Architecture and evolutionary conservation ofXenopus tropicalisosteoblast-specific regulatory regions shed light on bone diseases and early skeletal evolution

Abstract

Understanding the genetic mechanisms underpinning the differentiation of osteoblasts, the bone producing cells, has far reaching implications for skeletal diseases and evolution. To this end, it is crucial to characterize osteoblastic regulatory landscape in a diverse array of distantly-related vertebrate species. By comparing of the ATAC-seq profile of Xenopus tropicalis (Xt) osteoblasts to liver, heart and lung control tissues, we identified 524 promoters and 6,750 distal regions whose chromatin is specifically open in osteoblasts. Nucleotide composition, Gene Ontology, and RNA-Seq confirmed that the identified elements correspond to bona fide osteogenic transcriptional enhancers, and TFBS enrichment revealed a well-conserved regulatory logic with mammals. Amongst the 357 Xt osteoblast-specific enhancers aligning to homologous human loci, 127 map to regions annotated as enhancers. Phenotype predictions based on the genes neighbouring these conserved enhancers are tightly related to impaired skeletal development. In addition, six conserved enhancers are located at loci associated to craniosynostosis (mx2, tcf12), osteopoikilosis (lemd3), osteopenia (gorab), skeletal dysplasia (flnb) and craniofacial abnormalities (gpc4). From an evolutionary perspective, the elephant shark genome aligns to 53 Xt osteoblast-specific enhancers that are also conserved and annotated as enhancers in humans, revealing an ancestral osteogenic role for the ATOH8, IRX3, NFAT, NFIB and MEF2C transcription factors, as well as for the FGF, IHH and BMP/TGFb signalling pathways. As the absence of bone in sharks is a derived feature, we propose that, in this lineage, the osteogenic regulatory network has been maintained for its function in odontoblasts. Our data argues in favour of a common origin for dentine and bone, and provides a glimpse into the key regulatory elements and upstream activators that drove the formation of an ancient type of mineralized tissue in the vertebrates that inhabited the oceans more than 460 million years ago.