Deciphering Mechanisms of Limb Malformations Caused by Noncoding Variants In Vivo

项目总结/文摘

PROJECT SUMMARY/ABSTRACT Limb malformations are the second most common congenital abnormality, occurring in 1 in every 500 live births. Mounting evidence implicates rare noncoding mutations to underlie non-syndromic (isolated) limb malformations. Many of these variants map to transcriptional enhancers, regions of regulatory DNA that tune gene expression. However, a fundamental gap remains in our understanding of the mechanisms by which these variants alter enhancer activity and their role in causing limb defects. The most frequently affected noncoding loci is the limb-specific enhancer of Sonic hedgehog (Shh). With over 30 independent rare variants linked to limb malformations, the Shh limb enhancer is particularly susceptible to so-called Gain-Of-Function (GOF) variants. GOF variants cause enhancer overactivity that leads to ectopic expression of their target genes. However, why GOF variants only cause ectopic gene expression in specific cell types and why only a small subset of enhancers are susceptible to GOF variants are both unknown. GOF variants are among the least understood enhancer mutations that cause human disease. Much of our lack of understanding of how GOF variants contribute to disease is owed to a lack of suitable model systems. In vitro cell culture and organoid-based systems fail to recapitulate ectopic expression from GOF variants nor model their phenotypic consequences. Thus, it is essential to use in vivo systems to determine the functional and clinical significance of GOF variants. To address this major need, our group recently developed a novel mouse enhancer reporter assay that enables highly-reproducible detection of ectopic gene expression in the cells of the anterior limb domain where Shh is normally not expressed. The overall goal of this proposal is to determine the genetic factors mediating the unique susceptibility of anterior limb bud cells and the Shh limb enhancer to GOF variants. I will test the hypothesis that susceptibility to GOF variants is dictated by the regulatory landscape of anterior limb bud cells and a unique, stable higher-order chromatin structure of the Shh locus. To identify the genetic factors that mediate ectopic Shh expression, I will characterize the regulatory landscapes and local chromatin architecture of anterior limb bud cells in which Shh is ectopically active at single-cell resolution. To determine genetic factors that predispose specific enhancers to pathogenesis, I will test the requirement of higher-order chromatin structure for limb malformations resulting from GOF variants. By identifying targetable genetic factors mediating ectopic gene expression, these studies will provide mechanistic insights into how GOF variants in the limb-specific Shh enhancer contribute to limb malformations. Findings resulting from this proposal can also be applied to predict the clinical significance of noncoding variants from patient sequencing data and will have implications for other developmental disorders linked to GOF variants.