Obesity-associated variants within FTO form long-range functional connections with IRX3

Abstract

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type-2 diabetes (T2D) 1–3. While 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 phenotypes 4–6. Yet, no direct connection between the obesity-associated variants and FTO expression or function has been made 7–9. 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. Supporting this, obesity-associated SNPs are associated with expression of IRX3, but not FTO, in human brains. Directly linking IRX3 expression with regulation of body mass and composition, Irx3-deficient mice exhibit a 25–30% reduction in body weight, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Furthermore, hypothalamic expression of a dominant negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data posit that IRX3 is a functional long-range target of obesity-associated variants within FTO, and represents a novel determinant of body mass and composition.

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).…”

“…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). In the mouse embryo, Fto displays near ubiquitous expression, while Irx3 is specifically expressed in several brain regions (Diez‐Roux et al, 2011).…”

“…This was of special interest since increased IRX3 expression was related to the rs9939609 variant and Irx3 hypothalamic function was identified to regulate fat composition through control of energy homeostasis (Smemo et al ., 2014). Even though this was an extensive study, hypothalamic enhancers could not be identified in FTO intron 1.…”

“…In a comprehensive IRX3 / Irx3 genomic and functional analysis, Smemo et al . (2014) have provided compelling proof that the IRX3 promoter interacts directly with sequences in the LD block. In addition, disrupted function of Irx3 in the mouse hypothalamus led to increased energy expenditure accompanied by activation of brown adipose tissue in the genetically modified mice.…”

“…In addition, disrupted function of Irx3 in the mouse hypothalamus led to increased energy expenditure accompanied by activation of brown adipose tissue in the genetically modified mice. While Irx3 expression was detected in the arcuate nucleus of the hypothalamus, three enhancer sequences isolated from the LD block drove expression in lung and several brain regions, but not in hypothalamus (Smemo et al ., 2014). …”