Chromatin Dynamics of the CFTR locus

描述（由申请人提供）：自从发现编码囊性纤维化跨膜传导调节因子（CFTR）蛋白的囊性纤维化（CF）基因以来，已经出现了几种治疗方法，这些方法延长并提高了CF患者的生活质量。然而，用于治疗CF的临床方法中的这些进展仍然与我们目前涉及CFTR基因（CFTR）的表观遗传调控和转录控制的知识不一致。尽管患有CF的患者和家庭的突变具有分子和遗传特征，但充分表征的多态性在预测与CF相关的疾病结局方面仍然不一致。这一现实强调了对CFTR如何调节以及转录如何与CF表型相对应的理解的缺乏。本申请的目的是确定调节CFTR转录的染色质中的基本机制。基因调控发生在天然染色质环境中，通过改变核小体迁移率、组蛋白含量或变异以及染色质中组蛋白的翻译后修饰。此外，CFTR转录的三维（3D）特征需要其他基因位点之间的协同相互作用，从而产生用于时间和空间控制的高阶染色体组织，目前还知之甚少。尽管在表征许多共价翻译后组蛋白修饰及其对基因表达的表观遗传编程的贡献方面取得了进展，但在这一特定的科学里程碑中，缺乏ATP依赖性染色体-解旋酶结构域（CHD）蛋白的功能表征及其对特定基因表达程序的贡献。最近的遗传和生化检查只有少数哺乳动物CHD蛋白揭示了显着的重要性，这些基因产物在发展和疾病。因此，对CHD蛋白质家族的单个成员进行功能表征的努力可能会对人类健康和理解疾病病理生理学产生深远的影响。我们提出以下具体目标。首先，我们将研究CHD 6与其他转录蛋白如CTCF合作调节CFTR的作用。这些研究将表征与CHD 6的特异性核蛋白相互作用以调节CFTR基因转录。其次，我们将确定与小鼠中CHD 6的条件性破坏相关的病理生理学，并研究对CFTR的表观遗传特征和转录的影响。最后，我们建议表征CHD 6作为CTCF的狭窄参与的作用，用于多个位点与CFTR的会聚，以协调使用ChIP测序，3C/4C和RNA FISH方法的3D转录程序，以了解与CHD 6相关的修改或变体组蛋白签名和染色体交换的动态。 囊性纤维化（CF）是一种常见的人类遗传性疾病。这项工作的目标是研究如何CFTR，基因负责CF，是调节在表观遗传水平上通过染色质重塑蛋白称为chromodomain-helicase DNA -binding protein 6（CHD 6）。

DESCRIPTION (provided by applicant): Since the discovery of the gene responsible for cystic fibrosis (CF) encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, several therapeutic approaches have emerged that have extended and enhanced the quality of life for patients with CF. However, such advances in clinical approaches used for treating CF still remain at odds with our current knowledge involving the epigenetic regulation and transcriptional control of the CFTR gene (CFTR). Despite the molecular and genetic characterization of mutations in patients and families afflicted with CF, the relationship of well -characterized polymorphisms still remain inconsistent in predicting disease outcome associated with CF. This reality stresses a lack in understanding how CFTR is regulated and how transcription of corresponds with the CF phenotype. The objective of this application is to determine the fundamental mechanisms in chromatin that regulate CFTR transcription. Gene regulation occurs within the native chromatin environment through the alteration of nucleosome mobility, histone content or variation, and post-translational modification of histones in chromatin. Furthermore, less is still known of the three dimensional (3D) characteristics of CFTR transcription requiring the cooperative interactions between other gene loci that give rise to the high order chromosomal organization for temporal and spatial control. Despite the advance in the characterizing many covalent post-translational histone modifications and their contribution to the epigenetic programming of gene expression, absent among this particular scientific milestone has been the functional characterization of ATP - dependent chromo - helicase domain (CHD) proteins and their contribution to specific gene expression programs. Recent genetic and biochemical examination of only a few mammalian CHD proteins reveal the remarkable importance of these gene products during development and in disease. Therefore, efforts to functionally characterize the individual members of the CHD family of proteins will likely have profound impact on human health and understanding disease patho-physiology. We propose the following specific aims. First, we will investigate the role of CHD6 to cooperate with additional transcriptional proteins such as CTCF to regulate CFTR. These studies will characterize specific nuclear protein interactions with CHD6 to regulate CFTR gene transcription. Second, we will determine the patho -physiology associated with conditional disruption of CHD6 in mice and study the consequence on the epigenetic signature and transcription of CFTR. Finally, we propose to characterize the role of CHD6 to participate as a stricture with CTCF for the convergence of multiple loci with CFTR to coordinate a 3D transcriptional program using ChIP sequencing, 3C/4C, and RNA FISH approaches to understand the modified or variant histone signatures and dynamics of chromosomal exchange associated with CHD6. PUBLIC HEALTH RELEVANCE: Cystic fibrosis (CF) is a common human genetic disease. The goal of this work is to study how CFTR, the gene responsible for CF, is regulated at the epigenetic level through the chromatin remodeling protein called chromodomain -helicase DNA -binding protein 6 (CHD6).