Rapid compensatory evolution by secondary perturbation of a primary disrupted transcriptional network

The discrete steps of transcriptional rewiring have been proposed to occur neutrally to ensure steady gene expression under stabilizing selection over long time-scales, especially when a regulon is being transferred from one transcription factor (TF) to another. Cooperative DNA binding between redundant regulatory components at the intermediate transition stage is believed to mediate this process, enabling a conflict-free switch between two TFs without a disruptive change in gene expression. Here, we have performed an evolutionary repair experiment on the Lachancea kluyveri yeast sef1 Δ mutant by means of a suppressor development strategy. Complete loss of SEF1 forced cells to activate a rewiring process to compensate for the pleiotropic defects arising from misexpression of multiple TCA cycle genes. Using different selective conditions, we identified one generalist and one specialist suppressive loss-of-function mutation of IRA1 and AZF1 , respectively. Our subsequent analyses show that Azf1 is a weak transcriptional activator regulated by the Ras1-PKA pathway. Azf1 loss-of-function triggers extensive gene expression changes responsible for both the compensatory and trade-off phenotypes. Our results indicate that the pleiotropic effects of dual perturbation of transcriptional networks are a potential mechanism for rapid adaptive compensation, facilitating the process of incipient transcriptional rewiring, and formation of complex traits. ### Competing Interest Statement The authors have declared no competing interest.