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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.

BackgroundPaternal allele-specific DNA methylation of theH19imprinting control region (ICR) regulates imprinted expression of theIgf2/H19genes. The molecular mechanism by which differential methylation of theH19ICR is established during gametogenesis and maintained after fertilization, however, is not fully understood. We previously showed that a 2.9-kbH19ICR fragment in transgenic mice was differentially methylated only after fertilization, demonstrating that two separable events, gametic and post-fertilization methylation, occur at theH19ICR. We then determined that CTCF/Sox-Oct motifs and the 478-bp sequence of theH19ICR are essential for maintaining its maternal hypomethylation status and for acquisition of paternal methylation, respectively, during the post-fertilization period.ResultsUsing a series of 5′-truncatedH19ICR transgenes to dissect the 478-bp sequence, we identified a 118-bp region required for post-fertilization methylation activity. Deletion of the sequence from the paternal endogenousH19ICR caused loss of methylation after fertilization, indicating that methylation activity of the sequence is required to protect endogenousH19ICR from genome-wide reprogramming. We then reconstructed a synthetic DNA fragment in which the CTCF binding sites, Sox-Oct motifs, as well as the 118-bp sequence, were inserted into lambda DNA, and used it to replace the endogenousH19ICR. The fragment was methylated during spermatogenesis; moreover, its allele-specific methylation status was faithfully maintained after fertilization, and imprinted expression of the bothIgf2andH19genes was recapitulated.ConclusionsOur results identified a 118-bp region within theH19ICR that is required for de novo DNA methylation of the paternally inheritedH19ICR during pre-implantation period. A lambda DNA-based artificial fragment that contains the 118-bp sequence, in addition to the previously identifiedciselements, could fully replace the function of theH19ICR in the mouse genome.

Acheiropodia, congenital limb truncation, is associated with homozygous deletions in the LMBR1 gene around ZRS, an enhancer regulating SHH during limb development. How these deletions lead to this phenotype is unknown. Using whole-genome sequencing, we fine-mapped the acheiropodia-associated region to 12 kb and show that it does not function as an enhancer. CTCF and RAD21 ChIP-seq together with 4C-seq and DNA FISH identify three CTCF sites within the acheiropodia-deleted region that mediate the interaction between the ZRS and the SHH promoter. This interaction is substituted with other CTCF sites centromeric to the ZRS in the disease state. Mouse knockouts of the orthologous 12 kb sequence have no apparent abnormalities, showcasing the challenges in modelling CTCF alterations in animal models due to inherent motif differences between species. Our results show that alterations in CTCF motifs can lead to a Mendelian condition due to altered enhancer–promoter interactions., Acheiropodia is associated with homozygous deletions in the LMBR1 gene around ZRS, an enhancer regulating SHH during limb development, but how these deletions lead to this phenotype is unknown. Here the authors use whole-genome sequencing, ChIP-seq, 4C-seq and DNA FISH to show that alterations in CTCF motifs are responsible via altered enhancer–promoter interactions.

Adolescent idiopathic scoliosis (AIS), a sideways curvature of the spine, is the most common pediatric musculoskeletal disorder, affecting ∼3% of the population worldwide. However, its genetic bases and tissues of origin remain largely unknown. Several genome-wide association studies (GWAS) have implicated nucleotide variants in noncoding sequences that control genes with important roles in cartilage, muscle, bone, connective tissue and intervertebral discs (IVDs) as drivers of AIS susceptibility. Here, we set out to define the expression of AIS-associated genes and active regulatory elements by performing RNA-seq and ChIP-seq against H3K27ac in these tissues in mouse and human. Our study highlights genetic pathways involving AIS-associated loci that regulate chondrogenesis, IVD development and connective tissue maintenance and homeostasis. In addition, we identify thousands of putative AIS-associated regulatory elements which may orchestrate tissue-specific expression in musculoskeletal tissues of the spine. Quantification of enhancer activity of several candidate regulatory elements from our study identifies three functional enhancers carrying AIS-associated GWAS SNPs at theADGRG6andBNC2loci. Our findings provide a novel genome-wide catalog of AIS-relevant genes and regulatory elements and aid in the identification of novel targets for AIS causality and treatment.

The human spinal column is a dynamic, segmented, bony, and cartilaginous structure that protects the neurologic system and simultaneously provides balance and flexibility. Children with developmental disorders that affect the patterning or shape of the spine can be at risk of neurologic and other physiologic dysfunctions. The most common developmental disorder of the spine is scoliosis, a lateral deformity in the shape of the spinal column. Scoliosis may be part of the clinical spectrum that is observed in many developmental disorders, but typically presents as an isolated symptom in otherwise healthy adolescent children. Adolescent idiopathic scoliosis (AIS) has defied understanding in part due to its genetic complexity. Breakthroughs have come from recent genome-wide association studies (GWAS) and next generation sequencing (NGS) of human AIS cohorts, as well as investigations of animal models. These studies have identified genetic associations with determinants of cartilage biogenesis and development of the intervertebral disc (IVD). Current evidence suggests that a fraction of AIS cases may arise from variation in factors involved in the structural integrity and homeostasis of the cartilaginous extracellular matrix (ECM). Here, we review the development of the spine and spinal cartilages, the composition of the cartilage ECM, the so-called “matrisome” and its functions, and the players involved in the genetic architecture of AIS. We also propose a molecular model by which the cartilage matrisome of the IVD contributes to AIS susceptibility.

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a conprehensive view of intra- and inter-chromosomal contacts between specific genomic sites. The locus control region of the β-globin genes (β-LCR) is a super-enhancer that is capable of activating all of the β-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3’HS1 in the human β-globin locus, in this way thought to contribute to the formation of a “chromatin hub”. The β-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared “transcription factory” for co-transcription. Theoretically, enhancers could also activate target gene promoters on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the β-LCR and the β-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because a lack of suitable regulatory elements that might facilitate trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and β-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the β-globin gene in trans even in this idealized experimental context.

The renin-angiotensin system plays an essential role in blood pressure homeostasis. Because renin activity is reflected as a blood pressure phenotype, its gene expression in the kidney is tightly regulated by a feedback mechanism; i.e., renin gene transcription is suppressed in a hypertensive state. To address the molecular mechanisms controlling hypertension-responsive mouse renin (mRen) gene regulation, we deleted either 5′ (17-kb) or 3′ (78-kb) regions of the endogenous mRen gene and placed the animals in a hypertensive environment. While the mRen gene bearing the 3′ deletion was appropriately downregulated, the one bearing the 5′ deletion lost this hypertension responsiveness. Because the 17-kb sequence exhibited enhancer activity in vivo and in vitro, we narrowed down the enhancer to a 2.3-kb core using luciferase assays in As4.1 cells. When this 2.3-kb sequence was removed from the endogenous mRen gene in the mouse, its basal expression was dramatically reduced, and the hypertension responsiveness was significantly attenuated. Furthermore, we demonstrated that the angiotensin II signal played an important role in mRen gene suppression. We propose that in a hypertensive environment, the activity of this novel enhancer is attenuated, and, as a consequence, mRen gene transcription is suppressed to maintain blood pressure.

The renin-angiotensin system plays an essential role in blood pressure homeostasis. Because renin activity is reflected as a blood pressure phenotype, its gene expression in the kidney is tightly regulated by a feedback mechanism; i.e., renin gene transcription is suppressed in a hypertensive state. To address the molecular mechanisms controlling hypertension-responsive mouse renin (mRen) gene regulation, we deleted either 5' (17-kb) or 3' (78-kb) regions of the endogenous mRen gene and placed the animals in a hypertensive environment. While the mRen gene bearing the 3' deletion was appropriately downregulated, the one bearing the 5' deletion lost this hypertension responsiveness. Because the 17-kb sequence exhibited enhancer activity in vivo and in vitro, we narrowed down the enhancer to a 2.3-kb core using luciferase assays in As4.1 cells. When this 2.3-kb sequence was removed from the endogenous mRen gene in the mouse, its basal expression was dramatically reduced, and the hypertension responsiveness was significantly attenuated. Furthermore, we demonstrated that the angiotensin II signal played an important role in mRen gene suppression. We propose that in a hypertensive environment, the activity of this novel enhancer is attenuated, and, as a consequence, mRen gene transcription is suppressed to maintain blood pressure.

Genomic imprinting is a major monoallelic gene expression regulatory mechanism in mammals, and depends on gamete-specific DNA methylation of specialized cis-regulatory elements called imprinting control regions (ICRs). Allele-specific DNA methylation of the ICRs is faithfully maintained at the imprinted loci throughout development, even in early embryos where genomes undergo extensive epigenetic reprogramming, including DNA demethylation, to acquire totipotency. We previously found that an ectopically introduced H19 ICR fragment in transgenic mice acquired paternal allele-specific methylation in the somatic cells of offspring, while it was not methylated in sperm, suggesting its gametic and postfertilization modifications are separable events. We hypothesized that this latter activity might contribute to maintenance of the methylation imprint in early embryos. Here we demonstrate that methylation of the paternally inherited transgenic H19 ICR commences soon after fertilization in a maternal Dnmt3a- and Dnmt3L-dependent manner. When its germline methylation was partially obstructed by insertion of insulator sequences, the endogenous, paternal H19 ICR also exhibited postfertilization methylation. Finally, we refined the responsible sequences for this activity in transgenic mice, and found that deletion of the 5' segment of the endogenous paternal H19 ICR decreased its methylation after fertilization, attenuated Igf2 gene expression. These results demonstrate that this segment of the H19 ICR is essential for its de novo post-fertilization DNA methylation, and that this activity contributes to the maintenance of imprinted methylation at the endogenous H19 ICR during early embryogenesis

Renin is predominantly expressed in juxtaglomerular cells in the kidney and regulates blood pressure homeostasis. To examine possible in vivo functions of a mouse distal enhancer (mdE), we generated transgenic mice (TgM) carrying either wild-type or mdE-deficient renin BACs (bacterial artificial chromosome), integrated at the identical chromosomal site. In the kidneys of the TgM, the mdE contributed 80% to basal renin promoter activity. To test for possible physiological roles for the mdE, renin BAC transgenes were used to rescue the hypotensive renin-null mice. Interestingly, renal renin expression in the Tg(BAC):renin-null compound mice was indistinguishable between the wild-type and mutant BAC carriers. Surprisingly, however, the plasma renin activity and angiotensin I concentration in the mdE compound mutant mice were significantly lower than the same parameters in the control mice, and the mutants were consistently hypotensive, demonstrating that blood pressure homeostasis is regulated through transcriptional cis elements controlling renin activity.

Renin, a rate-limiting enzyme in the renin–angiotensin system, is regulated to maintain blood pressure homeostasis: renin gene expression in the kidney is suppressed in a hypertensive environment. We found that expression of a 15-kb human RENIN (hREN) transgene was aberrantly upregulated (>4.2-fold), while the endogenous mouse renin (mRen) gene was suppressed (>1.7-fold) in Tsukuba hypertensive mice (THM), a model for genetically induced hypertension. We then generated transgenic mice using a 13-kb mRen gene fragment that was homologous to the 15-kb hREN transgene and found that its expression was also upregulated (>3.1-fold) in THM, suggesting that putative silencing elements of the renin genes were distally located in the loci. We next examined the possible role of a previously identified mouse distal enhancer (mdE) located outside of the 13-kb mRen gene fragment. Deletion of the mdE in the context of a 156-kb mRen transgene did not affect its transcriptional repression in THM, implying that although the silencing element of the mRen gene is located within the 156-kb fragment tested, it is distinct from the mdE. Consistent with these results, deletion of the 63-kb region upstream of the mdE from the endogenous mRen gene locus abrogated its transcriptional repression in THM. We finally tested whether dysregulation of the short renin transgenes also occurred in the fetal or neonatal kidneys of THM and found that their expression was not aberrantly upregulated, demonstrating that aberrant regulation of short renin transgenes commences sometime between neonate and adult periods.