FTO Obesity Variant Circuitry and Adipocyte Browning in Humans

Abstract

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.).

Figure 1. Activation of a Superenhancer in Human Adipocyte Progenitors by the FTO Obesity Risk Haplotype

Panel A shows the genetic association with body-mass index (BMI) for all common FTO locus variants, including the reported single-nucleotide variant (SNV) rs1558902 (red diamond) and the predicted causal SNV rs1421085 (red square). Gray shading delineates consecutive 10-kb segments. CEU denotes a population of Utah residents with northern and western European ancestry, and LD linkage disequilibrium. Panel B shows chromatin state annotations for the locus across 127 reference epigenomes (rows) for cell and tissue types profiled by the Roadmap Epigenomics Project.^, For information on the colors used to denote chromatin states, see Figure S1A in the Supplementary Appendix. Vertical lines delineate the consecutive 10-kb segments shown in Panel A. ESC denotes embryonic stem cell, HSC hematopoietic stem cell, and iPSC induced pluripotent stem cell. Panel C shows human SGBS adipocyte enhancer activity, for 10-kb tiles, of the risk and nonrisk haplotypes with the use of relative luciferase expression. The boxes indicate means from seven triplicate experiments, and T bars indicate standard deviations.

Figure 2. Activation of IRX3 and IRX5 Expression in Human Adipocyte Progenitors by the FTO Obesity Risk Genotype

Panel A shows gene annotations and LD with array tag variant rs9930506 in a 2.5-Mb window; LD is expressed as r² values in the CEU population. Arrows indicate the direction of transcription of annotated genes in the locus. Panel B shows chromosome conformation capture (Hi-C) interactions contact probabilities in human IMR90 myofibroblasts, revealing a 2-Mb topologically associating domain, and LD mean r² statistics for all SNV pairs at 40-kb resolution. Panel C shows box plots for expression levels, after 2 days of differentiation, in human adipose progenitors isolated from 20 risk-allele carriers and 18 nonrisk-allele carriers, evaluated by means of a quantitative polymerase-chain-reaction analysis for all genes in the 2.5-Mb locus. The horizontal line within each box represents the median, the top and bottom of each box indicate the 75th and 25th percentile, and I bars indicate the range.

Figure 3. Regulation of Obesity-Associated Cellular Phenotypes in Human Adipocytes by IRX3 and IRX5

Panel A shows the mitochondrial and FXR and RXR activation genes with strongest positive (in red) or negative (in green) correlation with IRX3 and IRX5 in human perirenal adipose tissue from 10 participants. Panels B and C show box plots of the increased adipocyte diameter and decreased mitochondrial DNA content in isolated differentiated adipocytes from risk-allele carriers (16 and 8 participants, respectively) relative to nonrisk-allele carriers (26 and 8, respectively). The vertical line within each box represents the median, the left and right margins of each box indicate the interquartile range, and I bars indicate the range. Panel D shows box plots of the altered basal and isoproterenol-stimulated oxygen consumption rate (OCR) on small interfering RNA (siRNA) knockdown and doxycycline (DOX)–mediated overexpression of IRX3 and IRX5 in 8 risk-allele carriers and 10 nonrisk-allele carriers. The siRNA efficiency was 62% for IRX3 and 71% for IRX5. The horizontal line within each box represents the median, the top and bottom of each box indicate the interquartile range, and I bars indicate the range.

Figure 4. Disruption of a Conserved ARID5B Repressor Motif by Causal SNV rs1421085 in Humans

Panel A shows disruption of an ARID5B repressor motif in the evolutionarily conserved motif module surrounding rs1421085. The sequences shown at the top of the panel indicate the frequencies of each nucleotide, with the size scaled to indicate the information content (measured as entropy) at each position. Panel B shows adapted phylogenetic module complexity analysis (PMCA) scores in the FTO region for all 82 noncoding SNPs in LD (r²≥0.8) with tag SNV rs1558902, which was identified in a genomewide association study; rs1421085 had the maximal score. Chromatin state annotation is shown for Roadmap Epigenomics reference genome E025, which corresponds to adipose-derived mesenchymal stem cells; for information on the colors used to denote chromatin states, see Figure S1A in the Supplementary Appendix. Panel C shows increased endogenous expression of IRX3 and IRX5 on single-nucleotide T-to-C editing of rs1421085 in the nonrisk haplotype of a nonrisk-allele carrier, using CRISPR–Cas9 (five clonal expansions). CRISPR–Cas9 reediting from the engineered C risk allele back to a T nonrisk allele with the use of an alternative single guide RNA restores low endogenous IRX3 and IRX5 gene expression. Panel D shows reduced expression of IRX3 and IRX5 on C-to-T editing of the risk allele in adipocyte progenitors from a risk-allele carrier. Knockdown of ARID5B increases IRX3 and IRX5 levels, as compared with the nontargeting control (siNT), only in the rescued allele, but not in the risk allele.

Figure 5. Rescue of Metabolic Effects on Adipocyte Thermogenesis through Editing of SNV rs1421085 in a Risk-Allele Carrier

Panel A shows increased mean expression of IRX3 and IRX5 during early adipocyte differentiation specifically for the risk allele; increased expression levels are rescued by C-to-T genome editing. I bars indicate standard deviations. Panel B shows increased expression of thermogenic and mitochondrial genes on C-to-T endogenous single-nucleotide editing of rs1421085 in adipocyte progenitors from a patient with the risk allele. Panel C shows increased basal and isoproterenol-stimulated OCR on C-to-T single-nucleotide endogenous rescue of rs1421085 in adipocytes from a risk-allele carrier. Panel D shows a summary of our mechanistic model of the FTO locus association with obesity, implicating a developmental shift favoring lipid-storing white adipocytes over energy-burning beige adipocytes. At its core lies a single-nucleotide T-to-C variant, rs1421085, which disrupts a conserved ARID5B repressor motif and activates a mesenchymal superenhancer and its targets (IRX3 and IRX5), leading to reduced heat dissipation by mitochondrial thermogenesis (a process that is regulated by UCP1, PGC1α, and PRDM16) and to increased lipid storage in white adipocytes.