Negative feedback confers mutational robustness in yeast transcription factor regulation

Abstract

Organismal fitness depends on the ability of gene networks to function robustly in the face of environmental and genetic perturbations. Understanding the mechanisms of this stability is one of the key aims of modern systems biology. Dissecting the basis of robustness to mutation has proven a particular challenge, with most experimental models relying on artificial DNA sequence variants engineered in the laboratory. In this work, we hypothesized that negative regulatory feedback could stabilize gene expression against the disruptions that arise from natural genetic variation. We screened yeast transcription factors for feedback and used the results to establish ROX1 (Repressor of hypOXia) as a model system for the study of feedback in circuit behaviors and its impact across genetically heterogeneous populations. Mutagenesis experiments revealed the mechanism of Rox1 as a direct transcriptional repressor at its own gene, enabling a regulatory program of rapid induction during environmental change that reached a plateau of moderate steady-state expression. Additionally, in a given environmental condition, Rox1 levels varied widely across genetically distinct strains; the ROX1 feedback loop regulated this variation, in that the range of expression levels across genetic backgrounds showed greater spread in ROX1 feedback mutants than among strains with the ROX1 feedback loop intact. Our findings indicate that the ROX1 feedback circuit is tuned to respond to perturbations arising from natural genetic variation in addition to its role in induction behavior. We suggest that regulatory feedback may be an important element of the network architectures that confer mutational robustness across biology.

Fig. 1.

Screening yeast transcription factors for regulatory feedback. (A) Each part of the schematic represents one yeast strain. Green rectangles indicate the coding sequence of GFP, and the oval on the right represents a plasmid carrying an overexpression construct controlled by the GAL1-10 promoter (44). △, whole-gene deletion. (B) Each data point represents the results of two analyses of nuclear abundance of a transcription factor fused to GFP (26) measured by quantitative microscopy in diploid strains. Each analysis, as indicated on the x or y axis, compared fluorescence from a given tagged factor in two strains encoding different doses of the untagged version of the factor with strains named according to the schematic in A. (C) Each set of bars reports nuclear fluorescence measurements of the indicated factor as a fusion with GFP measured by quantitative microscopy and normalized with respect to WT levels. Each bar reports measurements from one strain with names as in A; error bars represent the SD over biological replicates and microscope fields. *Comparisons relative to WT that are significant at Wilcoxon P < 0.001.

Fig. 2.

Transcription factor binding sites in the ROX1 promoter are required for transcriptional feedback. Each set of bars reports expression from one ROX1 transcriptional reporter in a diploid yeast strain measured by flow cytometry. Shading represents the presence or absence of a single untagged copy of ROX1 in the strain background. Each set of bars labeled with Site corresponds to a reporter with the indicated Rox1 binding sites (Fig. S1) mutagenized in the ROX1 promoter; 4 site indicates mutagenesis of all four sites. Δ, whole-gene deletion of ROX1.

Fig. 3.

The ROX1 locus confers strong induction during oxygen exposure. Each set of points reports fluorescence of a haploid yeast strain bearing an ROX1-GFP fusion reporter gene measured by quantitative microscopy after a transfer of a culture from hypoxia to normoxia. Each data point represents median nuclear fluorescence across cells of one culture at the indicated time after oxygenation. Each color represents one reporter. FB mutant indicates a feedback mutant ROX1 promoter with all four Rox1 binding sites mutagenized, and subopt indicates that the ROX1 and GFP sequences were encoded with suboptimized codons.

Fig. 4.

Rox1 feedback as a mechanism for mutational robustness. Each panel shows ROX1 expression levels in haploid segregants from crosses between an Rox1-GFP strain and a tester strain: BY4741, a laboratory strain derived from a fig tree isolate; YPS606, an isolate from an oak tree (49); SK1, a laboratory strain derived from a soil isolate (49); and 22:3:b, a strain of hybrid laboratory and vineyard origin (50). In a given panel, each distribution reports culture medians of fluorescence measured by flow cytometry. Each median was taken across cells of an isogenic culture of one segregant and normalized with respect to the mean across all strains of the same cross. Each color represents strains bearing an ROX1-GFP fusion with the indicated modification. No feedback indicates the suboptimized feedback mutant (Figs. 2 and 3 and Fig. S2). p, Resampling P value evaluating the F statistic for differential variance between the feedback mutant and WT distributions. Coefficients of variation of distributions of WT and feedback mutant strains are 0.01 and 0.07, respectively, for the BY4741 cross; 0.07 and 0.31, respectively, for YPS606; 0.26 and 0.75, respectively, for SK1; and 0.10 and 0.18, respectively, for 22:3:b.

Fig. 5.

Transcription factors subject to feedback are toxic when overexpressed. Each panel represents analysis of growth rates across a panel of yeast strains, with each overexpressing a single transcription factor (31). Each distribution represents growth rates across the set of genes with or without evidence for feedback as indicated. The x axis reports growth rate in doublings per hour. (A) Blue represents factors emerging as screen hits in Fig. 1; red reports all other screened factors (n = 22). Distribution medians are 0.17 and 0.29, respectively. (B) Blue represents factors with significant sequence matches to their own binding motif in their own promoters; red reports all other tested factors (n = 83). Distribution medians are 0.24 and 0.27, respectively. p, Wilcoxon P value evaluating the difference in growth rates between factors with and without evidence for feedback.