Auxin Response Factor Specificity in Arabidopsis

描述(申请人提供)：目前的初级生长素反应模型主要基于关于生长素感知机制的最新发现、生长素反应因子(ARF蛋白)与DNA结合的体外研究以及生长素反应基因表达数据。生长素反应启动子的定义特征知之甚少，体内指导ARF-启动子相互作用的特异性决定因素也没有被研究过。解决这些问题是理解初级生长素反应和证实生长素在转录中的作用模式的关键的下一步。拟议的研究结果将对植物和动物的激素反应的研究具有广泛的实用价值，并将与植物生长和发育的研究特别相关。通过本方案中提出的实验，我的目标是1)通过分析生长素反应基因的表达和与转录因子MP/ARF5和NPH4/ARF7的染色质免疫沉淀(ChIP)来确定ARF靶基因；2)通过整合和对新的和现有的数据集的比较生物信息学分析来确定MP/ARF5-和NPH4/ARF7-招募启动子；3)通过定量启动子-报告分析和DNA序列分析确定ARF蛋白-启动子相互作用的特异性决定因素

DESCRIPTION (provided by applicant): The current model of the primary auxin response is based largely on recent findings regarding the mechanism of auxin perception, on in vitro studies of DNA binding by Auxin Response Factors (ARF proteins), and on auxin-responsive gene expression data. The defining features of auxin-responsive promoters are very poorly understood, and the determinants of specificity guiding in vivo ARF-promoter interactions have not been investigated. Addressing these questions is a critical next step in understanding the primary auxin response and substantiating the model of auxin action in transcription. The results of the proposed studies will be of broad utility to studies of hormone responses in plants and animals, and will be of particular relevance to studies of plant growth and development. With the experiments presented in this proposal, I aim to 1) identify ARF target loci through analysis of auxin-responsive gene expression and Chromatin Immunoprecipitation (ChIP) with the transcription factors MP/ARF5 and NPH4/ARF7; 2) define MP/ARF5- and NPH4/ARF7-recruiting promoters through consolidation and comparative bioinformatics analysis of new and existing datasets; 3) define determinants of specificity in ARF protein-promoter interactions using quantitative promoter-reporter assays and analysis of DNA sequences coimmunoprecipitating with truncated and chimeric ARF proteins