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Haploinsufficiency of the single-minded homology 1 (SIM1) gene in humans and mice leads to severe obesity, suggesting that altered expression ofSIM1, by way of regulatory elements such as enhancers, could predispose individuals to obesity. Here, we identified transcriptional enhancers that could regulateSIM1, using comparative genomics coupled with zebrafish and mouse transgenic enhancer assays. Owing to the dual role ofSim1in hypothalamic development and in adult energy homeostasis, the enhancer activity of these sequences was annotated from embryonic to adult age. Of the seventeen tested sequences, twoSIM1candidate enhancers (SCE2 and SCE8) were found to have brain-enhancer activity in zebrafish. Both SCE2 and SCE8 also exhibited embryonic brain-enhancer expression in mice, and time course analysis of SCE2 activity showed overlapping expression withSim1from embryonic to adult age, notably in the hypothalamus in adult mice. Using a deletion series, we identified the critical region in SCE2 that is needed for enhancer activity in the developing brain. Sequencing this region in obese and lean cohorts revealed a higher prevalence of single nucleotide polymorphisms (SNPs) that were unique to obese individuals, with one variant reducing developmental-enhancer activity in zebrafish. In summary, we have characterized two brain enhancers in theSIM1locus and identified a set of obesity-specific SNPs within one of them, which may predispose individuals to obesity.