Characterization of upstream factors regulating the C. elegans T-box gene tbx-2

秀丽隐杆线虫 T-box 基因 tbx-2 上游因子的表征

• 批准号: 7811556
• 负责人: Angenee Cary Milton
• 金额: $ 3.49万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-26 至 2013-01-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7811556
• 关键词: Binding; Boxing; Caenorhabditis elegans; Cis-Acting Sequence; Complex; Development; Ectopic Expression; Embryo; Embryonic Development; Enhancers; Family; Gene Expression; Genes; Genetic; Goals; Indium; Libraries; Molecular Genetics; Muscle; Muscle Development; Neurons; Pathway interactions; Pharyngeal structure; Primordium; RNA Interference; Regulation; Reporter; Reporter Genes; Specific qualifier value; Temperature; Tissues; Trans-Activators; Work; base; feeding; member; mutant; pharynx muscle; promoter; transcription factor

DESCRIPTION (provided by applicant): The C. elegans tbx-2 gene encodes a conserved member of the T-box transcription factor family essential for development of ABa-derived pharyngeal muscles. The tbx-2 promoter is active in pharyngeal precursors prior to formation ofthe pharyngeal primordium, as well as in a subset of neurons and in body wall muscle in later embryos. We are examining regulation of the tbx-2 promoter to understand how pharyngeal muscle is specified in the ABa lineage. Based on results from a feeding RNAi screen of an expanded transcription factor library, I have found that tbx-2 promoter activity is repressed by the NF-Y CCAAT-binding complex and by TBX-2 autoregulatory activity. RNAi knockdown of either NF-Y components or TBX-2 results in strong ectopic expression of tbx-2 promoter::gfp in tissues that do not normally express tbx-2. I plan to pursue these observations by asking if NF-Y and TBX-2 repress endogenous tbx-2 expression, by identifying cis-elements in the tbx-2 promoter targeted by these factors, and by determining if NF-Y and TBX-2 repress tbx-2 expression in the embryo. To identify additional mechanisms regulating tbx-2 expression I am also characterizing a tbx-2 transcriptional enhancer that is necessary and sufficient for expression in the embryonic pharynx. My objective is to identify cis-acting sequences important for enhancer activity, and to identify and functionally characterize candidate regulators of tbx-2 expression that target these sequences. Finally, I will carry out an RNAi-based screen to identify transcription factors regulating tbx-2 as genetic enhancers of a weak tbx-2 mutant. Collectively these aims will elucidate the developmental pathway regulating tbx-2 expression and specifying ABa-derived pharyngeal muscle fate.

描述(由申请人提供)：线虫TBX-2基因编码T-box转录因子家族的保守成员，对ABA来源的咽肌的发育至关重要。在咽原基形成之前，TBX-2启动子在咽前体细胞中活跃，在以后胚胎的神经细胞和体壁肌肉中也是活跃的。我们正在研究TBX-2启动子的调控，以了解咽肌是如何在ABA谱系中指定的。基于对一个扩展的转录因子文库进行RNAi筛选的结果，我发现TBX-2启动子的活性受到NF-Y CCAAT结合复合体和TBX-2自身调节活性的抑制。RNAi敲除NF-Y成分或TBX-2会导致TBX-2启动子：：GFP在不正常表达TBX-2的组织中强烈异位表达。我计划通过以下方式继续这些观察：通过确定这些因子靶向的TBX-2启动子中的顺式元件，以及确定NF-Y和TBX-2是否抑制胚胎中的TBX-2表达，来询问是否NF-Y和TBX-2抑制内源性TBX-2的表达。为了确定其他调节tbx-2表达的机制，我还鉴定了一种tbx-2转录增强子，它是在胚胎咽部表达所必需的，也是足够的。我的目标是识别对增强子活性重要的顺式作用序列，并识别和功能表征针对这些序列的TBX-2表达的候选调控因子。最后，我将进行基于RNAi的筛选，以确定调节tbx-2的转录因子是弱tbx-2突变体的遗传增强剂。总而言之，这些目标将阐明调控Tbx-2表达的发育途径，并确定ABA来源的咽肌的命运。