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（如造血细胞中的珠蛋白位点）表达的调控机制的详细知识相比，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。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，深埋在内含子或基因间序列中。此外，存在功能障碍来阻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位点的表达。囊性纤维化跨膜传导调节基因（CFTR）是一个对特化上皮细胞功能至关重要的基因，并显示出涉及内含子调控元件的复杂表达模式。CFTR氯离子通道的缺失会损害分泌上皮的功能，导致囊性纤维化（CF）。仅CFTR的基础启动子并不能确定该基因在时间和空间上的准确表达，对其他调控其表达的元件的功能也知之甚少。在这个项目中，我们将研究控制气道上皮中CFTR基因表达的组织特异性调控的新机制。我们将识别和表征顺式调控元件和决定气道特异性CFTR表达的交易因子。此外，我们将建立这些控制序列在染色质内的作用机制，并确定它们如何影响基因表达。这些实验很可能揭示在气道上皮基因表达中具有普遍重要性的调控途径。我们将验证CFTR基因表达的上皮特异性调控是通过不同机制在呼吸和肠上皮中实现的假设。我们认为，在这两种组织中，CFTR的表达都受到结合内含子元件的转录因子的控制，但在两种上皮中使用了不同的反式作用因子和顺式作用因子。我们进一步提出CFTR维持在染色质结构域中，其两侧有5‘和3’边界元件，促进在不同细胞环境下基因表达的协调调节。特异性目标1将定义组织特异性顺式调控元件，协调气道上皮中CFTR的表达，并确定与这些元件相互作用的反式作用因子。特异性Aim 2将确定气道上皮细胞中CFTR位点的染色质结构域，并确定参与维持该结构域活性或非活性配置的因素。这些研究将填补我们对CFTR理解的重要空白，并为设计调节CFTR在体内表达水平的新型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。公共卫生相关性。囊性纤维化跨膜传导调节基因（CFTR），当突变导致CF时，具有复杂的组织特异性和时间调控模式。一般来说，控制这一过程的元素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，并集中于阐明可能与CFTR在气道中的表达特别相关的新的调控机制。这项工作可能带来的转化机会包括调节CFTR在体内的表达和构建高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.