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

描述（由申请人提供）：秀丽隐杆线虫的tbx-2基因编码T-box转录因子家族的一个保守成员，这对aba衍生的咽肌的发育至关重要。在咽部原基形成之前，tpx -2启动子在咽部前体中活跃，在后期胚胎的一部分神经元和体壁肌中也活跃。我们正在研究tbx-2启动子的调控，以了解咽肌是如何在ABa谱系中被指定的。根据扩增转录因子库的RNAi筛选结果，我发现tbx-2启动子活性受到NF-Y ccaat结合复合体和tbx-2自调节活性的抑制。RNAi敲低NF-Y成分或TBX-2都会导致TBX-2启动子：：gfp在正常不表达TBX-2的组织中强烈异位表达。我计划通过询问NF-Y和TBX-2是否抑制内源性TBX-2表达，通过鉴定这些因子靶向的TBX-2启动子中的顺式元件，以及通过确定NF-Y和TBX-2是否抑制胚胎中的TBX-2表达来继续这些观察结果。为了确定调节tbx-2表达的其他机制，我还描述了一个tbx-2转录增强子，这是胚胎咽部表达所必需和充分的。我的目标是鉴定对增强子活性重要的顺式作用序列，并鉴定针对这些序列的tbx-2表达的候选调节因子并对其进行功能表征。最后，我将进行基于rnai的筛选，以确定作为tbx-2弱突变体遗传增强子的调节tbx-2的转录因子。总的来说，这些目标将阐明调节tbx-2表达的发育途径，并指定aba衍生的咽肌命运。

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.