Using a Yeast Inverse One-Hybrid System to Identify Functional Binding Sites of Transcription Factors

Abstract

Binding of transcription factors to promoters is a necessary step to initiate transcription. From an evolutionary standpoint, the regulatory proteins and their binding sites are considered to have molecularly coevolved. We developed an efficient yeast strategy, an “inverse one-hybrid system”, to identify binding targets of transcription factors globally in a genome of interest. The technique consists of a yeast strain expressing a ­transcription factor of interest mated to yeast containing a library of random genomic fragments cloned upstream of a reporter gene (URA3). Positive growth on media without uracil denotes a fragment being bound by the transcription factor, e.g., zebrafish FoxI1. The bound fragments in hundreds of positive clones are sequenced and retested for their binding activities using a colony PCR and sequencing strategy. The resulting tools allow for rapid and genomic-wide identification of transcriptional binding targets.