Abstract
RUNX2 encodes the master bone transcription factor driving skeletal development in vertebrates, and playing a specific role in craniofacial and skull morphogenesis. The anatomically modern human (AMH) features sequence changes in the RUNX2 locus compared with archaic hominins’ species. We aimed to understand how these changes may have contributed to human skull globularization occurred in recent evolution. We compared in silico AMH and archaic hominins’ genomes, and used mesenchymal stromal cells isolated from skull sutures of craniosynostosis patients for in vitro functional assays. We detected 459 and 470 nucleotide changes in noncoding regions of the AMH RUNX2 locus, compared with the Neandertal and Denisovan genomes, respectively. Three nucleotide changes in the proximal promoter were predicted to alter the binding of the zinc finger protein Znf263 and long-distance interactions with other cis-regulatory regions. By surface plasmon resonance, we selected nucleotide substitutions in the 3’UTRs able to affect miRNA binding affinity. Specifically, miR-3150a-3p and miR-6785-5p expression inversely correlated with RUNX2 expression during in vitro osteogenic differentiation. The expression of two long non-coding RNAs, AL096865.1 and RUNX2-AS1, within the same locus, was modulated during in vitro osteogenic differentiation and correlated with the expression of specific RUNX2 isoforms. Our data suggest that RUNX2 may have undergone adaptive phenotypic evolution caused by epigenetic and post-transcriptional regulatory mechanisms, which may explain the delayed suture fusion leading to the present-day globular skull shape.
Highlights
RUNX Family Transcription Factor 2 gene (RUNX2) encodes the master bone transcription factor driving skeletal development in vertebrates, and playing a specific role in craniofacial and skull morphogenesis
The sequences of the RUNX2 locus (Chr6:45,318,000–45,670,000 hg[38], for a total of 352.000 bases) have been aligned in anatomically modern humans (AMH) and ancient species’ (Neandertal and Denisovan) genomes to map the changes occurred during recent evolution
We have identified 459 and 470 changes acquired in AMH compared with Neandertal and Denisovan, respectively (Supplementary File 1 and Fig. 2)
Summary
RUNX2 encodes the master bone transcription factor driving skeletal development in vertebrates, and playing a specific role in craniofacial and skull morphogenesis. AMH has a more “globular” less elongated skull that, besides other features, displays a more prominent frontal bone, suggesting a different ossification pattern of the neurocranium and a delayed metopic suture fusion, compared with archaic s pecies[2,3] This globularization of the human skull resulted from an evolutionary path led by and coherent with the structural and functional evolution of the brain, at a pace that has been marked by subtle genetic changes intervened in the human genome. Upon the sequencing of the entire archaic hominins’ g enomes[4,5,6], numerous efforts have been devoted to shed light on the evolutionary genetic relationships among species by identifying the genomic changes that may have had functional consequences in the morphological patterning of AMH On this regard, the locus containing the RUNX Family Transcription Factor 2 gene (RUNX2) has been identified by Green and collaborators, as part of the genomic regions that underwent a positive selection in AMH after the evolutionary split from N eandertal[4]. It is worth noting that most of the known genes associated with either syndromic or nonsyndromic CS are involved in largely interacting pathways that merge on RUNX2 as the main downstream effector of the osteogenic c ascade[20]
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