An Animal Model for Cornelia de Lange Syndrome

描述（由申请人提供）：Cornelia de Lange综合征（CdLS）是由控制姐妹染色单体内聚的基因突变引起的。CdLS患者表现出产前和产后发育迟缓、智力迟钝、自闭症特征以及四肢和器官的结构异常。出乎意料的是，模式生物研究表明，多种CdLS缺陷是由控制发育的基因表达受到影响引起的，而不是染色单体内聚缺陷。本研究的长期目标是了解内聚因子如何调节基因表达和发育，以增加对CdLS和相关出生缺陷病因的了解。内聚蛋白复合物具有环状结构，主要观点是内聚蛋白通过包围姐妹染色单体介导内聚。NIPBL （Nipped-B-Like）蛋白将内聚蛋白装载到染色体上，大多数CdLS患者具有杂合性功能丧失的NIPBL突变。这些突变使NIPBL降低不到30%，并且不会引起内聚缺陷。一小部分较轻的CdLS病例是由内聚蛋白亚基的错义突变引起的。这些突变也不影响内聚。CdLS最令人困惑的方面是，如此微小的内聚因子变化如何对发育产生如此巨大的影响。在模式生物中，类似的微小变化会改变基因的表达和发育，而不会改变内聚力。在果蝇中，黏结蛋白优先与活性基因结合，细胞系之间的结合差异与基因转录的差异相关。这些数据表明了一种模型，在这种模型中，内聚蛋白包围了活性基因，而转录则解开了染色体。进一步提出，内聚蛋白通过多种机制影响转录。由于内聚蛋白结合如此紧密，它与基因的结合必须由NIPBL动态控制，以促进转录。因此，我们提出CdLS是由改变基因表达的内聚动力学变化引起的。人类和果蝇的内聚因子在结构和功能上有很强的相似性。这项工作将利用高度可适应的果蝇动物模型来阐明内聚因子如何调节基因表达。有三个目的：(1)确定内聚因子如何影响细胞和体内的转录延伸、基因激活和绝缘子功能；(2)利用染色质免疫沉淀确定基因表达如何调节内聚蛋白结合；(3)利用光漂白后的荧光恢复确定内聚因子的变化如何影响体内内聚蛋白染色体结合动力学。希望这些研究的见解将阐明内聚因子的微小变化导致CdLS的机制，并影响诊断和治疗方法的发展。该项目旨在确定在细胞分裂时控制染色体适当分裂的蛋白质如何同时控制果蝇的基因和发育。这些蛋白的人类版本的变化导致了科妮莉亚·德·兰格综合征（CdLS），这是一种严重的发育障碍。CdLS儿童在出生前和出生后都表现出缓慢的生长，并受到智力迟钝和四肢和心脏等器官缺陷的折磨；通过研究这些蛋白质如何在果蝇中起作用，我们将获得有助于寻找诊断和治疗CdLS患者的新方法的知识。

DESCRIPTION (provided by applicant): Cornelia de Lange syndrome (CdLS) is caused by mutations in genes that control sister chromatid cohesion. CdLS patients show slow pre- and postnatal growth, mental retardation, autistic features and structural abnormalities in limbs and organs. Unexpectedly, model organism studies indicate that the diverse CdLS deficits are caused by effects on expression of genes that control development, rather than defects in chromatid cohesion. The long-term goal of this proposal is to learn how cohesion factors regulate gene expression and development to increase understanding of the etiology of CdLS and related birth defects. The cohesin complex has a ring-like structure and the leading idea is that cohesin mediates cohesion by encircling the sister chromatids. The NIPBL (Nipped-B-Like) protein loads cohesin onto chromosomes, and most CdLS patients have heterozygous loss-of-function NIPBL mutations. These mutations reduce NIPBL by less than 30%, and do not cause cohesion defects. A small fraction of milder CdLS cases are caused by missense mutations in cohesin subunits. These mutations also do not affect cohesion. The most puzzling aspect of CdLS is how such small changes in cohesion factors have such dramatic effects on development. In model organisms, similar small changes alter gene expression and development without altering cohesion. In Drosophila, cohesin binds preferentially to active genes, and differences in binding between cell lines correlate with differences in gene transcription. These data suggest a model in which cohesin encircles active genes where transcription unwinds the chromosome. It is further proposed that cohesin affects transcription by multiple mechanisms. Because cohesin binds so tightly, its association with genes must be controlled dynamically by NIPBL to facilitate transcription. Thus it is proposed that CdLS is caused by changes in cohesin dynamics that alter gene expression. There are strong structural and functional parallels between human and Drosophila cohesion factors. The proposed work will take advantage of the highly amenable Drosophila animal model to elucidate how cohesion factors regulate gene expression. There are three aims: (1) determine how cohesion factors affect transcriptional elongation, gene activation, and insulator function in cells and in vivo, (2) determine how gene expression regulates cohesin binding using chromatin immunoprecipitation, and (3) determine how changes in cohesion factors affect cohesin chromosome-binding dynamics in vivo using fluorescence recovery after photobleaching. It is hoped that insights from these studies will shed light on the mechanisms by which small changes in cohesion factors cause CdLS, and impact the development of diagnostic and therapeutic methods. PUBLIC HEALTH RELEVANCE This project is to determine how proteins that control the proper division of chromosomes when cells divide also control genes and development in the fruitfly. Changes in the human versions of these proteins cause Cornelia de Lange syndrome (CdLS), a severe developmental disorder. CdLS children display slow growth before and after birth, and are afflicted with mental retardation and defects in limbs and organs such as the heart; by examining how these proteins work in the fruitfly, we will gain knowledge that will aid the search for new methods to diagnose and treat CdLS patients.