Tissue-specific regulation of a gene essential for airway epithelial function

摘要 囊性纤维化(CF)的新疗法已取得重大进展。这一毁灭性的继承 疾病是由囊性纤维化跨膜传导调节因子(CFTR)基因突变引起的， 它编码一种多功能蛋白(Cftr)，在调节阴离子跨细胞运输方面起着关键作用。 膜。CFTR在呼吸道、胰腺、肠道和男性生殖管中具有特别好的特征 上皮细胞。目前恢复有缺陷CFTR的正常活动的药理学方法侧重于纠正 错误折叠突变体的细胞转运和电导错误的离子通道的增强活性。 这两类突变都会增加CFTR基因的表达，从而增加CFTR蛋白的数量 底物，可能会提高体内的药物效果。此外，现有的治疗目标不会使 约15%的患者缺乏足够的功能性CFTR，原因是基因表达发生突变。一个详细的 了解控制基因的正常转录机制是成功的先决条件。 调控cftr表达的途径。在之前的R01资助期，我们实现了我们的目标 阐明CFTR基因在呼吸道和肠道上皮细胞中的组织特异性调控途径。这个 Cftr基因座位于CCCTC结合因子(CTCF)建立的拓扑相关结构域(TAD)内 绝缘体元件。在这种环境中，远端顺式作用增强子与 基因启动子通过由粘附素复合体稳定的环机制来实现。增强剂是细胞- 类型特定，并与不同的激活或抑制转录因子(TF)相关。建立在我们的 大大提高了对CFTR法规的理解，我们将追求三个具体目标，以解决 靶向调控CFTR基因表达将增加功能性CFTR的首要假设 细胞表面的蛋白质。单独或与药理作用联合提高转录水平 方法，将缓解疾病表型。此外，我们假设，对 协调细胞类型特异性基因表达的顺式元件和相互作用因子将揭示新的途径 增强cftr转录。在第一个目标中，我们将确定细胞特异性顺式调节元件如何 协调内源性CFTR基因的表达。实验将结合CRISPR/CAS9靶向 对这些元件进行基因表达分析和位点构型分析。在第二个目标中，我们将 破译在原代呼吸道上皮细胞中调节CFTR表达的转录网络。 候选转录因子将通过全基因组方法进行检查，以揭示它们对基因座的影响。第三个目标 我们将确定和表征激活CFTR基因表达的途径，以增强CFTR mRNA和 功能性CFTR蛋白的表达。这些结果将使新的方法能够在 呼吸道治疗方面的益处，并将为其他复杂遗传病的新疗法提供信息。

Summary Significant progress has been made towards new therapies for Cystic Fibrosis (CF). This devastating inherited disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a multifunctional protein (CFTR) with a pivotal role in regulating anion transport across cell membranes. CFTR is particularly well characterized in airway, pancreas, intestine and male genital duct epithelia. Current pharmacological approaches to restore normal activity to defective CFTR focus on correcting cellular trafficking of misfolding mutants and on potentiating activity of ion channels with faulty conductance. For both classes of mutation increasing CFTR gene expression and hence the amount of CFTR protein substrate, would likely enhance in vivo drug efficacy. Moreover, existing therapeutic goals will not benefit the ~15% of patients lacking sufficient functional CFTR due to mutations that disrupt gene expression. A detailed understanding of the normal transcriptional mechanisms controlling the gene is a prerequisite for successful approaches to modulate CFTR expression. In the previous R01 funding period we achieved our goal to elucidate the tissue-specific control pathways of the CFTR gene in airway and intestinal epithelial cells. The CFTR locus lies within a topologically associating domain (TAD) established by CCCTC-binding factor (CTCF) insulator elements. Within this environment, distal cis-acting enhancers are brought into close association with the gene promoter by a looping mechanism that is stabilized by the cohesin complex. The enhancers are cell- type specific and associate with different activating or repressing transcription factors (TFs). Building on our greatly improved understanding of CFTR regulation, we will pursue three specific aims addressing the overarching hypothesis that targeted modulation of CFTR gene expression will increase functional CFTR protein at the cell surface. Increasing transcript levels, alone or in combination with pharmacological approaches, will alleviate disease phenotype. Further, we hypothesize that an enhanced understanding of the cis-elements and interacting factors coordinating cell-type specific gene expression will reveal new ways to augment CFTR transcription. In the first aim we will determine how cell-specific cis-regulatory elements coordinate expression of the endogenous CFTR locus. Experiments will combine CRISPR/Cas9 targeting of these elements with gene expression assays and analysis of locus architecture. In the second aim we will decipher the transcriptional network that regulates CFTR expression in primary airway epithelial cells. Candidate TFs will be examined by genome-wide methods to reveal their impact on the locus. In the third aim we will identify and characterize pathways that activate CFTR gene expression to enhance CFTR mRNA and functional CFTR protein expression. These results will enable novel approaches to increase CFTR in the airway for therapeutic benefit and will inform new therapies for other complex genetic diseases.