Chromatin Dynamics of the CFTR locus

描述(申请人提供)：自从编码囊性纤维化跨膜传导调节蛋白的囊性纤维化相关基因被发现以来，已经出现了几种治疗方法，延长和提高了囊性纤维化患者的生活质量。然而，用于治疗CF的临床方法的这些进展仍然与我们目前涉及CFTR基因的表观遗传调控和转录控制(CFTR)的知识不一致。尽管患有CF的患者和家系的突变具有分子和遗传特征，但在预测与CF相关的疾病结果方面，特征明确的多态之间的关系仍然不一致。这一现实强调了对CFTR是如何调控的，以及转录如何与CF型相对应的了解不足。这项应用的目的是确定染色质中调控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、组蛋白的含量或变异，以及染色质中组蛋白的翻译后修饰。此外，CFTR转录的三维(3D)特征需要其他基因座之间的协同作用，从而导致高阶染色体组织的时间和空间控制，目前还知之甚少。尽管在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，缺乏对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CHD蛋白的遗传和生化检测表明，这些基因产物在发育和疾病中具有显着的重要性。因此，对CHD蛋白家族的个体成员进行功能表征的努力可能会对人类健康和对疾病病理生理的理解产生深远的影响。我们提出了以下具体目标。首先，我们将研究CHD6与其他转录蛋白(如CTCF)协同调控CFTR的作用。这些研究将表征特定的核蛋白与CHD6的相互作用，以调节CFTR基因的转录。其次，我们将确定与CHD6在小鼠中条件性中断相关的病理生理学，并研究其对CFTR表观遗传特征和转录的影响。最后，我们建议用芯片测序、3C/4C和RNA FISH方法来描述CHD6作为CTCF参与多个基因座收敛的狭窄的作用，以协调3D转录程序，以了解与CHD6相关的修改或变异的组蛋白签名和染色体交换的动力学。

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.