Tissue-specific regulation of a gene essential for normal airway epithelia

描述(由申请人提供)：与我们对基因簇表达调控机制的详细了解相比，例如在造血细胞中的珠蛋白基因位点，我们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。对许多基因的表达进行时间和空间调节的遗传元件位于基因启动子之外，并且深埋在内含子或基因间序列中。此外，存在功能障碍，以防止来自一个基因的调节信号干扰表现出不同组织特异性的侧翼基因的表达。囊性纤维化跨膜传导调节基因是囊性纤维化跨膜传导调节基因的一个例子，该基因对特化上皮细胞的功能至关重要，并显示出涉及内含子调控元件的复杂表达模式。CFTR型氯离子通道的缺失会损害分泌上皮细胞的功能，导致囊性纤维化。Cftr的基本启动子本身并不能在基因上提供准确的时间和空间表达，而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，控制组织特异性调节CFTR基因在呼吸道上皮细胞中的表达。我们将确定和表征顺式作用的调节元件和交易因素，以决定CFTR的气道特异性表达。此外，我们将建立这些控制序列在染色质中的作用机制，并确定它们如何影响基因表达。这些实验可能揭示对呼吸道上皮基因表达具有普遍重要性的调节途径。我们将检验这一假设，即上皮特异性调节CFTR基因表达是通过呼吸和肠道上皮中不同的机制实现的。我们认为，在这两种组织中，CFTR的表达是由与内含子元件结合的转录因子控制的，但两种上皮中使用的反式作用因子和顺式元件不同。我们进一步认为，CFTR维持在染色质结构域中，两侧有5‘和3’边界元件，有助于在不同细胞环境中协调基因表达的调节。具体目标1将定义组织特有的顺式调控元件，这些元件协调呼吸道上皮中CFTR的表达，并确定与这些元件相互作用的反式作用因子。具体目标2将确定呼吸道上皮细胞中CFTR基因的染色质结构域，并确定在活跃或不活跃的构型中维持该结构域所涉及的因素。这些研究将解决我们对CFTR理解上的一个重要差距，并为设计在体内调节CFTR表达水平的新型CF疗法提供机会。此外，对CFTR调控的更好了解有可能增加基因治疗方案的临床成功。与公共卫生相关。囊性纤维化跨膜传导调节基因(CFTR)，当突变时会导致CF，具有复杂的组织特异性和时间调节模式。总体而言，控制这一点的因素特征不佳。我们的团队在确定和阐明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，并专注于阐明可能与呼吸道CFTR表达特别相关的新的调节机制。这项工作可能带来的翻译机会包括在体内调节CFTR的表达，以及构建有效的、组织特异性的基因治疗载体。

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.