Integrated Approcahes to Find Hox-regulated DNA Elements

描述(由申请人提供)：该合作项目的目标是确定果蝇基因组中由HOX同源结构域蛋白家族直接调控的转录调控元件。为了实现这一目标，将进行三个实验步骤。首先，将使用最先进的基因表达谱方法来分析含有HOX基因突变的胚胎的基因表达差异。将用于基因图谱分析的基因类型包括多余的疼痛/老虎比较，以增加识别真正受Hox调控的基因的可能性。第二步将使用两种方法之一--DamID或染色质免疫沉淀(CHIP)--在体内确定果蝇基因组中哪些基因直接与Hox蛋白结合。与第一步相结合，这些实验将允许在全基因组范围内识别直接的HOX靶基因。这一分析的第三步将是使用计算方法来识别在直接调控的HOX靶基因的子集之间共享的顺式调控元件。计算机识别的调控元件将通过实验得到证实，包括体外蛋白质-DNA结合实验和体内报告基因分析。一旦这些方法被设计出来，它们将适用于生物学中的各种问题。

DESCRIPTION (provided by applicant): The goal of this collaborative project is to identify transcriptional regulatory elements in the Drosophila melanogaster genome that are directly regulated by the Hox family of homeodomain proteins. To accomplish this goal, three experimental steps will be carried out. In the first, state-of-the art gene expression profiling methods will be used to analyze gene expression differences in embryos containing Hox gene mutations. The genotypes that will be used for gene profiling analysis incorporate redundant pain/vise comparisons to increase the probability that bona fide Hox-regulated genes will be identified. The second step will be to use one of two methods -- DamID or chromatin immunoprecipitation (ChIP) - to determine which genes in the Drosophila genome are directly bound by Hox proteins in vivo. Combined with the first step, these experiments will allow the identification of direct Hox target genes on a genome-wide scale. The third step in this analysis will be to use computational approaches to identify cis-regulatory elements that are shared between subsets of the directly regulated Hox target genes. Computationally-identified regulatory elements will be confirmed experimentally, both by in vitro protein-DNA binding experiments and by in vivo reporter gene analysis. Once these methods are worked out, they will be applicable to a wide variety of problems in biology.