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. ? ?
