Principle of Y2H and Y1H assays. (Top) Yeast two-hybrid (Y2H) assays detect protein–protein interactions (PPIs). The “bait” protein is expressed as a fusion (or “hybrid”) with the DNA-binding domain (DB) of the yeast transcription factor Gal4, whereas the “prey” protein is expressed as a fusion with the Gal4 activation domain (AD). The DB-bait fusion protein can bind Gal4-binding sites artificially cloned upstream of reporter gene(s) that are integrated within the genome of the host yeast strain. Following an interaction with a prey protein, a functional Gal4 TF is created, and reporter gene expression is activated. (Bottom) Yeast one-hybrid (Y1H) assays detect protein–DNA interactions (PDIs). The “prey” is usually a transcription factor (TF) expressed as a fusion with the Gal4 AD, whereas the “bait” is a DNA fragment of interest. The DNA-bait is cloned upstream of reporters, and each DNA-bait::reporter cassette is integrated into the yeast genome. If the prey binds the bait, reporter gene expression is induced by the AD regardless of whether the prey TF is an activator or repressor in its native form.