Compensatory trans-regulatory alleles minimizing variation in TDH3 expression are common within Saccharomyces cerevisiae

Abstract

Heritable variation in gene expression is common within species. Much of this variation is due to genetic differences outside of the gene with altered expression and is trans-acting. This trans-regulatory variation is often polygenic, with individual variants typically having small effects, making the genetic architecture and evolution of trans-regulatory variation challenging to study. Consequently, key questions about trans-regulatory variation remain, including the variability of trans-regulatory variation within a species, how selection affects trans-regulatory variation, and how trans-regulatory variants are distributed throughout the genome and within a species. To address these questions, we isolated and measured trans-regulatory differences affecting TDH3 promoter activity among 56 strains of Saccharomyces cerevisiae, finding that trans-regulatory backgrounds varied approximately twofold in their effects on TDH3 promoter activity. Comparing this variation to neutral models of trans-regulatory evolution based on empirical measures of mutational effects revealed that despite this variability in the effects of trans-regulatory backgrounds, stabilizing selection has constrained trans-regulatory differences within this species. Using a powerful quantitative trait locus mapping method, we identified ∼100 trans-acting expression quantitative trait locus in each of three crosses to a common reference strain, indicating that regulatory variation is more polygenic than previous studies have suggested. Loci altering expression were located throughout the genome, and many loci were strain specific. This distribution and prevalence of alleles is consistent with recent theories about the genetic architecture of complex traits. In all mapping experiments, the nonreference strain alleles increased and decreased TDH3 promoter activity with similar frequencies, suggesting that stabilizing selection maintained many trans-acting variants with opposing effects. This variation may provide the raw material for compensatory evolution and larger scale regulatory rewiring observed in developmental systems drift among species.

Keywords: Compensation; eQTL; gene regulation; mapping; stabilizing selection.

Figure 1

Extensive trans‐regulatory variation affecting TDH3 expression is segregating among S. cerevisiae strains. (A) Variation in TDH3 trans‐regulatory backgrounds among yeast strains was measured using a reporter gene containing the TDH3 promoter from the BY strain and a yellow fluorescent protein (YFP). This reporter was integrated into the genome of 56 diverse S. cerevisiae strains. Twelve replicate populations were grown in YPD and analyzed by flow cytometry for YFP expression. (B) Variation among replicates relative to the BY reference strain was used to calculate the average effect of each strain's trans‐regulatory background on TDH3 promoter activity. Darker colors reflect higher TDH3 reporter activity. Strain names in blue are used in subsequent mapping experiments. (C) Frequency of trans‐regulatory effects relative to reference strain. (D) Phylogenetic relationships among strains as estimated from genome‐wide polymorphism data (MacLean et al. 2017). Color of branches corresponds to estimated trans‐regulatory effect from ancestral character estimation.

Figure 2

Natural selection has constrained TDH3 trans‐regulatory variation. (A) Effects of trans‐regulatory mutations on TDH3 promoter activity. Mutants were collected and analyzed in prior work (Metzger et al. 2016). (B) Simulated neutral trajectories for TDH3 promoter activity based on empirically measured effects of new mutations. Lighter colors reflect more extreme values after 30,000 mutations. (C) Comparison of observed differences in TDH3 promoter activity among S. cerevisiae strains with neutral expectation. The blue background represents the 95th, 90th, 80th, 70th, and 60th percentiles, from light to dark, for the simulated neutral trajectories. Green dots are differences in TDH3 promoter activity and estimated number of mutations based on the S. cerevisiae phylogeny. Dashed line indicates the point where the observed data depart significantly from expectation. (D) Same as (C), but using genetic distance instead of phylogenetic distance among strains. The green areas represent the 95th, 90th, 80th, 70th, and 60th percentiles, from light to dark, for the observed differences from sampling pairs of strains.

Figure 3

Compensatory alleles underlie the maintenance of TDH3 trans‐regulatory effects. (A) The genomic basis of TDH3 trans‐regulatory variation was mapped using an xQTL approach. Left: Three rounds of mating and sporulation were used to increase mapping resolution. Middle: Three rounds of FACS based selection were used to enrich for alleles increasing and decreasing TDH3 trans‐regulatory activity. In each round, the top or bottom 5% of the population was collected. Right: Comparisons of allele frequency from Illumina sequencing of FACS‐based pools was used to identify eQTL. Each block (dashed lines) represents a different genomic region. Colored lines represent allele frequencies. Black: Reference strain. Blue: Testing strain. For each block, the top bars are after selection for high YFP fluorescence, while the bottom bars are after selection for low YFP fluorescence. eQTLs are identified when allele frequencies among the high and low selected pools differ significantly. (B) G’ statistic for evidence of eQTL in each comparison. Effects are relative to the non‐BY reference allele. Dashed gray lines indicate chromosome boundaries. Dashed red lines gives threshold for statistical significance. Called eQTLs with 95% confidence intervals on the location are highlighted for each strain. Brown: M22 × BY. Blue: YPS1000 × BY. Green: SK1 × BY. (C) Relationship between G’ statistic for different mapping procedures. X‐axis—G’ statistic for high recombination and strong selection (three rounds of crossing and three rounds of selection). Y‐axis—(Black) G’ statistic for low recombination and strong selection (one round of crossing and three rounds of selection). (Red) G’ statistic for high recombination and weak selection (three rounds of crossing and one round of selection). Each point is for an eQTL identified with high recombination and strong selection (three rounds of crossing and three rounds of selection) from the M22 × BY cross. Solid lines show fits from a linear model for high recombination and low selection (red) or low recombination and high selection (black). (D) Number of non‐BY eQTL increasing or decreasing TDH3 promoter activity for each cross. (E) eQTL shared among the three crosses irrespective of direction of effect. Areas are proportional to the number of eQTL shared. Brown: eQTL identified only in the M22 × BY cross. Blue: eQTL identified only in the YPS1000 × BY cross. Green: eQTL identified only in the SK1 × BY cross. Black: eQTL identified in all three crosses. (F) Same as (E), but for non‐BY eQTL that increase TDH3 promoter activity. (G) Same as (E), but for non‐BY eQTL that decrease TDH3 promoter activity.