Protein moonlighting by a target gene dominates phenotypic divergence of the Sef1 transcriptional regulatory network in yeasts.

Transcriptional rewiring generates phenotypic novelty, acting as an important mechanism contributing to evolutionary development, speciation, and adaptation in all organisms. The phenotypic outcomes (functions) of transcription factor (TF) activity are determined by the combined effects of all target genes in the TF's regulatory network. Plastic rewiring of target genes accumulates during species divergence and ultimately alters phenotypes, indicating a TF functional switch. We define this phenomenon as 'disruptive rewiring', where the rewiring process disrupts the link between a TF and its original target genes that determine phenotypes. Here, we investigate if 'complete' disruptive rewiring is a prerequisite for a TF functional switch by employing chromatin immunoprecipitation sequencing, RNA expression, and phenotypic assays across yeast species. In yeasts where Sef1 targets TCA (tricarboxylic acid) cycle genes, we demonstrate that Sef1 orthologs can promote and inhibit respiratory growth by modulating the moonlighting function of their conserved target, NDE1. This modulation occurs without changing the overall association of Sef1 with TCA cycle genes. We propose that phenotypic masking by NDE1 promotes 'deceptive' disruptive rewiring of the Sef1 regulatory network in Saccharomyces cerevisiae, thereby potentially constraining future evolutionary trajectories.