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

描述（由申请人提供）：与我们对控制基因簇（如造血细胞中的珠蛋白基因座）表达的调控机制的详细了解相比，对其他分化细胞类型中单个基因的组织特异性表达知之甚少。赋予许多基因表达时间和空间调控的遗传元件位于基因启动子之外，并深埋在内含子或基因间序列中。此外，有功能障碍，以防止来自一个基因的调控信号干扰侧翼位点的表达，显示不同的组织特异性。对特化上皮细胞的功能至关重要并且显示出涉及内含子调控元件的复杂表达模式的基因的一个实例是囊性纤维化跨膜传导调节基因（CFTR）。CFTR氯离子通道的缺失损害分泌上皮的功能，引起囊性纤维化（CF）。单独的CFTR的基础启动子不赋予该基因准确的时间和空间表达，并且对调节其表达的其他元件的功能知之甚少。在这个项目中，我们将研究新的机制控制CFTR基因表达的组织特异性调节在气道上皮。我们将确定和表征决定CFTR气道特异性表达的顺式作用调节元件和反式作用因子。此外，我们将建立染色质内这些控制序列的作用机制，并确定它们如何影响基因表达。这些实验很可能揭示气道上皮细胞基因表达中具有普遍重要性的调控途径。我们将测试的假设，上皮特异性调节CFTR基因的表达是通过不同的机制，在呼吸道和肠上皮细胞。我们建议，在这两种组织中，CFTR的表达是由转录因子结合到内含子元件，但不同的反式作用因子和顺式元件中使用的两个上皮细胞。我们进一步提出CFTR维持在染色质结构域中，两侧是5'和3'边界元件，其促进不同细胞环境中基因表达的协调调节。具体目标1将定义组织特异性顺式调节元件，其在气道上皮中协调CFTR表达，并鉴定与这些元件相互作用的反式作用因子。具体目标2将确定气道上皮细胞中CFTR基因座的染色质结构域，并确定参与维持该结构域处于活性或非活性构型的因子。这些研究将解决我们对CFTR理解的一个重要空白，并为设计体内调节CFTR表达水平的新型CF疗法提供机会。此外，更好地了解CFTR调控有可能增加基因治疗方案的临床成功率。公共卫生相关性。囊性纤维化跨膜传导调节基因（CFTR），当突变时导致CF，具有组织特异性和时间调节的复杂模式。控制这一点的因素，一般来说，没有得到很好的描述。我们的团队在识别和阐明CFTR的控制机制方面取得了实质性进展。目前的研究计划建立在这一进展的基础上，并集中在阐明新的调节机制，可能是特别相关的CFTR在气道中的表达。这项工作可能产生的翻译机会包括调节CFTR在体内的表达和构建有效的组织特异性基因治疗载体。

DESCRIPTION (provided by applicant): In comparison to our detailed knowledge of the regulatory mechanisms governing the expression of gene clusters, such as the globin loci in hematopoietic cells, much less is known about the tissue specific expression of individual genes in other differentiated cell types. The genetic elements that confer temporal and spatial regulation on the expression of many genes lie outside the gene promoters and are buried deep within intronic or intergenic sequences. Moreover, there are functional barriers to prevent regulatory signals from one gene interfering with the expression of flanking loci that show divergent tissue-specificity. One example of a gene that is essential to the function of specialized epithelial cells and shows a complex expression pattern involving intronic regulatory elements, is the cystic fibrosis transmembrane conductance regulator gene (CFTR). Absence of the CFTR chloride ion channel impairs the function of secretory epithelia, causing cystic fibrosis (CF). The basal promoter of CFTR alone does not confer accurate temporal and spatial expression on the gene and little is known about the function of other elements regulating its expression. In this project we will investigate novel mechanisms controlling tissue-specific regulation of CFTR gene expression in the airway epithelium. We will identify and characterize cis-acting regulatory elements and transacting factors that determine airway-specific expression of CFTR. Moreover, we will establish the mechanisms of action of these control sequences within chromatin and determine how they influence gene expression. These experiments are likely to reveal regulatory pathways that are of general importance in gene expression in the airway epithelium. We will test the hypothesis that epithelial-specific regulation of CFTR gene expression is achieved by different mechanisms in the respiratory and intestinal epithelium. We propose that in both tissues, CFTR expression is controlled by transcription factors binding to intronic elements but that different trans-acting factors and cis-elements are utilized in the two epithelia. We further propose that CFTR is maintained in a chromatin domain, flanked by 5' and 3' boundary elements that facilitate coordinated regulation of gene expression in different cellular environments. Specific Aim 1 will define the tissue-specific cis-regulatory elements that orchestrate CFTR expression in the airway epithelium and identify the trans-acting factors that interact with these elements. Specific Aim 2 will determine the chromatin domain of the CFTR locus in airway epithelial cells and identify the factors that are involved in the maintenance of this domain in active or inactive configurations. These studies will address an important gap in our understanding of CFTR and provide opportunities to design novel CF therapies that modulate CFTR expression levels in vivo. Moreover, a better knowledge of CFTR regulation has the potential to increase the clinical success of gene therapy protocols. PUBLIC HEALTH RELEVANCE. The cystic fibrosis transmembrane conductance regulator gene (CFTR), that when mutated causes CF, has a complex pattern of tissue-specific and temporal regulation. The elements that control this are, in general, poorly characterized. Our team has made substantial progress on identifying and elucidating the control mechanisms for CFTR. The current research program builds upon this progress and concentrates on elucidating novel regulatory mechanisms that may be particularly relevant to CFTR expression in the airway. Translational opportunities that may arise from this work include modulating CFTR expression in vivo and the construction of efficient, tissue-specific gene therapy vectors.