Generation and in vivo validation of cis-regulatory maps in eukaryotic genomes

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DESCRIPTION (provided by applicant): The initial phases of the ENCODE project resulted in the development and establishment of several high throughput strategies for the rapid identification of predicted functional elements in the human genome. Adequate systems to experimentally validate these predictions are currently lacking, especially methods that can test for elements that act as transcriptional silencers and boundary elements. We propose to generate an in vitro and in vivo platform to allow the efficient testing of predicted enhancer, silencers and insulators in the human genome. We will target a panel of 4 human cell lines as well as mouse embryonic stem cells with reporter vectors integrated in site-specific manner through homologous recombination. By using various vector designs we will develop cell lines that can be used for testing genomic sequences both for transcriptional activation properties as well as silencing and boundary properties, independent of variegation and multiple-copy integration effects. We will establish a low cost, efficient in vivo zebrafish transgenic system to test these putative functional noncoding sequences. We will also generate a mouse model to test insulators and silencers, currently not afforded by standard mouse transgenesis technologies. Finally, we will implement a modification on ChIP chip strategies, by integrating epitope tags to various classes of DNA binding proteins, allowing their use in ChIP chip experiments independent of the availability of specific antibodies raised against each factor being tested. Combined, these strategies will generate a comprehensive and powerful toolkit that can be used for the generation of genome-wide cis-regulatory maps of virtually all DNA-binding proteins, providing an efficient system for the identification of their binding sites as well as a variety of experimental assays for the validation of the predictions made by ChIP chip and computational experiments.