Options and Considerations When Using a Yeast One-Hybrid System

Comprehensive mapping of protein–DNA interactions is essential to uncover the mechanisms involved in gene regulation. However, the data generated has been sparse given the number of regulatory elements and transcription factors (TFs) encoded in the genomes of metazoan organisms. Yeast one-hybrid (Y1H) assays provide a powerful “DNA-centered” method, complementary to “TF-centered” methods such as chromatin immunoprecipitation, to identify the TFs that can bind a DNA sequence of interest. Here, we present different technical variations that should be considered when using a Y1H system, including the type of DNA sequence to test, source of TF clones, as well as types of vectors and screening format. Finally, we discuss limitations of the assay and future challenges.