Transcriptional Derepression Uncovers Cryptic Higher-Order Genetic Interactions

Abstract

Disruption of certain genes can reveal cryptic genetic variants that do not typically show phenotypic effects. Because this phenomenon, which is referred to as 'phenotypic capacitance', is a potential source of trait variation and disease risk, it is important to understand how it arises at the genetic and molecular levels. Here, we use a cryptic colony morphology trait that segregates in a yeast cross to explore the mechanisms underlying phenotypic capacitance. We find that the colony trait is expressed when a mutation in IRA2, a negative regulator of the Ras pathway, co-occurs with specific combinations of cryptic variants in six genes. Four of these genes encode transcription factors that act downstream of the Ras pathway, indicating that the phenotype involves genetically complex changes in the transcriptional regulation of Ras targets. We provide evidence that the IRA2 mutation reveals the phenotypic effects of the cryptic variants by disrupting the transcriptional silencing of one or more genes that contribute to the trait. Supporting this role for the IRA2 mutation, deletion of SFL1, a repressor that acts downstream of the Ras pathway, also reveals the phenotype, largely due to the same cryptic variants that were detected in the IRA2 mutant cross. Our results illustrate how higher-order genetic interactions among mutations and cryptic variants can result in phenotypic capacitance in specific genetic backgrounds, and suggests these interactions might reflect genetically complex changes in gene expression that are usually suppressed by negative regulation.

Fig 1. Capacitance, higher-order genetic interactions, and genetic background effects might be related phenomena that involve interactions among capacitating mutations and cryptic variants.

‘YFG’ and ‘yfgΔ’ refer to the wild type and mutant alleles of a gene that can genetically interact with cryptic variants. The green yeast indicates the combination of a capacitating mutation and cryptic variants that shows a phenotypic effect.

Fig 2. Colony morphology phenotypes that occur in the BYx3S cross in the presence of ira2Δ2933.

BY, 3S, and most segregants show a smooth phenotype, while a small fraction of segregants show a rough phenotype.

Fig 3. Characterization of the six-way genetic interaction.

(A) Allele frequency plots for BY and 3S second iteration backcross populations of END3 ^3S rough strains. Fixed loci are denoted with a blue, orange, or grey bars depending on whether the BY, 3S, or mutant alleles, respectively, were detected at a locus. The allele frequencies were estimated by averaging data in sliding windows containing 10 SNPs. (B) Cryptic variants involved in the five- and six-way interactions. (C) Dependence of both genetic interactions on the ira2Δ2933 mutation.

Fig 4. FLO11 is required for rough morphology and shows differential expression across genetic backgrounds.

(A) Deletion of FLO11 leads to smooth morphology in both the five- and six-way genetic interaction backgrounds. (B) RT-PCR of FLO11 and the housekeeping gene ACT1 in multiple genetic backgrounds. FLO11 is not expressed in BY or 3S, but is expressed in recombinants that carry the five- and six-way genetic interactions. FLO11 is also expressed in 3S ira2Δ2933 and 3S sfl1Δ strains.

Fig 5. Deletion of SFL1 reveals interacting cryptic variants.

(A) Three phenotypic classes—smooth, bumpy, and rough—were observed among progeny from the BYx3S sfl1Δ cross. The proportion of segregants observed in each phenotypic class is shown below representative pictures for each class. (B) Genotypes observed among rough progeny from the BYx3S sfl1Δ cross.