Phenotypic Alteration and Target Gene Identification Using Combinatorial Libraries of Zinc Finger Proteins in

We have developed a method with prokaryotic organisms that uses randomized libraries of zinc finger-containing artificial transcription factors to induce phenotypic variations and to identify genes involved in the generation of a specific phenotype of interest. Combining chromatin immunoprecipitation experiments and in silico prediction of target DNA binding sequences for the artificial transcription factors, we identified ubiX, whose down-regulation correlates with the thermotolerance phenotype in Escherichia coli. Our results show that randomized libraries of artificial transcription factors are powerful tools for functional genomic studies. Engineered zinc finger proteins (ZFPs), either those by themselves or those that have been fused to transcriptional activation or inhibition domains, have been used to regulate the transcription of numerous target genes in eukaryotic cells (2, 4, 9, 10, 13). Recent advances in the field allow one to construct ZFP libraries that consist of tens of thousands of active transcription factors (3, 6, 7, 8). Such libraries have been used successfully to scan the yeast and mammalian genomes for genes that induce phenotypic alterations in cells when thei