Identification of conserved skeletal enhancers associated with craniosynostosis risk genes

Abstract

Craniosynostosis (CS) is a common congenital defect affecting more than 1/2000 infants. Infants with CS have a premature fusion of one or multiple cranial sutures resulting in restricted brain expansion. Single gene mutations account for 15-20% of cases, largely as part of a syndrome, but the majority are nonsyndromic with complex underlying genetics. Two noncoding genomic regions contributing to CS risk were previously identified by GWAS, one near BMP2 and one within BBS9. We hypothesized that the region within BBS9 contains distal regulatory elements controlling the neighboring gene encoding BMPER, a secreted modulator of BMP signaling. To identify regulatory sequences that might underlie disease risk, we surveyed conserved noncoding sequences from both risk loci identified from the GWAS for enhancer activity in transgenic Danio rerio. We identified enhancers from both regions that direct expression to skeletal tissues, consistent with the endogenous gene expression. Importantly, for each locus, we found a skeletal enhancer that also contains a sequence variant associated with CS risk. We examined the activity of each enhancer during craniofacial development and found that the BMPER-associated enhancer is active in the restricted region of cartilage closely associated with frontal bone initiation. We used an enhanced yeast one-hybrid assay to identify transcription factor interactions with several identified enhancers, implicating multiple signaling pathways in their regulation. In a targeted screen focused on risk-associated SNPs, we further identified differential binding to alternate and reference alleles. Additionally, we found that the risk allele of the BMPER enhancer directs significantly broader expression than the reference allele in transgenic zebrafish. Our findings support a specific genetic mechanism to explain the contribution of two risk loci to CS. More broadly, our combined in vivo approach is applicable to many complex genetic diseases to build a link between association studies and specific genetic mechanisms.

TMF suppresses chondrocyte hypertrophy in osteoarthritic cartilage by mediating the FOXO3a/BMPER pathway

“…In addition, in the presence of twisted gastrulation, full-length BMPER inhibits BMP-signaling in C2C12 cells (52). The results of a previous study have also demonstrated that BMPER exerts positive and negative effects on BMP activity, and these effects are context-dependent (53).…”

Transforming growth factor beta signaling and craniofacial development: modeling human diseases in zebrafish

“…Although murine studies have been crucial for understanding the biology of cranial sutures and how suture closure is regulated, zebrafish are emerging as a model organism for understanding this process; zebrafish have been used to model Saethre-Chotzen syndrome (caused by mutations in TWIST1 and TCF12 ) and craniosynostosis caused by alterations to retinoic acid synthesis ( Laue et al, 2011 ; Teng et al, 2018 ). A recently published zebrafish study identified putative enhancers of bmp2 and bmper (a regulator of BMP signaling) that drive reporter gene expression in the osteogenic fronts of transgenic zebrafish calvaria, suggesting that the function of BMP signaling in cranial development and suture homeostasis is conserved in zebrafish ( He et al, 2023 ).…”

“…Studies have shown that zebrafish are a tractable model for studying suture biology and craniosynostosis, and the prospect of using zebrafish to study the suture biology and the pathogenesis of craniosynostosis is exciting ( Laue et al, 2011 ; Teng et al, 2018 ). With respect to BMP signaling, a recent zebrafish study identified that GWAS variants associated with BMP2 lie in putative enhancer regions for BMP2 and BMPER, and that these enhancers likely drive the expression of these genes in the osteogenic fronts of the frontal bones, further implicating BMP signaling in suture homeostasis and craniosynostosis ( Justice et al, 2012 ; He et al, 2023 ). Furthermore, yeast 2 hybrid assays indicated that the GWAS variants in these enhancers abrogate transcription factor binding ability, further suggesting that altered BMP signaling contributes to the pathogenesis of craniosynostosis ( He et al, 2023 ).…”

“…With respect to BMP signaling, a recent zebrafish study identified that GWAS variants associated with BMP2 lie in putative enhancer regions for BMP2 and BMPER, and that these enhancers likely drive the expression of these genes in the osteogenic fronts of the frontal bones, further implicating BMP signaling in suture homeostasis and craniosynostosis ( Justice et al, 2012 ; He et al, 2023 ). Furthermore, yeast 2 hybrid assays indicated that the GWAS variants in these enhancers abrogate transcription factor binding ability, further suggesting that altered BMP signaling contributes to the pathogenesis of craniosynostosis ( He et al, 2023 ). Moreover, unlike human sutures, which fuse by adulthood, zebrafish sutures remain patent throughout life; investigations into the mechanisms by which suture patency is maintained in zebrafish could inform the development of treatments or therapies for individuals affected by craniosynostosis and advance our understanding of stem cells in regenerative medicine ( Topczewska et al, 2016 ).…”

“…Additional zebrafish studies on suture homeostasis are emerging. Laue et al (2011), Teng et al (2018), He et al (2023) Frontiers in Cell and Developmental Biology frontiersin.org (Cubbage and Mabee, 1996). Several of the cartilage elements ossify via endochondral ossification or are degraded after membrane bones form in their place (Cubbage and Mabee, 1996).…”