ChIP-chip: A genomic approach for identifying transcription factor binding sites

Abstract

Publisher Summary Transcription factors are key regulatory proteins that can influence the expression of hundreds of genes in response to a particular environmental condition or internal cue. The collective set of genes regulated by a transcription factor therefore defines the state of a cell and can determine cell fate. Among the 6200 predicted proteins in the yeast Saccharomyces cerevisiae, there are about 300 transcription factors. Approximately 85% of the yeast transcription factors have been characterized to some extent and some of these are known to play a critical role in cell cycle initiation, pheromone response, mating type switching, pseudohyphal growth, and nutrient and stress response. This chapter develops an approach in yeast that will comprehensively identify genomic sequences directly bound by transcription factors. This approach has been used to successfully identify targets of the yeast transcription factors SBF and MBF. Briefly, protein-DNA complexes are fixed in vivo with formaldehyde and lysed, and the lysate is sonicated to shear DNA. The transcription factor of interest is purified by immunoprecipitation; the associated DNA is extracted and then amplified and labeled for hybridization to a yeast intergenic array. Two important reagents for this method are specific immunoprecipitating antibodies and a yeast intergenic microarray.