Translating Human Height Genetics to Skeletal Biology by Functional Genomics of the Growth Plate

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Project Summary Disorders of growth plate chondrocyte maturation impact the growth of the skeleton, resulting in a spectrum of diseases from skeletal dysplasia to extreme short stature. These diverse conditions underscore the importance of the tightly regulated chondrocyte life cycle to normal epiphysial physiology, yet the genetic pathways directing chondrocyte maturation are poorly understood. The current proposal leverages an in vitro model of the growth plate to (1) conduct high-throughput, genome-wide functional knock-out (KO) screening of chondrocyte maturation, (2) prioritize screening hits with orthologues linked to human skeletal growth through genome-wide association studies (GWAS), and (3) investigate the mechanisms by which top targets act to affect chondrocyte maturation, beginning with top screening hit, Protein Inhibitor of Activated STAT1 (PIAS1). As preliminary data for the current proposal, I developed a screening assay in which a lentiviral library of 80,000 unique single-guide RNAs (sgRNAs) is transduced into Cas9+ chondrocytes to simultaneously KO 20,000 genes in replicate. This assay can robustly detect genetic determinants of chondrocyte maturation and has already identified genes highly relevant to skeletal biology, including members of the Indian hedgehog signaling family. In the present application, I intend to uncover new genetic determinants of chondrocyte maturation in the growth plate by adapting my preliminary screen to probe KOs leading to both delayed and early chondrocyte maturity and intersect these results with GWAS data from human limb length. Furthermore, I will investigate my hypothesis that PIAS1 acts to delay chondrocyte maturation through its regulation of chondrocyte transcription and protein SUMOylation, while establishing a pipeline for future mechanistic studies of top screening targets. Functional genomic screening can expedite discovery of new roles for genes previously unstudied in human growth plate chondrocytes. By identifying new functional genetic mediators of growth plate maturation, I hope to gain insight into the development of skeletal dysplasia and growth disorders while establishing targets for the design of future therapeutics.