Orthogonal Inducible Transcriptional Control Systems for Investigation of Gene-Gene Interactions

Genes do not act in isolation but through complex networks of interactions. While many double and triple gene interactions have been uncovered in Saccharomyces cerevisiae , most have been identified through overexpression or loss-of-function studies, leaving dosage-dependent interactions largely unexplored. Multi-gene control systems suffer from various drawbacks, including limited range and high cell-to-cell variation of expression across the population. To address this gap, we extend a recently developed inducible expression system that enables precise, tunable control of gene expression to additional repressor-inducer pairs: LacI/IPTG and LexA-hER/β-estradiol. We demonstrate that both new systems can control endogenous genes on their own by placing various low, medium and high expressed endogenous genes under their control. Using all three WTC systems, we show simultaneous and orthogonal double and triple gene control that confirms known gene-gene interactions within the anaphase signaling network and reveals new dosage-dependent phenotypes. Given their precision, orthogonality and compatibility with all commonly used growth media, this collection of WTC systems represents a new tool suitable for precise investigation of dosage-dependent gene-gene interactions in yeast.