Evolutionary regain of lost gene circuit function

The original PF gene circuit (6) consists of a doxycycline (Dox)-inducible rtTA transcriptional activator that identically up-regulates both its own expression and the expression of the Zeocin resistance gene, zeoR, fused to yEGFP (yEGFP::zeoR), by binding to 2 tetO2 operator sites upstream of the rtTA and the yEGFP::zeoR coding regions (Fig. 1A). The yEGFP::ZeoR bifunctional fusion protein (35) precisely reports ZeoR levels and protects cells by directly binding to Zeocin (36) to prevent its intercalation into DNA, which causes DNA breaks and thus cell cycle arrest or death (37). Previously, we evolved yeast populations carrying the PF circuit (9) in 2 μg/mL of Dox inducer without Zeocin antibiotic, a condition denoted “D2Z0,” where the first number represents Dox concentration in micrograms per milliliter, while the second number represents Zeocin concentration in milligrams per milliliter. Since the activation of the PF stress-response module is costly in the absence of stress, the rtTA protein apparently lost its function during these evolution experiments through different coding-sequence mutations (9). To test whether evolutionary repair of lost rtTA function was possible, we further evolved 7 of these loss-of-function rtTA mutants from the previous experiment (Fig. 1B) in the condition D2Z2 (2 µg/mL Dox and 2 mg/mL Zeocin), which should activate the original PF gene circuit or any reverting mutants, ensuring their survival in stress. We will refer to the 7 ancestral mutations by their genotype: “Missense 1, 2, 3, and 4” correspond to rtTA_+189C→G, rtTA_+562T→C, rtTA_+275G→A, and rtTA_+13G→T, respectively; “Nonsense” corresponds to rtTA_+442G→T; “Duplication” corresponds to rtTA_{+95, 30bp}; and “Deletion” corresponds to rtTA_{+651, 78bpΔ} (SI Appendix, Table S1).

To understand the dynamics of the original PF gene circuit (Fig. 1A) and its 7 rtTA mutants (Fig. 1B), we studied qualitatively a simple mathematical model of this genetic autoregulatory module (SI Appendix, Mathematical Modeling). In rate-balance plots from these models (Fig. 1C), intersections between rtTA synthesis (Fig. 1C, blue sigmoid) and loss (Fig. 1C, red elbow) rate curves correspond to stable (Fig. 1C, full circles) and unstable (Fig. 1C, open circles) rtTA gene expression states that impose corresponding cellular yEGFP::zeoR protein levels. For example, the original PF gene circuit (6) is monostable below a threshold concentration of ∼0.05 μg/mL Dox, with a single intersection corresponding to a single low rtTA expression state imposing low yEGFP::zeoR expression. As Dox concentrations increase, the elbow-shaped curve of Dox-free, inactive rtTA loss (from combined dilution and degradation) moves rightward, approaching the sigmoidal rtTA synthesis curve until they meet and intersect 3 times. Thus, for Dox concentrations above the threshold, a second stable high-expression state emerges, indicating a transition from monostable to bistable dynamics through a saddle-node bifurcation (Fig. 1C). Losing the high gene-expression peak during long-term evolution in D2Z0 (9) could indicate either that a mutant gene circuit became completely noninducible, or that the sigmoidal rtTA synthesis rate curve shifted somehow, elevating the bistability threshold beyond D2Z0. Still, the elbow-shaped loss curve slides rightward as Dox increases further, so it could still reach and intersect a right-shifted sigmoid curve 3 times, causing a high-expression peak to still emerge at sufficiently high Dox concentrations. To examine the possibility of such weaker but still present rtTA function, we looked for high expressors in flow cytometry histograms of each mutant in D2Z0. Indeed, upon closer examination, we noticed ∼1% high-expressing cells in Missense 1 and 2 populations, but not any other mutants (SI Appendix, Figs. S3–S8). Therefore, we deem the Missense 1 and 2 mutant gene circuits to be quasifunctional rather than completely nonfunctional (SI Appendix, Table S1).

To fully test which of the 7 loss-of-function PF mutants are still quasifunctional, we next hyperinduced them with excess Dox. We expected that hyperinduction would shift the elbow-shaped curve of rtTA loss farther rightward and move quasifunctional rtTA mutants into their bistable regime (Fig. 1C and SI Appendix, Mathematical Modeling), thus generating high-expressor subpopulations, while nonfunctional mutants would remain unimodal. To this end we grew clonal populations carrying each rtTA mutation in D6Z0 (Fig. 2 A and B), a 3-fold higher (6 µg/mL) Dox concentration than D2Z0 where they previously arose. These hyperinducing conditions confirmed that Missense 1 and 2 were indeed quasifunctional, while the remaining 5 mutants—Missense 3 and 4, Nonsense, Duplication, and Deletion—still appeared uninducible and thus nonfunctional (SI Appendix, Table S1). Excess Dox did not alter the growth rate of any clones, indicating that hyperinduction is not toxic for any mutant. Thus, any further mutations abolishing leftover rtTA function in Missense 1 and 2 would not be beneficial. The results did not change upon hyperinducing the mutants in 8 µg/mL Dox (SI Appendix, Fig. S1A). Hysteresis experiments also confirmed the upshift of bistability range for Missense 1 and 2 compared to PF (SI Appendix, Fig. S2).

Mathematical models suggested another mechanism besides hyperinduction that can cause stable high expression. Slow growth reduces dilution of cell contents and thus tilts downward the rtTA loss curve, besides shifting it rightward. This moves the quasifunctional PF mutants into the bistable regime (SI Appendix, Mathematical Modeling), similar to growth-mediated bistability observed in other systems (38, 39). To experimentally test this, we grew PF mutants as well as standard PF cells in D2Z0 with 7.5% ethanol that slowed the growth rate to a value similar to that in D2Z2 (SI Appendix, Figs. S3–S8). Interestingly, ethanol strongly enriched the high-expressor fraction of ancestral PF cells, from 71 to 91% in D2Z0. As expected, slow growth due to ethanol also enriched the high-expressor fractions of Missense 1 (more than 2-fold) and Missense 2 (about 9-fold). Most surprisingly, ethanol caused a few high-expressor cells to appear even in Missense 3, which failed to respond to hyperinduction in D6Z0 or D8Z0. Therefore, we classify Missense 3 as a dysfunctional mutant (SI Appendix, Table S1), unlike the nonfunctional mutants Nonsense and Deletion, which do not become bistable even in slow growth. We obtained comparable results using Cisplatin, which interferes with DNA (40) like Zeocin (37). In contrast, G418 did not have this effect, presumably because protein synthesis inhibition (41) abolishes gene circuit function (SI Appendix, Figs. S3–S8). Overall, these data and the model indicate that slow growth due to stressors can cause shifts in dynamics that generate high expressors in Missense 3 and can enrich the already present high expressor subpopulations of Missense 1 and 2.

Next, we set out to test if evolution could revert the 2 quasifunctional mutants (Missense 1 and 2) to regain stronger rtTA function. Thus, we evolved 3 replicates each of Missense 1 and 2 in D2Z2 medium, where regaining high rtTA activity would beneficially shift cells into the drug-resistant state of high yEGFP::ZeoR expression. For control purposes, we also propagated the same initial populations in D2Z0.

We observed qualitatively similar evolutionary dynamics for both Missense 1 and 2 (Fig. 3), which started with a minimal (0.47% and 0.63%) subpopulation of high-expressor cells immediately upon transfer from D2Z0 into D2Z2. Early on, population fitness levels dropped significantly in all D2Z2 cultures compared to the control D2Z0 cultures (Fig. 3 C and D). Soon afterward, drug exposure generated a substantial high-expressing subpopulation for a few days (Fig. 3 A and B). Growth rates started recovering in ∼4 d, as the high-expressor peak increased (Fig. 3 C and D). From day 1 onward, we also observed a slight upward fluorescence shift for low expressors, corresponding to elevated basal yEGFP::ZeoR levels compared to D2Z0 controls (Fig. 3 A and B). This slight upward expression shift is clearly distinguishable from “induced” high expression and resembles phenotypes arisen previously during evolution in Zeocin drug (D0Z2) (9), where various extracircuit mutations, often coupled with intracircuit synonymous and promoter mutations, elevated the low, basal yEGFP::ZeoR expression. Concurrently, the high-expressor population fraction reached a maximum around day 4 and then returned to the same low level as in D2Z0 within ∼8 d (Fig. 3 A and B).

Considering that high expressors were rare in D2Z0, while in D2Z2 their fraction initially increased and then later dropped back to the D2Z0 level, at least 2 hypotheses are possible. First, compensatory mutations in rtTA or elsewhere could improve rtTA activator function, thereby increasing the high-expressor fraction, and then later subsequent mutations could revert this effect. To identify such compensatory mutations, we performed whole-genome sequencing (WGS) on 1 replicate population each for both mutants on days 1, 3, and 14. The original rtTA mutations already present at the beginning of the experiment were identified at 100% frequency at all time points for both Missense 1 and 2. However, we identified no other intracircuit mutations. Since certain variant types (e.g., deletions and duplications) in the PF gene circuit are difficult to detect by WGS (24), we also performed Sanger sequencing of the PF region in 10 individual clones from each mutant at day 14 (SI Appendix, Table S2). All Missense 1 clones carried a deletion of the first tetO2 operator upstream of the yEGFP::zeoR coding region, whereas the other tetO2 site stayed intact. All Missense 2 clones carried only their original gene circuit mutation without any tetO2 or other circuit modifications, despite their identical phenotypes to Missense 1. Therefore, there is no phenotypic signature directly and specifically attributable to the tetO2 operator site deletion (SI Appendix, Table S3). Overall, the only de novo intracircuit mutation we found did not explain the observed gene-expression changes.

High expression requires rtTA function. How then could a substantial high-expressor peak emerge and later diminish without any changes in the rtTA coding sequence or its promoter? Modeling and experiments suggested a second hypothesis: By decelerating growth and reducing dilution, Zeocin tilts downward and shifts rightward the red elbow curve of rtTA loss (Fig. 3E). This causes a shift in dynamics (SI Appendix, Mathematical Modeling) that enriches the rare high-expressing drug-resistant cells that preexist in D2Z0 (SI Appendix, Figs. S3 and S4), which are then further enriched by phenotypic selection. At the same time, the low-expressing subpopulation will remain drug-sensitive, enabling any mutants that elevate basal expression to grow faster and spread in the population. Once such fast-growing mutants arise, their faster growth/dilution rate tilts upward and shifts leftward the red elbow curve (Fig. 3E), causing a return of dynamics toward monostability (SI Appendix, Mathematical Modeling). Concurrently, the benefits of high-expression also vanish, causing the high-expressor fraction to diminish back to the D2Z0 levels. Importantly, these events can occur without any mutations in the PF gene circuit, as long as extracircuit mutations can raise basal yEGFP::ZeoR expression to promote fast growth in Zeocin.

To identify such extracircuit mechanisms of yEGFP::zeoR-dependent Zeocin resistance, we looked for genomic, extracircuit mutations in the WGS data (Materials and Methods). In the Missense 1 population at day 14 we found moderate-frequency (∼15%), nonsynonymous mutations in 2 genes affecting protein stability and transcription, PAH1 and SET3, including a 1-bp frameshift deletion in the latter. Additional mutations in other genes and intergenic regions exist at lower frequencies (SI Appendix Table S3 and Dataset S1). In contrast to Missense 1, evolved Missense 2 populations carried multiple extracircuit mutations exceeding 90% in frequency at day 14 (SI Appendix, Table S3 and Dataset S2). Mutations in the genes PHB2, MDM32, and COX1 suggest alteration in mitochondrial metabolism and function. We also observed a synonymous mutation in the FG-nucleoporin NUP159 gene, which is involved in posttranscriptional regulation (42).

To test if elevated yEGFP::zeoR gene expression mediates drug resistance without rtTA activity, we compared the growth of Missense 1- and 2-evolved populations and clonal isolates to their unevolved Missense 1 and 2 ancestors in Zeocin-only media (D0Z2). Evolved Missense 1 and 2 populations and clonal isolates grew significantly faster than the ancestral PF or unevolved Missense 1 and 2 cells in D0Z2 (SI Appendix, Figs. S12 and S13), indicating that the evolved Missense 1 and 2 populations were Zeocin-resistant independently of rtTA.

If the rtTA coding sequence and PF gene circuit dynamics did not change during evolution, hyperinduction should affect Missense 1 and 2 similarly to their ancestors. To test this, we hyperinduced each final evolved Missense 1 and 2 replicate population in D6Z0. Indeed, the evolved Missense 1 and 2 populations developed high expression in D6Z0 (Fig. 3F) and in D8Z0 (SI Appendix, Fig. S1B) as their ancestors did. Similarly, hyperinduction did not affect the growth rates of the evolved populations. Overall, the Missense 1 and 2 evolution experiments indicate that slow growth and phenotypic selection initially enrich the high-expressor fraction nongenetically, but then extracircuit mutations accelerate growth by elevating basal stress-protective yEGFP::ZeoR expression, which shifts the dynamics to diminish the high-expressor fractions back to levels equivalent to D2Z0.

As the lack of high expression in D6Z0 indicates, the 4 other initial PF mutants (Missense 4, Nonsense, Duplication, and Deletion) had mutations that disrupted rtTA protein function such that it became completely uninducible, regardless of inducer amount (Fig. 2B and SI Appendix, Fig. S1A) or growth rate (SI Appendix, Figs. S6 and S7). To test if evolution could restore rtTA function in any of these nonfunctional mutants, we also evolved 3 replicates of each in D2Z2 where regaining rtTA activity would generate a beneficial high yEGFP::ZeoR expression peak.

Early in the evolution experiment (Fig. 4B and SI Appendix, Fig. S9 A–F), the growth rate in D2Z2 of each nonfunctional mutant population dropped significantly below the controls evolving in D2Z0. The growth rates of the Missense 4, Nonsense, and Duplication populations then recovered over ∼5 d, while the growth rates of the Deletion populations recovered within 3 d, approaching that of D2Z0 control cultures. Unlike quasifunctional mutants, nonfunctional mutants never gave rise to a high-expressor subpopulation (Fig. 4A and SI Appendix, Fig. S9 A–C). The yEGFP::zeoR expression distributions remained unimodal but shifted slightly upwards compared to the basal (D2Z0 control) expression in all experimental cultures. Consequently, these evolving cell populations relied again on yEGFP::zeoR expression to gain drug resistance and increase their fitness, without repairing rtTA function. Indeed, Sanger sequencing of 10 isolated clones from each mutant revealed no additional mutations inside the gene circuit, except for the deletion of the first tetO2 site upstream of yEGFP::zeoR in 0 of 10 Missense 4 clones, 1 of 10 Nonsense clones, 8 of 10 Deletion clones, and 2 of 10 Duplication clones (SI Appendix, Table S4). All mutants retained the original rtTA mutation and no other rtTA mutations were detected. As with Missense 1 and 2, there was no phenotype associated specifically with the tetO2 deletion (SI Appendix, Table S4).

To identify possible extracircuit mutations causing the slight basal expression increase, we analyzed the WGS data. We found mutations in or near genes controlling mRNA and protein levels through degradation or synthesis, such as the poly-A tail shortening CCR4, the TFIID subunit TAF2, the ribosomal subunit RPL41A, and others (Datasets S4–S7).

To examine rtTA nonfunctionality at the end of the evolution experiment, we hyperinduced final evolved populations in D6Z0. As expected, the yEGFP::ZeoR expression of nonfunctional mutants remained unimodal and low in D6Z0 and in D8Z0 (Fig. 4C and SI Appendix, Figs. S1B and S9G), indicating that rtTA and the gene circuit in these mutants remained nonfunctional at the end of evolution in D2Z2, as anticipated.

As we did for Missense 1 and 2, we also examined rtTA-independent Zeocin resistance of nonfunctional mutants by growing evolved populations and isolated clones in D0Z2 media for 24 h. The evolved Missense 4, Nonsense, and Duplication populations and isolated clones grew significantly faster than their unevolved ancestors (SI Appendix, Figs. S12 and S13). Interestingly, the unevolved Deletion mutant seemed to have some preexisting yEGFP::ZeoR-dependent resistance to Zeocin (SI Appendix, Figs. S12 and S13). This corroborates the faster Deletion recovery and slightly higher yEGFP::ZeoR basal expression compared to all other nonfunctional mutants upon Zeocin exposure, already at day 1 (SI Appendix, Fig. S10), and reinforces general reliance on slightly elevated yEGFP::ZeoR expression for drug resistance.

Surprisingly, during evolution in D2Z2, all 3 replicates of the dysfunctional rtTA mutant (Missense 3) temporarily gave rise to a significant high-expressor subpopulation that persisted for over a week for each replicate. Ultimately, toward the end of the evolution experiment, these high-expressor subpopulations diminished and the fluorescence shift of the low peak indicated a slight increase in basal yEGFP::zeoR expression similar to all other genotypes. The fitness trends of each Missense 3 replicate resembled those of the other mutants: The initially low population fitness levels recovered to normal (control) levels within ∼4 d (Fig. 5 A and B).

Once again, despite the marked appearance and subsequent gradual disappearance of the high-expressing peak in D2Z2, we found no additional rtTA coding sequence mutations in Missense 3 populations. Sanger sequencing from day 14 detected only 1 intracircuit mutation in some clones: The deletion of 1 tetO2 site upstream from yEGFP::zeoR, while the other tetO2 sites remained intact. Interestingly, all individual Missense 3 clones that acquired a tetO2 deletion had unimodal gene expression, whereas the clones that lacked this mutation were bistable in D2Z2 at the end (SI Appendix, Table S5).

Mathematical modeling (SI Appendix, Mathematical Modeling) suggested that gene circuit dysfunction in Missense 3 is due to a drop in rtTA activator capacity (Fig. 5C), rather than reduced inducer sensitivity as for Missense 1 and 2. This means that the rtTA synthesis rate curve collapses downward (Fig. 5C) instead of shifting rightward (Fig. 2C) in such dysfunctional mutants. Importantly, this collapse implies that the elbow-shaped rtTA loss curve will always be above the collapsed sigmoid during normal growth, so the curves will miss intersecting each other again regardless of the hyperinducing Dox level. Yet, a bistable region can still be reached if slow growth/dilution tilts downward and shifts rightward the rtTA loss curve, causing high-expressors to emerge, despite Missense 3 being completely unresponsive to hyperinduction. Indeed, adding ethanol or Cisplatin but not G418 to D2Z0 indicated that slow growth enables some high expression in Missense 3 (SI Appendix, Fig. S5). Zeocin in D2Z2 enriches this high-expressor fraction further by selection. Mutations elevating the entire sigmoidal synthesis curve could then accelerate growth, thus reestablishing and maintaining bistability (Fig. 5C), while also stabilizing the high-expressor fraction (SI Appendix, Mathematial Modeling). On the other hand, not all mutations elevate expression sufficiently to maintain bistability as growth accelerates, causing the high-expressor fraction to diminish in the evolving population, which can be a mixture of such mutation types.

To identify these mutations predicted by the model, we examined WGS data for 2 replicate populations of Missense 3. Relevant high-frequency mutations potentially related to transcriptional regulation in replicate 1 (r1) included 1-bp frameshift deletions in SIF2 and SSN3, as well as a nonsynonymous mutation in SSN2. Missense 3 replicate 3 (r3), on the other hand, carried multiple genomic mutations that spread in the entire population by day 14 of the experimental evolution, including missense mutations in mediator complex components SRB8 and MED6, both of which are involved in RNA polymerase II-dependent transcriptional regulation (43–45) and could elevate both basal and maximal rtTA levels (the entire rtTA synthesis curve) as the model suggested. In addition, a chaperonin subunit CCT7 mutation suggests effects on general protein folding (46), and mutations in the SAP185 and EXG1 coding regions suggest altered cell cycle regulation (47, 48) (SI Appendix, Table S5 and Dataset S3).

We also hyperinduced the final evolved replicate populations from Missense 3 in D6Z0 to examine PF function at the end of evolution. Surprisingly, 2 evolved Missense 3 replicates (r1 and r3) had bimodal distributions, indicating that gene circuits in some clones became quasifunctional or fully functional. Missense r2 had a unimodal distribution in D6Z0, indicating that it remained insensitive to hyperinduction (Fig. 5D). We observed the same in D8Z0 (SI Appendix, Fig. S1B).

Finally, as for other mutants, we assessed rtTA-dependence of Zeocin resistance in evolved Missense 3 populations and clonal isolates by culturing them in D0Z2. OD600 measurements of original and evolved populations (SI Appendix, Figs. S12 and S13) indicated that evolved Missense 3 replicate populations grew significantly faster than their unevolved ancestor. Interestingly, isolated bimodal Missense 3 clones with mutation-free gene circuits grew slower in D0Z2 than unimodal clones with a tetO2 site deletion upstream from yEGFP::zeoR. This further supports that the tetO2 site deletion contributes to rtTA-independent, but yEGFP::zeoR-dependent drug resistance, whereas bimodal Missense 3 clones rely on regained rtTA activity for resistance.

So far, a likely common explanation for the observed evolutionary dynamics is the appearance and spread of extracircuit mutations that accelerate growth of the drug-sensitive, low-expressor subpopulation by elevating yEGFP::zeoR basal expression, thereby returning the dynamics toward the unimodal low-expression regime. If this is the case, then separating the high- and low expressors around day 4 should result in different evolutionary dynamics. Since drug selects against ancestral low-expressor cells, the increased selection pressure should cause drug-resistance mutations to spread quickly among low-sorted cells, preventing them from generating a substantial high-expressing peak. On the other hand, high-sorted cells would be insensitive to Zeocin and mutations could spread among them only after they generate a low-expressing peak by phenotypic switching. Therefore, high-sorted cells should take longer to become unimodal than low-sorted cells. Also, as discussed above, some quasi- or fully functional PF revertant clones may exist. If any revertant strains arose, we may be able to isolate them among the high-sorted cells because high expression is the hallmark of PF functionality.

To test these hypotheses, we separated the low-expressor and high-expressor subpopulations by fluorescence-activated cell sorting (FACS) at the end of day 4 of the original evolution experiment and cultured these high- and low-sorted subpopulations separately in the D2Z2 condition for 12 d (Fig. 6). As predicted, Missense 1 and 2 low-sorted subpopulations remained in the low-expression state throughout this experiment, indicating rapid takeover by genomic mutations conferring drug resistance. Accordingly, the corresponding high-sorted subpopulations generated bimodal distributions that became unimodal only after a few days as expected.

As opposed to Missense 1 and 2, Missense 3 low-sorted subpopulations transiently gave rise to new high-expressor subpopulations (Fig. 6 A–C), indicating either that basal expression-elevating mutants spread much more slowly than in Missense 1 and 2, or that revertant strains arose. For low-sorted Missense 3 replicates r2 and r3, the high-expressor peak gradually diminished within ∼9 d. In contrast, low-sorted Missense 3 r1 remained stably bimodal from day 5 throughout the end of this 12-d postsort experiment (Fig. 6 C and F).

High-sorted subpopulations from all 3 Missense 3 replicates gave rise to low-expressor cells within 1 d after flow sorting. Eventually, the high-expressor peak diminished in all high-sorted subpopulations, except as above, for Missense 3 r1, which remained stably bimodal, biased toward the high-expression state for the last 7 d postsort (Fig. 6 A–C). Therefore, some Missense 3 r1 clones must be revertant strains with stable high expression.

To identify PF circuit genetic changes underlying these phenotypes, we Sanger-sequenced the yEGFP::zeoR and rtTA regions of 5 such individual revertant clones. Once again, the PF sequence in revertant clones was genetically identical to the original Missense 3 mutant. WGS (Dataset S3) revealed 1 mutation in the SSN2 gene that spread in the high-sorted Missense 3 r1 population over time (reaching 62.2% on day 12 postsort), suggesting that it confers a survival benefit and is associated with the stable high-expression peak in this population. This mutation was also present at lower frequencies (5.6% and 6.6%) in the respective unsorted and low-sorted populations, further supporting its association with functional reversion. The SSN2 gene encodes an RNA polymerase II mediator complex subunit essential for transcriptional regulation, resembling the general transcriptional-regulatory function of mutations in Missense 2 and 3 r3 (SI Appendix, Tables S3 and S5). However, the dynamic consequences of such gene-expression increases depend on the original mutation (i.e., whether it shrinks or right-shifts the sigmoidal rtTA-synthesis function).

If fully functional revertant strains arose from Missense 3, they should be clearly bimodal in D2Z0, without Zeocin. Thus, to further characterize revertant strains, we studied the yEGFP::zeoR fluorescence distribution in 5 individual Missense 3 r1 clones at the end of the postsort experiment (on day 12) in D2Z2, D0Z2, D2Z0, and D0Z0 for 4 d. Based on these experiments, Missense 3 r1 revertant clones fell into 2 phenotypic categories: Some had approximately equal peak heights in D2Z2 and D2Z0, whereas other clonal populations consisted almost exclusively of high expressors (Fig. 7 A and B and SI Appendix, Fig. S11) like the ancestral PF. Remarkably, the mutations conferred stable high expression even without Zeocin. Additionally, maximum expression shifted leftward, causing the peaks to approach each other compared to the ancestral PF, as predicted by sigmoid elevation in the SI Appendix model (SI Appendix, Mathematical Modeling). Remarkably, the distributions were very similar in D2Z2 and D2Z0, indicating that the revertant strains were drug resistant at all expression levels (SI Appendix, Fig. S14), so Zeocin did not select for high expression.

Data and Matlab code associated with the figures (56) and raw sequencing data (57) can be found at https://openwetware.org/wiki/CHIP:Data.