Functional analysis of the dCAMTA Transcription factor

描述(由申请人提供)：拟议研究的长期目标是了解钙这种通用信使是如何调节基因表达和细胞活动的。钙离子在调节多种细胞过程中起着关键作用。钙稳态异常与衰老和许多人类疾病有关，如阿尔茨海默病。钙的持久调节作用取决于基因表达的变化。细胞对钙信号携带的信息进行解码并将其转化为基因表达的明显变化的机制尚不清楚。一种流行的模型是，钙离子通过不同的离子通道进入激活不同的转录因子。钙/钙调素通过一系列蛋白激酶或磷酸酶的调节，刺激一系列转录因子。最近的生物信息学分析表明，存在由钙/钙调蛋白直接激活的转录因子，它们可以更快地对钙信号做出反应。最近在植物中发现了一组候选基因，即钙调蛋白结合转录激活因子(CAMTA)。它们在动物中的同源物还有待研究。我们已经分离了果蝇dCAMTA的两个突变等位基因，即苍蝇CAMTA基因，并在这些突变体中检测到一个强烈而一致的表型，表明苍蝇光刺激的钙通道Trp的失活受损。因此，我们计划利用苍蝇光感受器细胞作为检测系统来研究这组新的钙调控转录因子。将结合分子和细胞生物学、遗传学、电生理和钙成像方法：1.在体内验证dCAMTA通过钙/钙调素结合而激活的假说。2：验证dCAMTA存在于细胞质中并在激活时移位到细胞核的假设。3：检验dCAMTA蛋白形成同源多聚体的假设。4.验证光感受器细胞中dCAMTA依赖Trp通道激活的假设。5.验证dCAMTA功能丧失导致光感受器细胞内钙水平升高的假设。6.确定dCAMTA的靶基因。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand how calcium, a universal messenger, regulates gene expression and cell activities. Calcium ions are pivotal in the regulation of a variety of cellular processes. Abnormal calcium homeostasis has been implicated in aging and in numerous human diseases, such as Alzheimer's disease. The enduring regulatory effects of calcium depend on changes in gene expression. The mechanisms by which cells decode the information carried by calcium signals and convert them into distinct alterations in gene expression is poorly understood. A prevailing model is that Ca2+ entry through different ion channels activates distinct transcription factors. Through the mediation of a series of protein kinases or phosphatases, Ca2+/calmodulin stimulates a range of transcription factors. Recent bioinformatic analyses have suggested the existence of transcription factors that are activated directly by Ca2+/calmodulin, which could respond to Ca2+ signals more quickly. A group of candidates, the calmodulin-binding transcription activators (CAMTAs) were recently identified in plants. Their homologs in animals have yet to be studied. We have isolated two Drosophila mutant alleles of dCAMTA, the fly CAMTA gene, and detected a strong and consistent phenotype in these mutants that implies an impaired deactivation of the fly light-stimulated Ca2+ channel TRP. Therefore we plan to use the fly photoreceptor cells as an assay system to study this new group of Ca2+-regulated transcription factors. A combination of molecular and cell biological, genetic, electrophysiological and calcium imaging approaches will be used to: 1. Test the hypothesis that dCAMTA is activated through Ca2+/calmodulin-binding in vivo. 2: Test the hypothesis that dCAMTA exists in the cytoplasm and translocates into the nuclei upon activation. 3: Test the hypothesis that dCAMTA proteins form homomultimers. 4. Test the hypothesis that dCAMTA depends on TRP channels for activation in the photoreceptor cells. 5. Test the hypothesis that the loss of dCAMTA function leads to increased Ca2+ level in the photoreceptor cells. 6. Determine the target genes of dCAMTA.