. This work was funded by grants from the National Institutes of Health (DK093972, HL119967, HL114010 and DK020595) to M.A.N. and (MH101820, MH090937 and DK20595) to N.J.C. J.L.G.-S. was funded by grants from the Spanish Ministerio de Economía y Competitividad (BFU2010-14839, CSD2007-00008) and the Andalusian Government (CVI-3488). C.-C.H. was supported by a grant from the Canadian Institute of Health Research. K.-H.K. is supported by a fellowship from the Heart and Stroke Foundation of Canada. S.S. is supported by an NIH postdoctoral training grant (T32HL007381)
The authors declare no competing financial interests.
Extended data figures and tables
4C-seq data for the Fto-Irx3 locus, visualized with the UCSC Genome Browser. a, Data (also shown in the circular plot in Fig. 1) generated using whole mouse embryos (E9.5), showing the frequency of interactions with the promoter of Irx3 (blue, top) or Fto (magenta, bottom). The background signal corrects for the strong correlation between (nonspecific) ligation events and the linear distance along the chromosome. Poisson statistical significance (−log(P value)) of the 4C-seq interactions over the background is plotted. Significant interactions (P < 0.01), ‘targets’, are displayed in black. b, As above for a but for adult mouse brain (8 weeks). c, As above for a but for whole zebrafish embryos (24 h post fertilization). In all, the region orthologous to the obesity association interval in the first intron of Fto is highlighted in pink.
a, ENCODE data for ChIA-PET using RNA polymerase 2 (POL2) in MCF7 (human breast adenocarcinoma) cells shows interactions between IRX3 and the obesity association interval in the first intron of FTO. No interactions are observed between the FTO promoter and the association interval. These public data are available from and was visualized with the WashU EpiGenome Browser (http://epigenomegateway.wustl.edu/browser/). b, Hi-C data previously generated11 in human IMR-90 (fetal lung) cells. In the association interval, the IRX3 signal is stronger than the background (random) signal. However, the signal for FTO is not. c, 3C data generated with adult (8 weeks) mouse brain. Using bait (red circle) in the association interval (red rectangle), we observe more frequent interactions with the Irx3 promoter compared to control regions 1 and 2 that are 29 and 42 kb away, respectively, indicative of looping.
a, FTO expression in lung and brain, shown by RNA in situ hybridization for mouse Fto mRNA, in newborn (P1) mouse. Lungs and heart (left, whole organs) were processed simultaneously and in the same well as brain (right, sagittal section) so that the relatively higher expression in brain can be observed. b, LacZ staining for β-galactosidase expression driven from the human FTO promoter. Top, the promoter–LacZ fusion is in the context of 162-kb of human genomic sequence carried in a BAC containing the first three exons of FTO, the entire obesity-associated interval and any enhancers present. The broad expression is consistent with previous reports in human and mouse (see main text for references). At bottom, the promoter–LacZ construct is isolated: only the 1,237 bp proximal to the transcriptional start site are included. Broad expression is recapitulated, indicating the robust transcriptional competency of the human FTO promoter. c, In contrast, the 2,820-bp proximal human IRX3 promoter is not sufficient to drive LacZ expression, which is consistent with an enhancer-dependent transcriptional control mechanism.
a, IRX3 expression in human tissues including brain. Expression data, measured on Affymetrix HG-U133 arrays, were obtained from the Body Atlas, Tissues (http://www.nextbio.com). The median expression across all 128 human tissues from 1,068 arrays is shown by the red line. b, IRX3 expression in 11 different regions of human brain. Data were retrieved from Human Brain Transcriptome data (http://www.molecularbrain.org). Amyg: amygdala; Caud nuc: caudate nucleus; Cere: cerebellum; Corp Call: corpus callosum; DRG: dorsal root ganglion; Frnt Cort: frontal cortex; Hippo: hippocampus; Hypo: hypothalamus; Pit: pituitary; Spine: spinal cord; Thal: thalamus.
Linkage disequilibrium (LD) plot of (logarithm (base 10) of odds) (LOD) score from HapMap phase II European data set, visualized in the UCSC browser. LD blocks are outlined in black. Obesity-associated SNPs from the National Human Genome Research Institute (NHGRI) GWAS catalogue are shown above, in green, demonstrating why this LD block is considered to define the ‘association interval’.
a, Representative photograph of WT and Irx3 KO mice fed ND at 18 weeks of age. b, Representative anatomical views of WT and Irx3 KO mice fed ND. Yellow dotted lines depict subcutaneous IWAT (left) and visceral PWAT (right). c, Tissue weights as a percentage of body weight showed smaller fat pad sizes in Irx3 KO mice, compared to WT mice, in both ND and HFD conditions. (ND, WT/KO, n = 20/12; HFD, WT/KO, n = 8/5.) Data are mean ± s.e.m. (*P < 0.05 versus WT, ND; †P < 0.05 versus WT, HFD). d, Representative H&E sections of PWAT, IWAT and BAT from ND mice demonstrated smaller adipocyte size in Irx3 KO mice than control mice. e, Quantitative PCR of WT and Irx3 KO PWAT for the indicated marker genes: leptin (lep) and adiponectin (adipoq) are adipogenic markers, positively and negatively associated with adiposity, respectively; Mcp1 correlates positively with adiposity. (*P < 0.05 versus WT value.) (WT/KO, n = 10/7).
a, Body weight (BW) changes of WT and Irx3 KO female mice fed a normal diet (ND). (WT/KO: n = 15/14). b, BMI, calculated by BW/BL2 (BL, body length), is lower in Irx3 KO female mice. (WT/KO, n = 7/7). c, d, Body composition analysis showed reduced fat mass and to a lesser extent reduced lean mass in Irx3 KO female mice compared to WT mice, leading to decreased fat mass ratio (WT/KO, n = 9/8). e, Representative H&E-stained sections of mammary gland (MG) WAT and periovarian (PO) WAT revealed smaller adipocyte size in Irx3 KO female mice, compared to WT. f, MGWAT and BAT weights as a percentage of body weight (WT/KO, n = 4/5). Data are mean ± s.e.m. (*P < 0.05 versus WT value.)
a, Energy expenditure over a 24-h period, corrected for lean mass (kcal kg−1 h−1), for 18-week-old WT and Irx3 KO mice fed with ND and HFD (ND WT/KO, n = 7/5; HFD WT/KO, n = 8/4). b, Locomotor activity of WT and Irx3 KO mice. c, Average amount of food intake over a 24-h period with or without normalization to lean mass. d, Average locomotor activity measured over 24 h. e, f, Elevated Ucp1 gene and protein expression in BAT (WT/KO, n = 7/6). Data are mean ± s.e.m. *P < 0.05 versus WT value.
Extended Data Figure 9 Hypothalamic-specific Irx3 dominant-negative mice are leaner with reduced adiposity.
a, Schematic diagram of generation of transgenic mice overexpressing dominant-negative Irx3 in the hypothalamus. b, Immunoblot analysis showed EnR-Irx3 expression in the hypothalamus of mutant mice without affecting endogenous Irx3 expression, compared to control mice. c, Tissue weights as a percentage of body weight showed that fat pad sizes are smaller in mutant mice, compared to control mice. d, Reduced leptin expression and increased adiponectin gene expression in PWAT of mutant mice (control/mutant, n = 5/7). Data are expressed as mean ± s.e.m. *P < 0.05 compared to control group.
a, Energy expenditure over a 24-h period, corrected for lean mass (kcal kg−1 h−1), for 18-week-old mice. b, Locomotor activity for mice in panel a. c, d, Average amount of food intake over a 24-h period with or without normalization to lean mass. e, Average locomotor activity measured over 24 h. f, g, Elevated gene and protein expression of Ucp1 in BAT of mutant mice (control/mutant, n = 5/7). Data are expressed as mean ± s.e.m. *P < 0.05 compared to control group.
About this article
Cite this article
Smemo, S., Tena, J., Kim, K. et al. Obesity-associated variants within FTO form long-range functional connections with IRX3. Nature 507, 371–375 (2014). https://doi.org/10.1038/nature13138
Nature Communications (2020)
Current Opinion in Pharmacology (2020)
FTO Intronic SNP Strongly Influences Human Neck Adipocyte Browning Determined by Tissue and PPARγ Specific Regulation: A Transcriptome Analysis
Estimation of non-null SNP effect size distributions enables the detection of enriched genes underlying complex traits
PLOS Genetics (2020)
Identifying causal variants and genes using functional genomics in specialized cell types and contexts
Human Genetics (2020)