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

Abstract

SummarySingle Nucleotide Polymorphisms in FTO intron 1 have been associated with obesity risk, leading to the hypothesis that FTO is the obesity‐related gene. However, other studies have shown that the FTO gene is part of the regulatory domain of the neighboring IRX3 gene and that enhancers in FTO intron 1 regulate IRX3. While Irx3 activity was shown to be necessary in the hypothalamus for the metabolic function of Irx3 in mouse, no enhancers with hypothalamic activity have been demonstrated in the risk‐associated region within FTO. In order to identify potential enhancers at the human FTO locus in vivo, we tested regulatory activity in FTO intron 1 using BAC transgenesis in zebrafish. A minimal gata2 promoter‐GFP cassette was inserted 1.3 kb upstream of the obesity associated SNP rs9939609 in a human FTO BAC plasmid. In addition to the previously identified expression domains in notochord and kidney, human FTO BAC:GFP transgenic zebrafish larvae expressed GFP in the ventral posterior tuberculum, the posterior hypothalamus and the anterior brainstem, which are also expression domains of zebrafish irx3a. In contrast, an in‐frame insertion of a GFP cassette at the FTO start codon resulted in weak ubiquitous GFP expression indicating that the promoter of FTO does likely not react to enhancers located in the obesity risk‐associated region. genesis 53:640–651, 2015. © 2015 The Authors. genesis Published by Wiley Periodicals, Inc.

Technologies, strategies, and cautions when deconvoluting genome‐wide association signals: FTO in focus

“…In the same study, in vivo promoter probing in mice of the FTO proximal promoter, and the FTO proximal promoter plus the FTO obesity‐associated intronic region, showed comparable reporter expression, whereas promoter probing of the IRX3 proximal promoter did not show any expression patterns. These results, and similar studies in zebrafish, 57 suggest that distinct enhancer elements are required for IRX3 expression but not for FTO . FTO and RPGRIP1L expression is ubiquitous but regulated by nutritional state in the hypothalamus 18,58 .…”

Family‐based Association between Allele T of rs4646536 in CYP27B1 and vitamin D deficiency

“…The function of rs4646536 is unknown. It has been reported that gene expression could be influenced by variants in intron via affecting the binding of transcription factors and splicing of mRNA . Thus, allele variation of rs4646536 from C to T may result in abnormal expression of CYP27B1 , which would lead to disorder of 25(OH)D concentration.…”

The importance of considering regulatory domains in genome-wide analyses – the nearest gene is often wrong!

“…3 ). In addition, studies have found that the FTO promoter does not respond to long-range regulation during zebrafish development ( Rinkwitz et al, 2015 ). Recently, it has been shown that rs1421085 is located within an ARID5B binding site, leading to impaired ARID5B -mediated repression of IRX3 and IRX5 during early adipocyte differentiation ( Claussnitzer et al, 2015 ).…”