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

描述（由申请人提供）：C。线虫tbx-2基因编码T-box转录因子家族的保守成员，该家族对于ABA衍生的咽肌的发育是必需的。tbx-2启动子在咽原基形成前的咽前体中，以及在后期胚胎的神经元和体壁肌肉中具有活性。我们正在研究tbx-2启动子的调节，以了解咽肌在阿坝谱系中是如何被指定的。基于对扩增的转录因子文库进行喂食RNAi筛选的结果，我发现tbx-2启动子活性受到NF-Y CCAAT结合复合物和TBX-2自身调节活性的抑制。NF-Y组分或TBX-2的RNAi敲低导致tbx-2启动子：：gfp在通常不表达tbx-2的组织中的强烈异位表达。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表达来进行这些观察。为了确定额外的机制，调节TBX-2的表达，我也表征TBX-2的转录增强子，这是必要的和足够的表达在胚胎咽。我的目标是确定顺式作用序列的增强子活性的重要性，并确定和功能特性的候选调节tbx-2表达，这些序列的目标。最后，我将进行基于RNAi的筛选，以确定调节tbx-2的转录因子作为弱tbx-2突变体的遗传增强子。总的来说，这些目标将阐明调节TBX-2表达和指定ABA衍生的咽肌命运的发育途径。

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.