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Adolescent idiopathic scoliosis (AIS) is a common and progressive spinal deformity in children that exhibits striking sexual dimorphism, with girls at more than five-fold greater risk of severe disease compared to boys. Despite its medical impact, the molecular mechanisms that drive AIS are largely unknown. We previously defined a female-specific AIS genetic risk locus in an enhancer near thePAX1gene. Here we sought to define the roles ofPAX1and newly-identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 9,161 individuals with AIS and 80,731 unaffected controls, significant association was identified with a variant inCOL11A1encoding collagen (α1) XI (rs3753841; NM_080629_c.4004C>T; p.(Pro1335Leu); P=7.07e−11, OR=1.118). Using CRISPR mutagenesis we generatedPax1knockout mice (Pax1−/−). In postnatal spines we found that Pax1 and collagen (α1) XI protein both localize within the intervertebral disc (IVD)-vertebral junction region encompassing the growth plate, with less collagen (α1) XI detected inPax1−/−spines compared to wildtype. By genetic targeting we found that wildtypeCol11a1expression in growth plate cells (GPCs) suppresses expression ofPax1and ofMmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, this suppression was abrogated in the presence of the AIS-associatedCOL11A1P1335Lmutant. Further, we found that either knockdown of the estrogen receptor geneEsr2, or tamoxifen treatment, significantly alteredCol11a1andMmp3expression in GPCs. These studies support a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering aPax1-Col11a1-Mmp3signaling axis in the growth plate.

Adolescent idiopathic scoliosis (AIS), a sideways curvature of the spine, is sexually dimorphic, with increased incidence in females. A GWAS identified a female-specific AIS susceptibility locus near thePAX1gene. Here, we used mouse enhancer assays, three mouse enhancer knockouts and subsequent phenotypic analyses to characterize this region. Using mouse enhancer assays, we characterized a sequence, PEC7, that overlaps the AIS-associated variant, and found it to be active in the tail tip and intervertebral disc. Removal of PEC7 or Xe1, a known sclerotome enhancer nearby, and deletion of both sequences led to a kinky phenotype only in the Xe1 and combined (Xe1+PEC7) knockouts, with only the latter showing a female sex dimorphic phenotype. Extensive phenotypic characterization of these mouse lines implicated several differentially expressed genes and estrogen signaling in the sex dimorphic bias. In summary, our work functionally characterizes an AIS-associated locus and dissects the mechanism for its sexual dimorphism.

Over 500 noncoding genomic loci are associated with obesity. The majority of these loci reside near genes that are expressed in the hypothalamus in specific neuronal subpopulations that regulate food intake, hindering the ability to identify and functionally characterize them. Here, we carried out integrative single-cell analysis (RNA/ATAC-seq) on both mouse and human male and female hypothalamus to characterize genes and regulatory elements in specific cell subpopulations. Utilizing both transcriptome and regulome data, we identify over 30 different neuronal and non-neuronal cell subpopulations and a shared core of transcription factors that regulate cell cluster-specific genes between mice and humans. We characterize several sex-specific differentially expressed genes and the regulatory elements that control them in specific cell subpopulations. Overlapping cell-specific scATAC peaks with obesity-associated GWAS variants, identifies potential obesity-associated regulatory elements. Using reporter assays and CRISPR editing, we show that many of these sequences, including the top obesity-associated loci (FTOandMC4R), are functional enhancers whose activity is altered due to the obesity-associated variant and regulate known obesity genes. Combined, our work provides a catalog of genes and regulatory elements in hypothalamus cell subpopulations and uses obesity to showcase how integrative single-cell sequencing can identify functional variants associated with hypothalamus-related phenotypes.

Fat distribution differences between males and females are a major risk factor for metabolic disease, but their genetic etiology remains largely unknown. Here, we establish ADGRG6 as a major factor in adipogenesis and gender fat distribution. Deletion of ADGRG6 in human adipocytes impairs adipogenesis due to reduced cAMP signaling. Conditionally knocking out Adgrg6 in mouse adipocytes or deleting an intronic enhancer associated with gender fat distribution generates males with female-like fat deposition, which are protected against high-fat-diet-induced obesity and have improved insulin response. To showcase its therapeutic potential, we demonstrate that CRISPRi targeting of the Adgrg6 promoter or enhancer prevents high-fat-diet-induced obesity. Combined, our results associate ADGRG6 as a gender fat distribution gene and highlight its potential as a therapeutic target for metabolic disease.