Chondrogenic Enhancer Landscape of Limb and Axial Skeleton Development

Abstract The precisely orchestrated differentiation of chondrocytes during skeleton development is a critical determinant of human height and body shape and disruptions of this process can cause severe skeletal abnormalities. The ultimate size and shape of each of over 200 bones depends on the intricate spatiotemporal regulation of chondrogenic and chondrocyte differentiation genes, but the genomic architecture coordinating these events remains poorly defined. Here we provide a comprehensive map of transcriptional enhancers specifically active in chondrocytes and show that they provide a mechanistic framework through which noncoding genetic variants can influence human stature. We isolated limb and trunk fetal chondrocytes from mice with a Col2a1 fluorescent regulatory sensor and used RNA-seq to identify 780 genes that are specifically expressed during chondrogenesis. To create cell type-specific enhancer maps, we performed ATAC-seq to map open chromatin regions and ChIP-seq for H3K27ac, an enhancer-associated histone modification, and identified 2’704 putative chondrogenic enhancer regions. Most (74%) of these enhancers showed pan -chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. We found that chondrogenic enhancers are enriched for the binding of several chondrogenic transcription factors including SOX9. Finally, we find that genetic variation overlapping chondrogenic enhancers explains a higher fraction of the heritability of human adult height than the one overlapping non-chondrogenic enhancers. This data provides a comprehensive mapping of the chondrogenic enhancer repertoire, paving the way to interpreting the role of non-coding sequence polymorphisms in phenotypic variation and bone diseases.