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White adipose tissue, once regarded as morphologically and functionally bland, is now recognized to be dynamic, plastic and heterogenous, and is involved in a wide array of biological processes including energy homeostasis, glucose and lipid handling, blood pressure control and host defence 1 . High-fat feeding and other metabolic stressors cause marked changes in adipose morphology, physiology and cellular composition 1 , and alterations in adiposity are associated with insulin resistance, dyslipidemia and type 2 diabetes 2 . Here we provide detailed cellular atlases of human and mouse subcutaneous and visceral white fat at single-cell resolution across a range of body weight. We identify subpopulations of adipocytes, adipose stem and progenitor cells, vascular and immune cells and demonstrate commonalities and differences across species and dietary conditions. We link specific cell types to increased risk of metabolic disease and provide an initial blueprint for a comprehensive set of interactions between individual cell types in the adipose niche in leanness and obesity. These data comprise an extensive resource for the exploration of genes, traits and cell types in the function of white adipose tissue across species, depots and nutritional conditions. A single-cell atlas of white adipose tissue from mouse and human reveals diverse cell types and similarities and differences across species and dietary conditions.

The fat mass and obesity-associated ( FTO ) gene plays a pivotal role in regulating body weight and fat mass; however, the underlying mechanisms are poorly understood. Here we show that primary adipocytes and mouse embryonic fibroblasts (MEFs) derived from FTO overexpression ( FTO-4 ) mice exhibit increased potential for adipogenic differentiation, while MEFs derived from FTO knockout ( FTO-KO ) mice show reduced adipogenesis. As predicted from these findings, fat pads from FTO-4 mice fed a high-fat diet show more numerous adipocytes. FTO influences adipogenesis by regulating events early in adipogenesis, during the process of mitotic clonal expansion. The effect of FTO on adipogenesis appears to be mediated via enhanced expression of the pro-adipogenic short isoform of RUNX1T1, which enhanced adipocyte proliferation, and is increased in FTO-4 MEFs and reduced in FTO-KO MEFs. Our findings provide novel mechanistic insight into how upregulation of FTO leads to obesity. Mutations in the FTO gene have been linked to obesity. Here, Merkestein et al. provide in vitro and in vivo evidence that FTO directly regulates adipogenesis in mice at the stage of mitotic clonal expansion, likely by modulating the expression of the transcription factor RUNX1T1.

IRX3 is linked to predisposition to obesity through the FTO locus and is upregulated during early adipogenesis in risk-allele carriers, shifting adipocyte fate toward fat storage. However, how this elevated IRX3 expression influences later developmental stages remains unclear. Here we show that IRX3 regulates adipocyte fate by modulating epigenetic reprogramming. ChIP-sequencing in preadipocytes identifies over 300 IRX3 binding sites, predominantly at promoters of genes involved in SUMOylation and chromatin remodeling. IRX3 knockout alters expression of SUMO pathway genes, increases global SUMOylation, and inhibits PPARγ activity and adipogenesis. Pharmacological SUMOylation inhibition rescues these effects. IRX3 KO also reduces SUMO occupancy at Wnt-related genes, enhancing Wnt signaling and promoting osteogenic fate in 3D cultures. This fate switch is partially reversible by SUMOylation inhibition. We identify IRX3 as a key transcriptional regulator of epigenetic programs, acting upstream of SUMOylation to maintain mesenchymal identity and support adipogenesis while suppressing osteogenesis in mouse embryonic fibroblasts. Here, the authors show that IRX3 is a transcriptional master regulator of multiple histone- and chromatin-remodeling enzymes and the SUMOylation pathway in adipocyte precursor cells. IRX3 ablation results in a SUMOylation-dependent switch from adipogenic to osteogenic identity.

Genetic studies have recently highlighted the importance of fat distribution, as well as overall adiposity, in the pathogenesis of obesity-associated diseases. Using a large study (n = 1,288) from 4 independent cohorts, we aimed to investigate the relationship between mean adipocyte area and obesity-related traits, and identify genetic factors associated with adipocyte cell size. To perform the first large-scale study of automatic adipocyte phenotyping using both histological and genetic data, we developed a deep learning-based method, the Adipocyte U-Net, to rapidly derive mean adipocyte area estimates from histology images. We validate our method using three state-of-the-art approaches; CellProfiler, Adiposoft and floating adipocytes fractions, all run blindly on two external cohorts. We observe high concordance between our method and the state-of-the-art approaches (Adipocyte U-net vs. CellProfiler: R2visceral = 0.94, P < 2.2 × 10-16, R2subcutaneous = 0.91, P < 2.2 × 10-16), and faster run times (10,000 images: 6mins vs 3.5hrs). We applied the Adipocyte U-Net to 4 cohorts with histology, genetic, and phenotypic data (total N = 820). After meta-analysis, we found that mean adipocyte area positively correlated with body mass index (BMI) (Psubq = 8.13 × 10-69, βsubq = 0.45; Pvisc = 2.5 × 10-55, βvisc = 0.49; average R2 across cohorts = 0.49) and that adipocytes in subcutaneous depots are larger than their visceral counterparts (Pmeta = 9.8 × 10-7). Lastly, we performed the largest GWAS and subsequent meta-analysis of mean adipocyte area and intra-individual adipocyte variation (N = 820). Despite having twice the number of samples than any similar study, we found no genome-wide significant associations, suggesting that larger sample sizes and a homogenous collection of adipose tissue are likely needed to identify robust genetic associations.

Obesity-associated morbidity is exacerbated by abdominal obesity, which can be measured as the waist-to-hip ratio adjusted for the body mass index (WHRadjBMI). Here we identify genes associated with obesity and WHRadjBMI and characterize allele-sensitive enhancers that are predicted to regulate WHRadjBMI genes in women. We found that several waist-to-hip ratio-associated variants map within primate-specific Alu retrotransposons harboring a DNA motif associated with adipocyte differentiation. This suggests that a genetic component of adipose distribution in humans may involve co-option of retrotransposons as adipose enhancers. We evaluated the role of the strongest female WHRadjBMI-associated gene, SNX10 , in adipose biology. We determined that it is required for human adipocyte differentiation and function and participates in diet-induced adipose expansion in female mice, but not males. Our data identify genes and regulatory mechanisms that underlie female-specific adipose distribution and mediate metabolic dysfunction in women. The identification of genes and enhancers associated with waist-to-hip ratio adjusted for body mass index (WHRadjBMI) in women highlights variants within Alu retrotransposons. The WHRadjBMI-linked gene SNX10 plays a role in human adipocyte differentiation and diet-induced adipose expansion in female mice.

Recent large-scale genomic association studies found evidence for a genetic link between increased risk of type 2 diabetes and decreased risk for adiposity-related traits, reminiscent of metabolically obese normal weight (MONW) association signatures. However, the target genes and cellular mechanisms driving such MONW associations remain to be identified. Here, we systematically identify the cellular programmes of one of the top-scoring MONW risk loci, the 2q24.3 risk locus, in subcutaneous adipocytes. We identify a causal genetic variant, rs6712203, an intronic single-nucleotide polymorphism in the COBLL1 gene, which changes the conserved transcription factor motif of POU domain, class 2, transcription factor 2, and leads to differential COBLL1 gene expression by altering the enhancer activity at the locus in subcutaneous adipocytes. We then establish the cellular programme under the genetic control of the 2q24.3 MONW risk locus and the effector gene COBLL1 , which is characterized by impaired actin cytoskeleton remodelling in differentiating subcutaneous adipocytes and subsequent failure of these cells to accumulate lipids and develop into metabolically active and insulin-sensitive adipocytes. Finally, we show that perturbations of the effector gene Cobll1 in a mouse model result in organismal phenotypes matching the MONW association signature, including decreased subcutaneous body fat mass and body weight along with impaired glucose tolerance. Taken together, our results provide a mechanistic link between the genetic risk for insulin resistance and low adiposity, providing a potential therapeutic hypothesis and a framework for future identification of causal relationships between genome associations and cellular programmes in other disorders. Glunk et al. explore target genes and cellular mechanisms related to a metabolic obesity with normal-weight phenotype, and identify a non-coding variant that affects actin remodelling in subcutaneous adipocytes, which in turn affects the capacity of these cells to accumulate lipids.

Genome-wide association studies have repeatedly shown that the strongest association with obesity arises from variants in the first intron of FTO. The intronic FTO variant rs1421085 is within an adipocyte-specific enhancer and that risk allele carriers have increased IRX3 and IRX5 expression in early adipogenesis (Claussnitzer et al., 2015). Additionally, the same human risk variant was linked to decreased AKTIP, RPGRIP1L and FTO expression in iPSC-derived neurons (Stratigopoulos et al., 2016). These data point towards several likely causal transcripts and tissues at the FTO locus and essentially, several likely mechanisms. Importantly, whether any of the high-risk variants at the FTO locus has any effect on the organismal level has not been addressed so far. The aim of my DPhil project was to use novel gene manipulation strategies in vivo to mechanistically dissect the Fto regulatory circuitry in mouse to pinpoint causal transcripts their effector tissues and to unravel their physiological role in body weight regulation . Using publicly available as well as my own genomic data (ATAC-seq) revealed that the intronic FTO regulatory element in human adipocytes is conserved in mouse pre-adipocytes. Manipulation of the corresponding motif in mouse (by deleting 82 nucleotides at the mouse orthologous region around rs1421085) resulted in depot- and sex-specific alteration of target genes Irx3 and Irx5 in pre-adipocytes. In addition to recapitulating many of the human findings in mouse, my results further unravelled a new level of regulatory complexity at the FTO/Fto locus. When these mutant mice were put on a high fat diet, I found a reduction on overall fat-mass that could be linked to altered mRNA levels of Irx3 and Irx5 in pre-adipocytes. Using a number of genetic techniques, I further showed that Irx3 regulates several processes during adipocyte development, amongst which is modulation of mitochondrial function. In summary, my findings provide new insight into how variants in FTO intron 1 affect adipocyte development and more specifically how IRX3 affects early adipocyte differentiation.

The locus is the strongest genetic association with coronary artery disease (CAD), yet its causal mechanisms remain unresolved. We map the regulatory architecture of in disease-relevant vascular cells, identifying 12 enhancers within the CAD risk haplotype that respond dynamically to inflammatory and metabolic stress in fibroblasts and smooth muscle cells. These activated states are enriched for CAD heritability, implicating stress-responsive vascular wall cells in disease pathophysiology. Dense CRISPRi tiling integrated with fine-mapping and genomic constraint across >500,000 individuals nominates as the effector gene, with rs1537371 as a likely causal variant. Perturbation and multi-modal analyses show that loss induces pro-fibrotic and angiogenic programs and sensitizes vascular cells to TGF- -driven pathological transitions. Our findings reveal a vascular-specific enhancer network through which noncoding variation at modulates CAD risk via -a previously unrecognized regulator of vascular remodeling located 269 kb from the risk haplotype.

IRX3 is implicated in genetic predisposition to obesity via the FTO variant locus. IRX3 shows FTO risk allele-dependent upregulation specifically during early adipogenesis, leading to a shift from energy-dissipation to fat storage in mature adipocytes. However, how changes in IRX3 expression at one developmental stage affect cellular phenotype at a later stage remains unclear. We here hypothesize that IRX3 regulates adipocyte development via transcriptional modulation of epigenetic reprogramming factors. We combined ChIP-, ATAC- and RNA-sequencing to map direct Irx3 target genes in regions of open chromatin during early adipogenesis of wild-type and Irx3-KO preadipocytes. Gene ontology analyses was performed to identify significantly enriched biological pathways. Denaturing western blotting was used to assess sumoylation levels, and the inhibitor ML-792 was used to specifically block sumoylation. Luciferase assays were performed to estimate effects of ML-792 on Pparγ activity. Bodipy lipid staining, immunofluorescence and qPCR were employed to assess adipogenic differentiation in 3D culture. Alkaline phosphatase and Alizarine Red S staining, as well as immunofluorescence and qPCR were used to assess osteogenic differentiation in 3D culture. We identified more than 300 Irx3 binding sites in preadipocytes, and these were almost exclusively restricted to promoter regions, with a strong enrichment of genes related to sumoylation, histone modifications and chromatin remodeling. Genes from every step of the sumoylation cycle were bound by Irx3 and differentially expressed in response to Irx3-KO, leading to increased global sumoylation levels in the KO cells. Irx3 ablation and elevated sumoylation inhibited Pparγ activity and adipogenic differentiation in preadipocytes, both of which could be restored by pharmacological inhibition of sumoylation. The Irx3-KO cells demonstrated reduced epigenetic suppression against osteogenesis, resulting in increased osteogenesis in 3D culture. Finally, osteogenesis induced by Irx3 ablation could partially be reversed by inhibition of sumoylation. Our study has uncovered IRX3 as a novel upstream regulator of sumoylation, and a potent controller of epigenetic regulators, both directly and indirectly via suppressing global sumoylation levels. This study indicates that the FTO locus promotes obesity via IRX3-mediated suppression of sumoylation, which promotes adipogenic commitment and differentiation through epigenetic programming. biorxiv;2023.10.17.562662v1/FIGU1F1figu1