Genetic variation in a non-protein-coding region of the gene FTO is implicated in obesity. A study finds evidence that, rather than affecting FTO itself, variations in this region influence expression of a distant gene, IRX3. See Letter p.371
Genome-wide association studies have identified more than 75 locations in the human genome at which changes in DNA sequence are linked to obesity and traits closely related to the condition, such as body mass index. In these studies, single nucleotide changes in a non-protein-coding region (an intron) of the gene FTO often have the strongest association with obesity1,2,3. It is generally thought that this association arises because of a genetic defect that alters the function or expression level of FTO, which encodes an enzyme known to be involved in the control of body weight and metabolism in mice4,5. However, evidence of a direct link between the single nucleotide changes and alteration of FTO function or expression level has been lacking. In this issue, Smemo et al.6 (page 371) present compelling evidence that the association between obesity and FTO actually involves another gene called IRX3, which encodes a transcription factor involved in multiple developmental processes.
IRX3 is located a long way — roughly 500 kilobases — from FTO's first intron, in which the single nucleotide changes (also known as single nucleotide polymorphisms, or SNPs) associated with obesity are located. Despite previous research7 suggesting that long-range enhancer sequences in FTO's first intron might regulate the expression of IRX3, attention has continued to focus on FTO, partly owing to a lack of more-definitive evidence linking these enhancers to IRX3 expression. Now, Smemo and colleagues have confirmed and expanded on this link.
Verifying a functional relationship between an enhancer sequence and a distant target gene requires a demonstration that the enhancer interacts physically with the target gene's promoter in the cell nucleus (a promoter is a DNA sequence immediately adjacent to the beginning of a gene that is involved in regulating transcription). Smemo and colleagues investigated long-range interactions between FTO and IRX3 using a technique called chromatin conformation capture, which measures how frequently different regions of the genome interact8. The authors found that the promoter of IRX3 physically interacts with the first intron of FTO (Fig. 1). This interaction was found in mouse and zebrafish embryos, adult mouse brains and human cell lines, suggesting that interaction between these two regulatory regions is an evolutionarily conserved feature of genome organization. The authors also found that sequences in the first intron of FTO show enhancer activity in several of the tissues in which IRX3 is expressed, and that the promoter of IRX3 alone cannot account for the gene's observed expression pattern, indicating that IRX3 relies on long-range regulatory input.
Smemo and co-workers reasoned that, if the association between obesity and FTO does result from genetic alteration of an IRX3 enhancer, then the same SNPs linked with obesity should also be associated with changes in IRX3 expression. To investigate this, the authors examined IRX3 expression in 153 human brain samples. Obesity-linked SNPs were associated with IRX3 expression in these samples, but not with expression of FTO, directly linking these variants to IRX3 regulation.
In a final, but crucial, set of experiments, Smemo et al. found that mice lacking a functional copy of Irx3 have significantly lower body weight than control animals, along with a higher base metabolic rate, less body fat and a predisposition to form brown over white adipose tissue (the latter being more likely to accumulate in obese individuals). This clearly demonstrates a previously unappreciated role for Irx3 in controlling body weight. Furthermore, the authors report that these same traits are also seen in mice when a 'dominant-negative' mutant form of Irx3, which interferes with the function of normal Irx3, is expressed in the hypothalamus, a region of the brain that has a key role in regulating food intake and metabolism. These data indicate that control of body weight relies specifically on Irx3 activity in the hypothalamus.
Smemo and colleagues' data suggest a model in which a particular genetic variant (or variants) in the first intron of FTO disrupts an enhancer of IRX3, thus altering IRX3 expression and, in turn, influencing body weight. However, important questions remain. First, what is the true functional variant (or variants) that causes the association between this region and obesity? Although many SNPs identified in this region have been associated with obesity, it is likely that these associations reflect a single genetic defect in the region. Without pinpointing this defect, it will be difficult to determine whether the genetic mechanism involves disrupted regulation of FTO, IRX3 or perhaps both. Second, what tissues are most relevant to this association? The current study does an admirable job of examining appropriate tissues wherever possible, but there are missing links. For example, the authors observed that loss of Irx3 function in the hypothalamus causes changes in body weight in mice, but the human brain samples were from the cerebellum. Additional studies are needed to answer these and other questions about the connection between obesity, FTO and IRX3.
The implications of Smemo and co-workers' study extend far beyond obesity. SNPs associated with other human traits frequently occur at or near transcriptional enhancers9, and linear genomic distance is often an unreliable predictor of enhancer–promoter interactions10,11. The connection between IRX3 and FTO is particularly instructive because the enhancers of one gene reside within an intron of the other. However, this is not an isolated case. For example, an enhancer in an intron of the gene LMBR1 regulates the developmental gene SHH located more than 1 megabase away, and mutations in this enhancer can cause limb malformations owing to altered SHH expression12. Similarly, an enhancer within an intron of the gene HERC2 contains a SNP that influences human eye, hair and skin colour by modulating the expression of a neighbouring gene called OCA2 (ref. 13). Through their study, Smemo et al. provide an excellent example of how detailed functional analyses, particularly those that link enhancers to their target genes, can help to evaluate the biological consequences of variation in non-coding sequences.
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Gorkin, D., Ren, B. Closing the distance on obesity culprits. Nature 507, 309–310 (2014). https://doi.org/10.1038/nature13212
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