Deletion of Pax1 scoliosis-associated regulatory elements leads to a female-biased tail abnormality.

Adolescent idiopathic scoliosis (AIS), a sideways curvature of the spine, is sexually dimorphic, with increased incidence in females. A genome-wide association study identified a female-specific AIS susceptibility locus near the PAX1 gene. Here, we use mouse enhancer assays, three mouse enhancer knockouts, and subsequent phenotypic analyses to characterize this region. Using mouse enhancer assays, we characterize a sequence, PEC7, which overlaps the AIS-associated variant, and find it to be active in the tail tip and intervertebral disc. Removal of PEC7 or Xe1, a known sclerotome enhancer nearby, or deletion of both sequences lead to a kinky tail 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 implicates 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.

Association of genetic variation in COL11A1 with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common and progressive spinal deformity in children that exhibits striking sexual dimorphism, with girls at more than fivefold 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 the PAX1 gene. Here, we sought to define the roles of PAX1 and newly identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 10,519 individuals with AIS and 93,238 unaffected controls, significant association was identified with a variant in COL11A1 encoding collagen (α1) XI (rs3753841; NM_080629.2_c.4004C>T; p.(Pro1335Leu); p=7.07E-11, OR = 1.118). Using CRISPR mutagenesis we generated Pax1 knockout mice (Pax1-/-). In postnatal spines we found that PAX1 and collagen (α1) XI protein both localize within the intervertebral disc-vertebral junction region encompassing the growth plate, with less collagen (α1) XI detected in Pax1-/- spines compared to wild-type. By genetic targeting we found that wild-type Col11a1 expression in costal chondrocytes suppresses expression of Pax1 and of Mmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, the latter suppression was abrogated in the presence of the AIS-associated COL11A1P1335L mutant. Further, we found that either knockdown of the estrogen receptor gene Esr2 or tamoxifen treatment significantly altered Col11a1 and Mmp3 expression in chondrocytes. We propose a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering a PAX1-COL11a1-MMP3 signaling axis in spinal chondrocytes.

Adolescent idiopathic scoliosis (AIS) is a twisting deformity of the spine that occurs during periods of rapid growth in children worldwide. Children with severe cases of AIS require surgery to stop it from getting worse, presenting a significant financial burden to health systems and families. Although AIS is known to cluster in families, its genetic causes and its inheritance pattern have remained elusive. Additionally, AIS is known to be more prevalent in females, a bias that has not been explained. Advances in techniques to study the genetics underlying diseases have revealed that certain variations that increase the risk of AIS affect cartilage and connective tissue. In humans, one such variation is near a gene called Pax1, and it is female-specific. The extracellular matrix is a network of proteins and other molecules in the space between cells that help connect tissues together, and it is particularly important in cartilage and other connective tissues. One of the main components of the extracellular matrix is collagen. Yu, Kanshour, Ushiki et al. hypothesized that changes in the extracellular matrix could affect the cartilage and connective tissues of the spine, leading to AIS. To show this, the scientists screened over 100,000 individuals and found that AIS is associated with variants in two genes coding for extracellular matrix proteins. One of these variants was found in a gene called Col11a1, which codes for one of the proteins that makes up collagen. To understand the relationship between Pax1 and Col11a1, Yu, Kanshour, Ushiki et al. genetically modified mice so that they would lack the Pax1 gene. In these mice, the activation of Col11a1 was reduced in the mouse spine. They also found that the form of Col11a1 associated with AIS could not suppress the activation of a gene called Mmp3 in mouse cartilage cells as effectively as unmutated Col11a1. Going one step further, the researchers found that lowering the levels of an estrogen receptor altered the activation patterns of Pax1, Col11a1, and Mmp3 in mouse cartilage cells. These findings suggest a possible mechanism for AIS, particularly in females. The findings of Yu, Kanshour, Ushiki et al. highlight that cartilage cells in the spine are particularly relevant in AIS. The results also point to specific molecules within the extracellular matrix as important for maintaining proper alignment in the spine when children are growing rapidly. This information may guide future therapies aimed at maintaining healthy spinal cells in adolescent children, particularly girls.

Association of genetic variation in COL11A1 with adolescent idiopathic scoliosis.

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 the PAX1 gene. Here we sought to define the roles of PAX1 and newly-identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 10,519 individuals with AIS and 93,238 unaffected controls, significant association was identified with a variant in COL11A1 encoding collagen (α1) XI (rs3753841; NM_080629.2_c.4004C>T; p.(Pro1335Leu); P=7.07e-11, OR=1.118). Using CRISPR mutagenesis we generated Pax1 knockout 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 in Pax1-/- spines compared to wildtype. By genetic targeting we found that wildtype Col11a1 expression in costal chondrocytes suppresses expression of Pax1 and of Mmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, this suppression was abrogated in the presence of the AIS-associated COL11A1P1335L mutant. Further, we found that either knockdown of the estrogen receptor gene Esr2, or tamoxifen treatment, significantly altered Col11a1 and Mmp3 expression in chondrocytes. We propose a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering a Pax1-Col11a1-Mmp3 signaling axis in spinal chondrocytes.

Deletion of Pax1 scoliosis-associated regulatory elements leads to a female-biased tail abnormality.

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 the PAX1 gene. 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.

Deletion of CTCF sites in the SHH locus alters enhancer-promoter interactions and leads to acheiropodia.

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.

Genomic characterization of the adolescent idiopathic scoliosis-associated transcriptome and regulome.

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 non-coding sequences that control genes with important roles in cartilage, muscle, bone, connective tissue and intervertebral disks (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 chromatin immunoprecipitation-sequencing against H3 lysine 27 acetylation 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 the ADGRG6 and BNC2 loci. 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 cartilage matrisome in adolescent idiopathic scoliosis.

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.

Recapitulation of gametic DNA methylation and its post-fertilization maintenance with reassembled DNA elements at the mouse Igf2/H19 locus.

BACKGROUND: Paternal allele-specific DNA methylation of the H19 imprinting control region (ICR) regulates imprinted expression of the Igf2/H19 genes. The molecular mechanism by which differential methylation of the H19 ICR is established during gametogenesis and maintained after fertilization, however, is not fully understood. We previously showed that a 2.9-kb H19 ICR fragment in transgenic mice was differentially methylated only after fertilization, demonstrating that two separable events, gametic and post-fertilization methylation, occur at the H19 ICR. We then determined that CTCF/Sox-Oct motifs and the 478-bp sequence of the H19 ICR are essential for maintaining its maternal hypomethylation status and for acquisition of paternal methylation, respectively, during the post-fertilization period. RESULTS: Using a series of 5'-truncated H19 ICR 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 endogenous H19 ICR caused loss of methylation after fertilization, indicating that methylation activity of the sequence is required to protect endogenous H19 ICR 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 endogenous H19 ICR. The fragment was methylated during spermatogenesis; moreover, its allele-specific methylation status was faithfully maintained after fertilization, and imprinted expression of the both Igf2 and H19 genes was recapitulated. CONCLUSIONS: Our results identified a 118-bp region within the H19 ICR that is required for de novo DNA methylation of the paternally inherited H19 ICR during pre-implantation period. A lambda DNA-based artificial fragment that contains the 118-bp sequence, in addition to the previously identified cis elements, could fully replace the function of the H19 ICR in the mouse genome.

Transvection-like interchromosomal interaction is not observed at the transcriptional level when tested in the Rosa26 locus in mouse.

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 comprehensive view of intra- and interchromosomal contacts between specific genomic sites. The locus control region of the ß-globin genes (ß-LCR) is a super-enhancer that is capable of activating all of the ß-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 ß-globin locus, in this way thought to contribute to the formation of a "chromatin hub". The ß-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 at the identical loci, yet on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the ß-LCR and the ß-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because of a lack of suitable regulatory elements that might facilitate such 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 ß-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 ß-globin gene in trans even in this idealized experimental context.

Synthetic DNA fragments bearing ICR cis elements become differentially methylated and recapitulate genomic imprinting in transgenic mice.

BACKGROUND: Genomic imprinting is governed by allele-specific DNA methylation at imprinting control regions (ICRs), and the mechanism controlling its differential methylation establishment during gametogenesis has been a subject of intensive research interest. However, recent studies have reported that gamete methylation is not restricted at the ICRs, thus highlighting the significance of ICR methylation maintenance during the preimplantation period where genome-wide epigenetic reprogramming takes place. Using transgenic mice (TgM), we previously demonstrated that the H19 ICR possesses autonomous activity to acquire paternal-allele-specific DNA methylation after fertilization. Furthermore, this activity is indispensable for the maintenance of imprinted methylation at the endogenous H19 ICR during the preimplantation period. In addition, we showed that a specific 5' fragment of the H19 ICR is required for its paternal methylation after fertilization, while CTCF and Sox-Oct motifs are essential for its maternal protection from undesirable methylation after implantation. RESULTS: To ask whether specific cis elements are sufficient to reconstitute imprinted methylation status, we employed a TgM co-placement strategy for facilitating detection of postfertilization methylation activity and precise comparison of test sequences. Bacteriophage lambda DNA becomes highly methylated regardless of its parental origin and thus can be used as a neutral sequence bearing no inclination for differential DNA methylation. We previously showed that insertion of only CTCF and Sox-Oct binding motifs from the H19 ICR into a lambda DNA (LCb) decreased its methylation level after both paternal and maternal transmission. We therefore appended a 478-bp 5' sequence from the H19 ICR into the LCb fragment and found that it acquired paternal-allele-specific methylation, the dynamics of which was identical to that of the H19 ICR, in TgM. Crucially, transgene expression also became imprinted. Although there are potential binding sites for ZFP57 (a candidate protein thought to control the methylation imprint) in the larger H19 ICR, they are not found in the 478-bp fragment, rendering the role of ZFP57 in postfertilization H19 ICR methylation a still open question. CONCLUSIONS: Our results demonstrate that a differentially methylated region can be reconstituted by combining the activities of specific imprinting elements and that these elements together determine the activity of a genomically imprinted region in vivo.

Homeostatic Response of Mouse renin Gene Transcription in a Hypertensive Environment Is Mediated by a Novel 5' Enhancer.

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.

Long-Range Control of Renin Gene Expression in Tsukuba Hypertensive Mice.

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.

De novo DNA methylation through the 5'-segment of the H19 ICR maintains its imprint during early embryogenesis.

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, whereas it was not methylated in sperm, suggesting that its gametic and postfertilization modifications were 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 and attenuated Igf2 gene expression. These results demonstrate that this segment of the H19 ICR is essential for its de novo postfertilization DNA methylation, and that this activity contributes to the maintenance of imprinted methylation at the endogenous H19 ICR during early embryogenesis.

A mouse renin distal enhancer is essential for blood pressure homeostasis in BAC-rescued renin-null mutant mice.

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.