Mechanisms and Pathology of Long Range Regulation in Limb Development

基因表达在空间和时间上的精确控制是基因功能的重要组成部分，也是人类疾病的重要原因。在这里，我们建议探索与人类肢体发育的调节病理学相关的一些机制，基于我们以前的发现，涉及保守的非编码序列（CNEs）的拷贝数变异（CNVs）可导致先天性畸形。使用一种创新的体内染色体策略（GROMIT），我们将在与肢体畸形相关的整个区域设计一系列靶向缺失和重复的小鼠。我们将产生Cooks综合征的小鼠模型，这是一种短指（趾）畸形伴指甲发育不良，由SOX 9的1Mb基因沙漠5的重复引起，以及与MSX2基因座周围重复相关的锁骨颅骨发育不良。这些小鼠将使我们能够研究这些综合征的发育病理学，并缩小相关的调控因素和机制。我们将使用染色质分析和构象捕获来识别增强子，并描述它们定义的监管格局。通过使用生物信息学工具和湿实验室技术的组合，我们的目标是确定参与肢体发育的大多数调控序列。这些资源和通过详细研究特定模型获得的知识将有助于识别和理解肢体畸形患者中的新调控突变。

The precise control of gene expression in space and time is an essential component of gene function, and as such an important cause of human diseases. Here we propose to explore some of the mechanisms associated with regulatory pathologies of limb development in humans based on our previous finding that copy number variations (CNVs) involving conserved non-coding sequences (CNEs) can result in congenital malformations. Using an innovative in vivo chromosomal strategy (GROMIT), we will engineer mice with targeted series of deletions and duplications throughout regions associated with limb deformities. We will produce mouse models for Cooks syndrome, a brachydactyly with nail dysplasia, caused by duplications of a 1Mb gene desert 5 of SOX9, and for cleidocranial dysplasia associated with duplications around the MSX2 locus. These mice will enable us to study the developmental pathology of these syndromes and to narrow down the associated regulatory elements and mechanisms. We will use chromatin profiling and conformation capture to identify enhancers and characterize the regulatory landscape they define. By using a combination of bioinformatic tools and wet lab techniques, we will aim at identifying most regulatory sequences involved in limb development. These resources and the knowledge gained by studying in detail specific models will facilitate the identification and understanding of new regulatory mutations in patients with limb malformations.