Auxin Response Factor Specificity in Arabidopsis

描述（由申请人提供）：主要生长素应答的当前模型主要基于关于生长素感知机制的最新发现、生长素应答因子（ARF蛋白）与DNA结合的体外研究以及生长素应答基因表达数据。生长素应答启动子的定义特征知之甚少，指导体内ARF-启动子相互作用的特异性决定因素尚未研究。解决这些问题是理解主要生长素反应和证实生长素在转录中的作用模型的关键下一步。拟议研究的结果将广泛用于植物和动物激素反应的研究，并将与植物生长和发育的研究特别相关。本论文的主要目的是：1）通过对MP/ARF 5和NPH 4/ARF 7转录因子的生长素应答基因表达和染色质免疫沉淀（ChIP）分析，确定ARF的靶位点; 2）通过整合和比较现有数据集，确定MP/ARF 5和NPH 4/ARF 7募集启动子; 3）使用定量启动子-报告基因测定和与截短的和嵌合的ARF蛋白共免疫沉淀的DNA序列的分析来确定ARF蛋白-启动子相互作用中特异性的决定因素

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