BACKGROUND Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. METHODS We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR–Cas9 genome editing in samples from patients. RESULTS Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR–Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7. CONCLUSIONS Our results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.)
Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms.
Objective COL6A3 may modulate adipose tissue function in obesity and insulin resistance. A role for human adipocytes linking COL6A3 with insulin resistance warrants exploration. Methods COL6A3 mRNA in abdominal subcutaneous adipose samples was compared between (1) BMI‐matched obese subjects resistant or sensitive to insulin (surgical whole tissue biopsies, n = 30/group), (2) lean/overweight and obese subjects (isolated adipocytes from collagenase‐treated surgical biopsies, n = 11/group), (3) developing primary human adipocytes with/without knockdown of the insulin‐sensitizing adipogenic gene PPARG (collagenase‐treated lipoaspirate, n = 5), and (4) small and large adipocytes from lean/overweight subjects (collagenase‐treated surgical biopsies or lipoaspirate, n = 10). Insulin resistance and sensitivity were assessed by euglycemic‐hyperinsulinemic clamp (glucose infusion rate <60 and >70 μmol kg−1 min−1, respectively) (1), or by HOMA‐IR and TG/HDL ratio (2). Results Whole tissue COL6A3 mRNA was 2.6‐fold higher in insulin resistant compared to sensitive subjects (P < 0.001). In isolated adipocytes, COL6A3 mRNA correlated positively with BMI (P = 0.007), HOMA‐IR (P = 0.039), and TG/HDL (P = 0.004). PPARG knockdown in developing adipocytes increased COL6A3 mRNA 1.5‐fold (P = 0.043). The inverse relationship with adipocyte development was further supported by 2.8‐fold higher COL6A3 mRNA in small compared to large adipocytes (P = 0.004). Conclusion Increased adipocyte COL6A3 expression associates with insulin resistance in humans, which may involve impaired PPARγ‐mediated adipocyte development.
Genome-wide association studies identified numerous disease risk loci. Delineating molecular mechanisms influenced by cis-regulatory variants is essential to understand gene regulation and ultimately disease pathophysiology. Combining bioinformatics and public domain chromatin information with quantitative proteomics supports prediction of cis-regulatory variants and enabled identification of allele-dependent binding of both, transcription factors and coregulators at the type 2 diabetes associated PPARG locus. We found rs7647481A nonrisk allele binding of Yin Yang 1 (YY1), confirmed by allele-specific chromatin immunoprecipitation in primary adipocytes. Quantitative proteomics also found the coregulator RING1 and YY1 binding protein (RYBP) whose mRNA levels correlate with improved insulin sensitivity in primary adipose cells carrying the rs7647481A nonrisk allele. Our findings support a concept with diverse cis-regulatory variants contributing to disease pathophysiology at one locus. Proteome-wide identification of both, transcription factors and coregulators, can profoundly improve understanding of mechanisms underlying genetic associations.
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