Transcription Network Controlling Drosophila Development

描述（由申请人提供）：本资助的主要目的是建立研究动物完整转录网络的策略。“后基因组时代”的一个基本挑战是破译包含在广泛的顺式作用DNA序列中的转录信息，这些序列指导复杂生物体中复杂的基因表达模式。我们认为，如果没有对转录调控网络的所有组成部分进行系统的表征，就无法完全应对这些挑战。早期果蝇胚胎中的转录网络非常适合这种系统范围的分析，因为它提供了强大的分子和遗传工具，相对简单，并且包含已知的，可处理的调节因子数量。利用该系统，我们建议开发方法和策略来收集四类基本数据，并使用这些数据进行生物信息学分析，预测转录因子在体内结合的调控序列，决定因子与启动子结合后如何相互作用的组合代码，以及特定调控序列驱动的表达模式。在整个项目中，数据收集方法将根据我们对数据的分析进行细化和修改。我们的具体目标是：

DESCRIPTION (provided by applicant): The chief aim of this grant is to establish a strategy to study complete transcription networks in animals. A fundamental challenge in the "post genome era" is to decipher the transcriptional information contained in the extensive cis-acting DNA sequences that direct intricate patterns of gene expression in complex organisms. We argue that these challenges cannot be fully met without a systematic characterization of all the components of the transcription regulatory network. The transcriptional network in the early Drosophila embryo is uniquely suited for such system-wide analyses because it offers powerful molecular and genetic tools, is relatively simple, and contains a known, tractable number of regulators. Using this system, we propose to develop methods and strategies to collect four essential classes of data and to use these data to develop bioinformatic analyses that predict which regulatory sequences transcription factors bind in vivo, the combinatorial code determining how factors interact once bound to promoters, and the patterns of expression driven by particular regulatory sequences. Throughout the project, the data collection methods will be refined and modified in response to our analysis of the data. Our specific aims are to: 1. Develop a strategy to obtain a new, more detailed understanding of the in vitro DNA binding specificities of transcription factors using a modified binding site selection protocol and other methods. 2. Optimize in vivo crosslinking and genomic microarrays to measure binding of endogenous factors to thousands of DNA elements in living embryos. 3. Develop advanced imaging methods to quantitate gene expression patterns in 3D with single cell resolution and use this information to identify transcription factor target genes. 4. Explore a novel transgenic promoter based strategy to test large numbers of predicted functional cis-regulatory sequences and determine the expression patterns they drive to aid future predictions. 5. Use engineering solutions to increase the throughout of Aims 1-4 to enable comprehensive analysis of not only the early Drosophila network, but also larger networks in other animals, including mammals. 6. Develop bioinformatic tools that utilize the data in Aims 1-4 to analyze the transcriptional network, and make the data and algorithms available to the community at large.