The gene-regulatory evolution of the human skeleton.

Skeletal modifications were central to human evolution, enabling adaptations for bipedalism, large cranial vaults and childbirth1. Despite their importance, the genetic changes that gave rise to the unique human form remain mostly unknown2. Here we systematically map the gene-regulatory changes that shaped human skeletal evolution. Using massively parallel reporter assays (MPRAs) in chondrocytes, we assayed 561,410 human-derived substitutions in promoters and enhancers, identifying 15,077 loci with human-specific regulatory activity. We then generated human-ape hybrid cells and differentiated them into osteochondral progenitors. Integrating the hybrid cells with MPRA measurements produced genome-wide atlases of human-specific changes in cis-regulatory expression, and the sequence variants that drive them. These atlases reveal an extensive rewiring of the extracellular matrix (ECM), including a marked suppression of glycosaminoglycan (GAG) biosynthesis, leading to an approximately three-to-fourfold reduction in joint GAG content in humans compared with non-human apes. We find that this human-specific shift bears signatures of selection, and is likely to be a key contributor to the exceptional susceptibility of humans to degenerative skeletal diseases3-5. Together, our results reveal a coordinated evolutionary remodelling of the human skeletal ECM, and establish a comprehensive framework for dissecting the genetic basis of human skeletal biology.