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

项目摘要 生长板软骨细胞成熟障碍影响骨骼的生长，导致一系列 从骨骼发育不良到极矮身材的疾病。这些不同的情况强调了 从严格调控的软骨细胞生命周期到正常的骨表生理，然而遗传途径 指导软骨细胞成熟的机制还知之甚少。目前的方案利用了一种体外模型 生长板用于(1)进行高通量、全基因组功能敲除(KO)筛选 软骨细胞成熟，(2)优先筛选与人类骨骼生长相关的同源基因，通过 全基因组关联研究(GWAS)，以及(3)调查TOP靶标作用于 影响软骨细胞成熟，从顶级筛选命中激活的蛋白抑制因子1(PIAS1)开始。 作为当前提议的初步数据，我开发了一种筛选试验，其中慢病毒文库 将8万个独特的单引导RNA(SgRNAs)导入Cas9软骨细胞，同时进行KO 有2万个基因在复制。该检测方法可以可靠地检测软骨细胞成熟的遗传决定因素和 已经确定了与骨骼生物学高度相关的基因，包括印度刺猬的成员 发信号的家庭。在目前的应用中，我打算发现软骨细胞的新的遗传决定因素 通过调整我的初步筛选来探测KO，从而在生长板中成熟，从而导致延迟和 并将这些结果与来自人类肢体长度的GWA数据进行交叉。此外，我 我将调查我的假设，即PIAS1通过调节 软骨细胞转录和蛋白质SUMO化，同时为未来的机制研究建立管道 排名靠前的筛选对象。功能基因组筛选可以加速发现基因的新角色 以前从未在人类生长板软骨细胞中进行过研究。通过鉴定新的功能性遗传介体 生长板的成熟，我希望能洞察骨骼发育不良和生长障碍的发展 同时为未来疗法的设计建立目标。

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.