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蛋白（CFTR）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的临床方法的这些进展仍然与我们目前关于CFTR基因（CFTR）的表观遗传调控和转录控制的知识不一致。尽管对CF患者及其家族的突变进行了分子和遗传表征，但在预测CF相关的疾病结局方面，特征化的多态性的关系仍然不一致。这一现实强调了缺乏对CFTR如何调节以及其转录如何与CF表型相对应的理解。本应用程序的目的是确定在染色质中调节CFTR转录的基本机制。基因调控发生在天然染色质环境中，通过改变核小体的流动性、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，对CFTR转录的三维（3D）特征知之甚少，该特征需要其他基因位点之间的合作相互作用，从而产生高阶染色体组织以进行时间和空间控制。尽管在描述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，但在这一特殊的科学里程碑中，缺乏ATP依赖的染色质解旋酶结构域（CHD）蛋白的功能表征及其对特定基因表达程序的贡献。最近对少数哺乳动物冠心病蛋白的遗传和生化检查揭示了这些基因产物在发育和疾病中的显着重要性。因此，研究冠心病蛋白家族成员的功能特征可能会对人类健康和了解疾病的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CHD6与其他转录蛋白（如CTCF）合作调节CFTR的作用。这些研究将描述特定的核蛋白与CHD6相互作用来调节CFTR基因转录。其次，我们将在小鼠中确定与CHD6条件性破坏相关的病理生理学，并研究其对CFTR表观遗传特征和转录的影响。最后，我们建议描述CHD6作为CTCF的一个结构参与CFTR与多个位点的聚合，以协调3D转录程序，使用ChIP测序，3C/4C和RNA FISH方法来了解与CHD6相关的修饰或变异组蛋白特征和染色体交换动力学。

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.