Orthogonal inducible transcriptional control systems for investigation of gene-gene interactions in Saccharomyces cerevisiae

Abstract Genes function within dense networks of interactions, where phenotypes often depend on the relative expression levels rather than loss or gain of function. Most genetic interactions in Saccharomyces cerevisiae have been identified using deletions or overexpression, leaving dosage-dependent interactions underexplored. Progress in this area requires transcriptional control systems that enable simultaneous, precise regulation of multiple genes with minimal cross-talk. Here, we expand the recently developed Well-tempered Controller (WTC 846 ), a TetR-repressible system that provides precise, graded control of gene expression in response to anhydrotetracycline. We extend this framework to two orthogonal repressor-inducer pairs: LexA-hER/β-estradiol and LacI/IPTG, establishing three compatible transcriptional controllers. We demonstrate robust, tunable regulation of endogenous genes spanning low to high expression levels. We achieve simultaneous, graded, and precise double and triple gene control, validating known dosage-dependent interactions within the anaphase signaling network and uncovering additional phenotypes. These orthogonal controllers provide a versatile toolkit for dissecting dosage-sensitive gene-gene interactions and facilitate systematic exploration of complex genetic networks in yeast.