The molecular mechanism of a cis-regulatory adaptation in yeast

Abstract

Despite recent advances in our ability to detect adaptive evolution involving the cis-regulation of gene expression, our knowledge of the molecular mechanisms underlying these adaptations has lagged far behind. Across all model organisms, the causal mutations have been discovered for only a handful of gene expression adaptations, and even for these, mechanistic details (e.g. the trans-regulatory factors involved) have not been determined. We previously reported a polygenic gene expression adaptation involving down-regulation of the ergosterol biosynthesis pathway in the budding yeast Saccharomyces cerevisiae. Here we investigate the molecular mechanism of a cis-acting mutation affecting a member of this pathway, ERG28. We show that the causal mutation is a two-base deletion in the promoter of ERG28 that strongly reduces the binding of two transcription factors, Sok2 and Mot3, thus abolishing their regulation of ERG28. This down-regulation increases resistance to a widely used antifungal drug targeting ergosterol, similar to mutations disrupting this pathway in clinical yeast isolates. The identification of the causal genetic variant revealed that the selection likely occurred after the deletion was already present at high frequency in the population, rather than when it was a new mutation. These results provide a detailed view of the molecular mechanism of a cis-regulatory adaptation, and underscore the importance of this view to our understanding of evolution at the molecular level.

Figure 1. A polygenic gene expression adaptation in the ergosterol biosynthesis (ERG) pathway.

(A) The final steps of the ERG pathway. Eight genes whose down-regulation contributes to a polygenic gene expression adaptation are colored red; the six previously implicated genes are underlined. Erg28 is shown next to its strongest interaction partner, Erg27. (B) Allelic bias of ERG genes, as measured by pyrosequencing in the RM/BY hybrid. The allelic bias indicates the magnitude of cis-regulatory divergence between RM and BY for each gene. Red color indicates genes that are part of the polygenic adaptation. Asterisks indicate those that interact strongly with Erg28 , all of which have stronger allelic bias than those that do not.

Figure 2. Pinpointing the causal mutation affecting ERG28 cis-regulation.

(A) Sequence divergence between RM and BY in the ERG28 promoter region. No other differences exist for 590 bp upstream of the gene, or in the 5′ UTR. (B) Genotypes at the two variable positions for RM, BY, and the two engineered strains. (C) The mRNA levels of ERG28 in each of the two engineered strains compared to wildtype RM, assayed by qPCR. The causal mutation is expected to result in a ∼1.30-fold difference, matching the allelic bias observed in the RM/BY hybrid (Figure 1B), whereas any non-causal mutation will not alter the RM expression level (∼1-fold change). (D) Allelic expression bias in hybrids between each engineered strain and BY, assayed by pyrosequencing. Any non-causal mutation will not alter the 1.30-fold RM/BY allelic bias, whereas the causal mutation is expected to be expressed at the same level as the BY allele (∼1-fold allelic bias).

Figure 3. Determining the molecular mechanism of the causal mutation.

(A) Two predicted transcription factor (TF) binding sites flanking the deletion. (B) The expected fold-change in ERG28 expression level when deleting TFs under different scenarios. Left: if a TF does not regulate ERG28, its deletion should have no effect on ERG28 levels. Center: If a TF regulates ERG28 and acts independently of the two-base deletion, then deleting the TF should result in some fold-change X, which will be observed in both the wildtype RM and RM AA112Δ backgrounds. Right: If a TF regulates the wildtype ERG28 promoter, but the deletion abolishes this regulation, then the TF deletion may only affect ERG28 mRNA levels in the wildtype background (A fourth possible scenario, not shown, is where the TF only regulates ERG28 in RM AA112Δ). (C) qPCR data showing changes in ERG28 mRNA levels upon deleting either SOK2 or MOT3. In both cases, a difference is observed in the wildtype background (p = 7.5×10⁻⁵ for SOK2 and 5.0×10⁻³ for MOT3), but not the RM AA112Δ background (p = 0.28 for SOK2 and 0.67 for MOT3), consistent with the TF regulation being entirely abolished by the deletion. (D) Chromatin immunoprecipitation data showing the difference in binding for Sok2 and Mot3 to the ERG28 promoter in wildtype RM/RM AA112Δ. In both cases a significant decrease in binding is observed in RM AA112Δ.

Figure 4. Fitness effect of the causal mutation.

(A) In rich synthetic defined (SD) media, the RM AA112Δ strain and RM T229C strains show no significant difference from RM. (B) In the presence of the antifungal drug amphotericin B, the RM AA112Δ strain shows a growth rate advantage over RM, whereas the RM T229C strain shows no difference from RM. Bars represent the mean log₂ ratios of log-phase growth rates from 48 replicate cultures, +/−1 S.E.