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Negative Feedback Confers Mutational Robustness in Yeast Transcription Factor Regulation

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Negative Feedback Confers Mutational Robustness in Yeast Transcription Factor Regulation Negative feedback confers mutational robustness in yeast transcription factor regulation Charles M. Denbya, Joo Hyun Ima, Richard C. Yub, C. Gustavo Pesceb, and Rachel B. Brema,1 aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3220; and bMolecular Sciences Institute, Berkeley, CA 94704 Edited* by Susan Lindquist, Whitehead Institute for Biomedical Research, Cambridge, MA, and approved January 26, 2012 (received for review October 5, 2011) Organismal fitness depends on the ability of gene networks to determinant of quantitative behaviors of inducible circuits (20– function robustly in the face of environmental and genetic 24), we also investigated the role of Rox1 feedback in expression perturbations. Understanding the mechanisms of this stability is regulation during oxygen response. one of the key aims of modern systems biology. Dissecting the basis of robustness to mutation has proven a particular challenge, Results with most experimental models relying on artificial DNA sequence We set out to establish a tractable model system for the study of variants engineered in the laboratory. In this work, we hypothe- feedback and robustness using yeast transcription factors. For sized that negative regulatory feedback could stabilize gene this purpose, we first screened transcription factor genes for expression against the disruptions that arise from natural genetic feedback on protein abundance. We used fluorescence micros- variation. We screened yeast transcription factors for feedback copy (25) to measure protein expression from a single genomic and used the results to establish ROX1 (Repressor of hypOXia) as copy of each factor tagged with GFP in a diploid strain (26) while a model system for the study of feedback in circuit behaviors and varying levels of an untagged copy of the factor (Fig. 1A). To its impact across genetically heterogeneous populations. Muta- maximize signal to noise, we analyzed the 23 most highly abun- genesis experiments revealed the mechanism of Rox1 as a direct dant factors in rich medium (Table S1) and found evidence for transcriptional repressor at its own gene, enabling a regulatory negative feedback in four cases (Fig. 1B). As expected (27, 28), program of rapid induction during environmental change that these screen hits included Rox1 and Swi4; we also found EVOLUTION reached a plateau of moderate steady-state expression. Addition- uncharacterized feedback loops by Mbp1 and Mot3. Independent ally, in a given environmental condition, Rox1 levels varied widely replicate experiments confirmed the ability of each factor to re- across genetically distinct strains; the ROX1 feedback loop regu- press its own abundance (Fig. 1C). lated this variation, in that the range of expression levels across To investigate the role of feedback in mutational robustness genetic backgrounds showed greater spread in ROX1 feedback and systems-level network behaviors, we focused on the tran- mutants than among strains with the ROX1 feedback loop intact. fi scription factor Rox1. This master regulator is repressed in Our ndings indicate that the ROX1 feedback circuit is tuned to hypoxic conditions and induced under normoxia to regulate respond to perturbations arising from natural genetic variation in biosynthetic pathways that use molecular oxygen as a substrate addition to its role in induction behavior. We suggest that regula- (29). Anticipating that Rox1 would act directly at the ROX1 tory feedback may be an important element of the network archi- promoter (27), we identified four candidate Rox1 binding sites in tectures that confer mutational robustness across biology. the 500 bp upstream of the ROX1 coding start site (Fig. S1). Mutagenesis confirmed the role of these sites in ROX1 feedback, obustness of organismal function in the face of perturbations with each site contributing incrementally to the strength of fi Ris critical for tness. Since the seminal work of Waddington autoregulation in an ROX1 transcriptional reporter (Fig. 2). (1), biologists have remarked on the stability of phenotypes Promoter response to changes in dose of ROX1 was markedly against environmental and genetic variation, and understanding reduced when all four sites were mutated in combination, in- how organisms achieve robustness remains one of the major dicating a near-complete abrogation of feedback (Fig. 2). We – challenges in systems biology (2 4). Much of the search for used these mutations to engineer a version of ROX1 in which the molecular mechanisms of robustness has focused on gene regu- feedback mutant promoter drove expression of Rox1 fused to lation. Characteristics of regulatory networks that confer ro- GFP; to avoid potentially confounding effects from elevated bustness include pathway redundancy and master regulatory Rox1-GFP steady-state levels in the presence of the feedback – organization (5), phenotypic capacitors (6 8), paired activating mutations, we manipulated the use of optimal codons (30) in the and inhibiting inputs (9), and cooperative and feed-forward ROX1-GFP coding region. The suboptimized sequence, in regulation (10). Additionally, negative regulatory feedback, in conjunction with mutated Rox1 binding sites in the ROX1 pro- which a biomolecule represses its own abundance, can buffer moter, gave rise to steady-state expression levels comparable variation in gene expression (11, 12), and negative feedback with those levels of the WT (Fig. S2). We refer to this version of loops have been shown to underlie robustness to variable envi- ROX1 as the suboptimized feedback mutant; a strain harboring – ronmental conditions and stochastic intracellular change (13 this gene grew indistinguishably from the WT across environ- 15). Negative feedback may also confer network stability against mental conditions (Fig. S3). the effects of mutations (3, 16), but evidence for negative feed- back as a driver of mutational robustness in vivo has been at a premium (17); the relevance of this principle to natural genetic Author contributions: C.M.D. and R.B.B. designed research; C.M.D. and J.H.I. performed variation remains largely unknown. research; R.C.Y. and C.G.P. contributed new reagents/analytic tools; C.M.D. and R.B.B. In this work, we focused on negative feedback in yeast hypoxia analyzed data; and C.M.D. and R.B.B. wrote the paper. regulation motivated by the extensive evidence for feedback in The authors declare no conflict of interest. oxygen response pathways across biology (18, 19). We charac- *This Direct Submission article had a prearranged editor. terized the feedback loop at the yeast hypoxia regulator ROX1 in Freely available online through the PNAS open access option. molecular detail, and we harnessed this system as a test bed to 1To whom correspondence should be addressed. E-mail: [email protected]. study how feedback confers stability against naturally occurring This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mutations. Given the precedent for negative feedback as a 1073/pnas.1116360109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1116360109 PNAS Early Edition | 1of5 Downloaded by guest on September 30, 2021 A B C 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. To analyze the role of feedback during ROX1 induction, we reporter construct (Fig. 3). We conclude that the ROX1 locus grew WT and feedback mutant strains under hypoxic conditions harbors regulatory information encoding strong activation during and measured Rox1-GFP expression during oxygen exposure. oxygen exposure. In WT cells, repression by Rox1 serves as For the feedback mutant promoter driving expression of the WT a brake on this induction signal, avoiding the deleterious effects ROX1-GFP reporter, levels during induction far overshot the of elevated expression. Thus, the feedback loop tunes the ki- steady state of the strain harboring a WT promoter (Fig. 3). Such netics of ROX1 induction, enabling a rapid approach to a mod- elevated expression was toxic to cells: this strain displayed erate level of steady-state expression during the transition from growth defects under a variety of environmental conditions (Fig. hypoxia to normoxia. S3). As expected, in the suboptimized feedback mutant, protein We next sought to evaluate Rox1 feedback as a mechanism for levels reached those of the WT at steady state but with slower robustness of gene expression to naturally occurring genetic fi kinetics owing to the
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