Why haploinsufficiency persistsSummer A. Morrilla,b and Angelika Amona,b,c,d,1

aDavid H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; bDepartment of Biology,Massachusetts Institute of Technology, Cambridge, MA 02139; cHoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA02139; and dPaul F. Glenn Center for Biology of Aging Research, Massachusetts Institute of Technology, Cambridge, MA 02139

Edited by Douglas Koshland, University of California, Berkeley, CA, and approved May 5, 2019 (received for review January 10, 2019)

Haploinsufficiency describes the decrease in organismal fitnessobserved when a single copy of a gene is deleted in diploids. Weinvestigated the origin of haploinsufficiency by creating a com-prehensive dosage sensitivity data set for genes under their nativepromoters. We demonstrate that the expression of haploinsufficientgenes is limited by the toxicity of their overexpression. We furthershow that the fitness penalty associated with excess gene copynumber is not the only determinant of haploinsufficiency. Haploin-sufficient genes represent a unique subset of genes sensitive tocopy number increases, as they are also limiting for important cellularprocesses when present in one copy instead of two. The selectivepressure to decrease gene expression due to the toxicity of over-expression, combined with the pressure to increase expression dueto their fitness-limiting nature, has made haploinsufficient genesextremely sensitive to changes in gene expression. As a consequence,haploinsufficient genes are dosage stabilized, showing much morenarrow ranges in cell-to-cell variability of expression comparedwith other genes in the genome. We propose a dosage-stabilizinghypothesis of haploinsufficiency to explain its persistence overevolutionary time.

haploinsufficiency | gene dosage | dosage sensitivity

For nearly a century, scientists and mathematicians have workedto formulate a theory to explain the origin of haploinsufficiency.

Why do these genes exhibit an abnormal phenotype upon deletionof one of their two homologous copies, when the majority of genesdo not? Early theories considered haploinsufficiency to be anartifact of diploidy, a rare failure of the wild-type allele to main-tain protective dominance (1). This idea was ultimately disprovenby the observation that equivalent rates of haploinsufficiency arepresent in organisms that primarily exist in the haploid state (2).Later theories evoked a more physiological explanation, wherebythe specific function of the gene dictates its sensitivity to changesin dosage (3). For example, genes encoding enzymes are sparseamong haploinsufficient (HI) genes, but genes encoding proteinsthat perform structural and regulatory functions in the cell areenriched among them (4). More recent studies suggest that thecontext of gene function is also important. In particular, geneswhose products function as members of macromolecular com-plexes or cellular signaling networks may be especially vulnerableto changes in gene dosage (5).

High-throughput screens, metadata analyses, and computationalpredictions have been applied to define which genes are haploin-sufficient. In budding yeast, about 3% of the genome is consideredhaploinsufficient under maximal growth conditions, resulting insubstantial defects in cellular proliferation when heterozygouslydeleted (6). In humans, ∼300 genes are known to be haploinsufficient,contributing to a wide range of human health issues including neu-rodevelopmental disorders and tumorigenesis when heterozygouslydeleted (7) although computational predictions estimate this numberto be much higher (8, 9). Importantly, haploinsufficiency of manygenes is conserved from yeast to humans (10) indicating that strongselective forces exist that prevent the up-regulation of their expression.

Two theories have been put forth to explain the cause of haploin-sufficiency: the dosage balance hypothesis and the insufficientamounts hypothesis. The dosage balance hypothesis (Fig. 1A) statesthat growth defects caused by changes to gene dosage—either over—

or underexpression - are due to stoichiometric imbalances of proteincomplexes interfering with cellular functions (11, 12). This hypothesispredicts that haploinsufficient genes also confer a growth defect whenpresent in excess by as little as one copy. In other words, haplo-insufficiency and sensitivity to increased gene dosage are mutuallydefined. This hypothesis elegantly explains why haploinsufficiency haspersisted over evolutionary time. Up-regulation of the gene is notpossible because too much protein, like too little protein, disruptsprotein complex stoichiometries that interfere with cellular function.The “insufficient amounts” hypothesis (Fig. 1B) postulates that haploin-sufficiency is the physiological result of reduced levels of proteinproduct being insufficient to perform its cellular function (6). Thishypothesis, unlike the dosage balance hypothesis, makes neither pre-dictions about the effects of overexpressing haploinsufficient genesnor explains why haploinsufficiency persisted over evolutionary time.

In this study, we set out to experimentally test the dosage balanceand insufficient amount hypotheses of haploinsufficiency, and concludethat neither adequately explains the persistence of haploinsufficiency.We find that while all haploinsufficient genes confer a growth dis-advantage when subtly overexpressed, the reverse is not true. Manygenes exist, including genes encoding known protein complex mem-bers, that impair proliferation when subtly overexpressed but not whenheterozygously deleted, arguing against the dosage balance hypothesesas a general explanation for the persistence of haploinsufficiency.Instead, our analyses of the growth defects of strains heterozygouslydeleted for haploinsufficient genes indicate that HI genes are limitingfor cellular growth and proliferation when present in one copy insteadof two. Based on these observations, we propose an expansion of thecurrent hypotheses for haploinsufficiency. Our “dosage-stabilizing”hypothesis stipulates that haploinsufficiency persists in organismsover evolutionary time because a balance must be struck between a

Significance

For most genes, a single copy is enough to support normalgrowth and development of diploid organisms, but a smallsubset of genes known as haploinsufficient (HI) genes exhibitextreme sensitivity to decreased gene dosage. Given the rela-tively high frequency of gene-inactivating mutations over thelifespan of an organism, and cell-to-cell variability in gene ex-pression, haploinsufficiency represents a significant barrier toorganismal fitness. Why the expression of these genes has notbeen modulated over evolutionary time to eliminate theirhaploinsufficiency remains unexplained. We find that the limitof haploinsufficient genes on organismal fitness cannot beovercome by an increase in expression because haploinsufficientgenes also confer a fitness disadvantage when encoded in extracopy, leaving these genes evolutionarily “stuck.”

Author contributions: S.A.M. and A.A. designed research; S.A.M. performed research;S.A.M. contributed new reagents/analytic tools; S.A.M. analyzed data; and S.A.M. andA.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: angelika@mit.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900437116/-/DCSupplemental.

Published online May 29, 2019.

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gene product being limiting for a biological process, while avoiding thetoxicity of its overproduction.

ResultsHaploinsufficient Genes Are Sensitive to Increased Copy Number. Thedosage balance hypothesis of haploinsufficiency predicts that HIgenes are toxic when subtly overexpressed; that is, they should alsobe sensitive to increased copy number (SIC; Fig. 1A). The budding yeast,Saccharomyces cerevisiae, is an ideal system to explore this predictionbecause several tools exist to generate comprehensive dosage-alteredlibraries of genes that are haploinsufficient or are toxic when over-expressed. The heterozygous deletion collection (13)—where onecopy of each of the ∼6000 yeast genes has been systematically de-leted in diploid strains—allowed us to study haploinsufficiency atgenome-wide resolution. To study sensitivity to increased copynumber, we utilized the previously constructed MoBY-CEN plasmidlibrary, which is composed of centromeric plasmids that expressnearly all yeast genes (4981/5915 confirmed ORFs) from their en-dogenous promoters (14). For the purposes of this study we considergenes introduced via MoBY-CEN vectors as present in “single-extracopy,” though of course the copy number of CEN vectors can vary,depending on ploidy and the type of selection (15).

We generated a high confidence data set of haploinsufficientgenes in yeast. Deutschbauer et al. (2005) used the heterozygousdeletion collection to identify 184 genes that are haploinsufficientunder maximal growth conditions—in YEP medium containing 2%glucose at 30 °C (6). Of these, we chose 100 highly haploinsufficientgenes to pursue further (henceforth top_HI) based on the followingcriteria: (i) accuracy of the gene deletion in the heterozygous deletioncollection, (ii) a confirmed growth defect (>5%) in heterozygousknockout strains, and (iii) presence of the gene in the MoBY-CENlibrary of plasmids. We note that the growth defect we measured wasin excellent agreement with previously defined fitness values (SIAppendix, Fig. S1B), except for a small number of strains that harbordeletions of ribosomal subunits, which are known to cause genomicalterations (16). The complete list of top_HI genes is shown in SIAppendix, Table S2, with excluded genes in SI Appendix, Table S3.The majority of genes that exhibit severe haploinsufficiency encoderibosomal proteins, and proteins required for transcription andtranslation as well as proteostasis (Fig. 2A).

Having created a high-confidence haploinsufficient gene set, wethen compared growth rates of strains heterozygous for haploinsufficientgenes with growth rates of strains harboring an extra copy of the top_HIgenes. Remarkably, most top_HI genes interfered with proliferationwhen expressed on a CEN plasmid, with 85/100 strains exhibiting astatistically significant growth defect under maximal growth condi-tions (Fig. 2B and SI Appendix, Fig. S1A, Dunn’s multiple compar-ison test P < 0.05, Dataset S1). Of the 15 strains that did not meetstatistical significance, nine strains displayed highly variable doublingtimes (identified as ‡ in SI Appendix, Fig. S1A). Six strains onlyshowed a very slight increase in doubling time (identified as † in SIAppendix, Fig. S1A), despite evidence that the genes were expressedand that their coding sequences harbored no mutations. We proposethat the genes present in excess in these six strains cause toxicity insituations other than maximal growth that yeast cells encounter aspart of their natural life cycle. Growth defects were also observed indiploid cells expressing top_HI CEN plasmids, though the effect wassmaller, indicating that increased ploidy buffers against phenotypescaused by copy number alteration (SI Appendix, Fig. S1C).

Comparison of the growth defect of strains heterozygously deletedfor a haploinsufficient gene (henceforth 2N-1) with the growth de-fect of strains harboring an extra copy of the same gene (henceforth1N+1) showed that the magnitude of the growth defect was pro-portional, with the phenotype of 2N-1 strains being generally moresevere (Fig. 2C Spearman correlation P = 0.0008). This is inagreement with a previous study which found that out of ∼100 genesanalyzed, the majority of genes whose fitness was affected by bothoverexpression and underexpression had more severe consequencesfor decreased expression than increased expression (17). We notethat for several genes the growth of 2N-1 and 1N+1 strains was notparticularly correlated (black data points in Fig. 2C). This is most

likely due to significant variability in the doubling time measure-ments for these 1N+1 strains (black data points in Fig. 2D). Our datafurther suggest that this variability is a consequence of copy numbervariation between different strain isolates (SI Appendix, Fig. S1D),which disproportionately affected the doubling times of strainscontaining genes that are most sensitive to increased copy number.

Why Are Haploinsufficient Genes Toxic upon Dosage Increase? Ourresults lead to the conclusion that, under maximal growth conditions,most genes that confer a significant growth defect in diploids whendeleted in single copy also cause a growth disadvantage when in extracopy. Why are haploinsufficient genes toxic when overproduced? Wecan envision two nonmutually exclusive possibilities: (i) increasedlevels of the gene could interfere with a specific cellular function, or(ii) production and potential subsequent degradation of the excessgene product could be costly.

A well-known example for gene-specific toxicity (option 1) inbudding yeast is the β-tubulin encoding gene TUB2. Expression of asingle extra copy of the gene leads to severe growth defects, as doesdeletion of the two α-tubulin encoding genes TUB1 and TUB3 indiploids (18, 19). Is there evidence that production and degradationof HI genes is generally costly (option 2)? Looking at median ex-pression, the collection of RNAs and proteins produced from the100 most haploinsufficient genes are 11-fold more abundant thanthose for the rest of the genome (SI Appendix, Fig. S2 A and B). Thispreponderance of highly expressed genes among top_HI genes isdriven by genes encoding ribosomal proteins (SI Appendix, Fig. S2 Aand B). While producing large amounts of excess protein is known toplace a burden on the cell’s transcription and translation machinery(20), more recent studies suggest that it is the demand for proteindegradation that most impacts cellular growth when additionalcopies of highly expressed genes are introduced into cells (21). Wefound that ∼65% of HI genes produce proteins known to be degradedby the proteasome when encoded in single copy excess, compared with26% of non-HI genes (Fig. 2E, Student’s t test P < 0.0001). The en-richment for proteasomal targets is again driven by ribosomal proteins(Fig. 2E). This observation raises the possibility that degradation ofexcess HI proteins could be costly to cells. Consistent with this idea,we found that 98/100 haploid strains bearing an extra copy of top_HIgenes exhibited increased sensitivity to the proteasome inhibitorMG132 (Fig. 2F). Ninety-six of one hundred strains were more sen-sitive to the Hsp90 inhibitor radicicol (Fig. 2G). When we excludedribosomal proteins, the majority of strains were still sensitive to theseproteotoxicity-inducing agents (Fig. 2 F and G). These observationsindicate that the toxicity of HI gene overexpression is in part due toexcess proteins placing a burden on the cell’s protein homeostasismachinery.

Prediction: HI genes are SIC hypothesisDosage balance hypothesis

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Fig. 1. Models of haploinsufficiency. Theoretical plots relating gene dosageto the fitness of strains for haploinsufficient genes. HI = haploinsufficiency,SIC = sensitivity to increased copy number. (A) The dosage balance hypothesis.Strains exhibiting changes in HI gene dosage show decreased fitness for bothunderexpression and overexpression, due to altered stoichiometry of proteincomplexes or cellular pathways. (B) The insufficient amounts hypothesis: HIgenes cause decreased fitness of cells as gene dosage decreases. HI geneproducts are limiting for growth.

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Most Dosage-Sensitive Genes Are Not Haploinsufficient. Our obser-vation that top_HI genes are toxic when subtly overexpressed supportsthe dosage balance hypothesis of haploinsufficiency. An additionalprediction of this theory is that haploinsufficiency and sensitivity toincreased copy number are mutually defined, at least for members ofprotein complexes. To test this prediction, we needed to create agenome-wide data set defining the genes that confer a fitness dis-advantage when present in single extra copy. While previous studieshad characterized the sensitivity of genes to high level overexpression(22, 23) or tested their copy number limit (21), none had defined thegenes that confer a fitness defect when expressed in single extra copyunder conditions of maximal growth. Again utilizing the MoBY-CENcollection of yeast plasmids, we generated two independent trans-formant pools of haploid strains, where each strain contained a singleextra copy of a gene for all genes in the genome. We competed poolsin liquid culture and monitored plasmid representation by sequencingplasmid-specific tags every 8 h for a period of 48 h (∼30 generations).In following each tag’s abundance over time, we were able to extract alinear slope for 2646 strains (from 4981 plasmids), of which 1588passed criteria for reproducibility and were assigned a fitness score(SI Appendix, Fig. S3 and Table S1). We converted each slope into arelative fitness value where 1 represents a zero slope with neutralfitness, and values <1 or >1 have negative or positive slopes, re-spectively. The distribution of fitness values showed approximatelyequal numbers of strains increasing and decreasing in the population(Fig. 3A and Dataset S2). Genes detected in both transformant poolsshowed good correlation between relative fitness values (Fig. 3B,Pearson correlation P < 0.0001). A gene was considered to have anegative impact on fitness, and defined as SIC, when its relativefitness was <1 and 1 SD below the population average. Using thiscriterion, we defined 251 genes to be SIC (FDR = 0.072, SI Appendix,Fig. S3C). Conversely, there were 247 genes which fell 1 SD above themean and had a relative fitness>1.We note that strains whose abundance

increases in the population likely do not have a growth advantage, basedon a comparison with known reference points (SI Appendix, Fig. S3D).Rather, these strains increase in abundance because of the relative de-crease of other strains in the culture.

Under identical growth conditions, Deutschbauer et al. (2005)found 3% of the yeast genome to be haploinsufficient, defined asheterozygous gene deletions that cause a decrease in fitness greaterthan 1 SD below the population mean (6). By the same definition, wefound 251/1588 genes to confer a fitness defect when present in singleextra copy, which when extrapolated to the entire yeast genome sug-gests that SIC genes may represent up to 15% of the genome. Thisobservation indicates that genes are much more likely to be sensitiveto increased copy number than gene copy loss, though more com-prehensive studies in diploid yeast will be needed to make an absolutecomparison. Strikingly, while 85% of haploinsufficient genes were SIC,only 10% of the genes identified as highly SIC were haploinsufficient(n = 26). When we restricted our analysis to members of proteincomplexes found in our competition data set, we observed a similarresult: 15% of SIC genes (22/89) are HI, while 83% of HI genes (33/40)were identified as SIC by doubling time measurements. This is true evenwhen we only considered genes whose products are known proteincomplex members (Fig. 3C). We conclude that while haploinsufficientgenes are toxic when present in excess, the converse is not true—manygenes which are found to be highly SIC are not haploinsufficient. Thisobservation also leads to the conclusion that dosage imbalance amongprotein complex members alone cannot explain haploinsufficiency.

Dosage Imbalance Does Not Fully Explain Haploinsufficiency. Thefinding that haploinsufficiency and sensitivity to increased copy numberare not mutually defined prompted us to test additional predictions ofthe dosage balance model.

The dosage imbalance hypothesis predicts that deletion of a HIgene in a haploid cell results in the same phenotype as deleting one

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Fig. 2. Toxicity of haploinsufficient gene copy num-ber increase. (A) Functional categories that definehighly haploinsufficient genes (top_HI). (B) Doublingtime of strains containing dosage-altered top_HI genes,with measurements made at 30 °C in YPD. 2N-1 arediploids with heterozygous deletions of HI genes (wild-type diploid) and 1N+1 are haploids with a singleextra copy of HI genes, contained on a CEN plasmid(wild-type haploid plus empty vector). (C) The rela-tionship between 2N-1 and 1N+1 relative growthrates. The black dots identify genes that are dispro-portionately toxic when in excess, which are thoughtto be correlation outliers due to high variability asshown in D. (D) A plot comparing doubling time andvariability in growth measurements for 1N+1 cells.Note that data points highlighted in black are thesame as black data points in C. (E) Degradation of HIgenes when encoded in single extra copy (32). Thefraction of excess protein degraded is the relativeprotein expression in 1N+1 compared with WT cells(1-log2 ratio), where a value of 0 represents no deg-radation and 1 represents complete degradation (two-tailed t test with Welch correction P < 0.0001). Thepercent of proteins in each category considered de-graded (>0.4) is indicated below each bar. Centralline =median. Plot whiskers 10–90th percentile. (F andG) Growth of strains containing top_HI genes on a CENplasmid were treated with 100 μMMG132 (F) or 25 μMradicicol (G), alongside untreated controls (DMSO) inYPD at 30 °C. Green bars represent the doubling timeof strains harboring an empty vector control. The num-ber of strains that are more sensitive to drug treat-ment than expected is indicated below each condition.

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copy in a diploid cell because both strains experience the same degreeof stoichiometric imbalance. This is not what we observe. Deletion ofthe nonessential top_HI genes in haploid cells causes an increase indoubling time of 11–100% compared with wild-type cells (Fig. 4A andDataset S1). Doubling time increased only 5–29% in heterozygouslydeleted diploid cells (Fig. 2B and Dataset S1). Importantly, the growthdefect of the haploid strain lacking the HI gene was invariably moresevere than the growth defect of the corresponding heterozygouslydeleted diploid strain (Fig. 4A).

Another prediction of the dosage balance hypothesis is that deletingall subunits of a protein complex should alleviate the haploinsufficiencyof deletions of individual complex members. We tested this predictionfor the eIF2 complex. eIF2 is an initiation factor for translation withthree obligate subunits, two of which (SUI2 and SUI3) are known to behaploinsufficient. If stoichiometry were the cause of the genes’haploinsufficiency, a strain heterozygously deleted for all three subunitgenes should not exhibit a growth defect. This is not what we observed.The dosage-balanced strain for eIF2 exhibited a strong growth defect(13 min increase in doubling time), on par with that of strains harboringsingle eIF2 gene deletions (9–18 min) (Fig. 4B). While the tripledeletion strain grew significantly slower than the wild-type strain, thephenotype was not as severe as that of strains with single deletions ofthe SUI2 and SUI3 genes. Based on the relative differences indoubling time, we estimate that protein stoichiometry imbalance canexplain only ∼33% of the haploinsufficient growth defect for thisprotein complex.

Finally, our results show that subtle overexpression of top_HIgenes causes increased sensitivity to proteotoxic agents such asradicicol (Fig. 2G). According to the dosage balance hypothesis, thisproteotoxicity should arise from stoichiometric imbalance of functional

complexes (24) and should thus also occur in strains heterozygouslydeleted for HI genes. This is not the case. Strains containing hetero-zygous deletions of HI genes are not, on average, more sensitive toradicicol, although a subtle increase in sensitivity to radicicol mighthave been missed in this bulk measurement (SI Appendix, Fig. S4A).Taken together, our data indicate that stoichiometric imbalance is notthe sole cause of haploinsufficiency.

Haploinsufficient Genes Are Rate Limiting for Cellular Fitness. Theconclusion that the dosage imbalance hypothesis alone cannot ex-plain haploinsufficiency prompted us to test elements of the in-sufficient amounts hypothesis. We hypothesized that HI genes arelimiting for organismal function, such as cell growth or proliferationwhen heterozygously deleted. To test the growth-limiting nature ofhaploinsufficiency, we examined the importance of HI genes for keyprocesses in cell growth and proliferation. Ribosomal proteins areparticularly enriched among HI genes as a class (6), and it is wellestablished that ribosomes are rate limiting for cell growth (25).Consistent with previous reports (26), we found that deletion of onecopy of ribosomal genes and of other haploinsufficient genes re-quired for translation caused a reduction in mass accumulation indiploid strains, as judged by a smaller average cell size (Fig. 4C andDataset S1). Strains carrying heterozygous deletions in HI genes notinvolved in protein synthesis were the same size or larger thancontrol strains (Fig. 4C and Dataset S1). Importantly, introducing anextra copy of these ribosomal genes did not lead to a decrease in cellsize (SI Appendix, Fig. S4B), arguing that stoichiometric imbalancesamong ribosome subunits are not responsible for the small-cell sizephenotype observed in diploid cells heterozygously deleted for theseribosomal protein genes. Instead, as small size is a characteristic ofreduced protein synthesis (25), our data indicate that insufficientamounts of ribosomal proteins result in the growth defect of strainsheterozygously deleted for their genes.

Is there evidence that other haploinsufficient genes are rate limitingin processes critical for cell proliferation? The top_HI genes include5/8 subunits of the CCT chaperone complex known to fold actin andtubulin. Strains harboring heterozygous deletions in these genes aresensitive to the actin and microtubule assembly inhibitors latrunculinA and benomyl, respectively (Fig. 4 D and E), suggesting CCT subunitsare indeed rate limiting for folding these cytoskeleton constituents. Wenote that strains with excess CCT subunits are not sensitive to benomyl(SI Appendix, Fig. S4C), again arguing against stoichiometric imbalancesand for insufficient amounts of protein as the source of this benomylsensitivity. We predict that the remaining top_HI genes are alsolimiting for processes important for growth or proliferation undermaximal growth conditions.

Haploinsufficient Genes Have a Narrow Expression Range. Takentogether, our results lead to the conclusion that two properties de-termine whether a gene is haploinsufficient in a specific environmentor growth condition: (i) the gene product is rate limiting for maximalorganismal fitness and at the same time (ii) the gene confers a fitnessdisadvantage when in excess. We propose that the fitness penaltywhen in excess prevents up-regulation of the gene to counteracthaploinsufficiency, and the rate-limiting nature of the gene causesthe observed fitness defect in heterozygotes. This model of dual, coun-teracting selective pressures over evolutionary time makes a very strongprediction: the expression range of HI genes, in particular its variationbetween cells in a population, should be narrow relative to that of othergenes in the genome. Using a previously published set of single-cell geneexpression data (27), we observe that the cell-to-cell variability in geneexpression is significantly more narrow among HI genes compared withother genes in the genome (Fig. 5A). While low variation can be drivenby high expression (the example in Fig. 5A shows the cell-to-cell vari-ability in the expression of 100 highly expressed, nonhaploinsufficientgenes), we still observed narrow ranges of expression among morelowly expressed HI genes (Fig. 5A and SI Appendix, Fig. S4D). Weconclude that haploinsufficient genes are narrowly expressedirrespective of their expression level. This conclusion is consistentwith recent data showing that variability in gene expression across a

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Fig. 3. Identification of genes that are sensitive to increased copy number(SIC). (A) The relative fitness distribution for 1588 strains across two in-dependent pools (n = 6). Each strain contains a single extra copy of a geneon a centromeric vector. Strains were competed for ∼30 generations in YPDat 30 °C. Bins = 0.005. (B) Reproducibility between the two independentpools of transformants. (C) The percentage of genes encoding proteincomplex members that are haploinsufficient among top SIC genes—definedas those genes which show decreased fitness of greater than 1 SD in pooledcompetition. For comparison, the percentage of genes that are SIC amongknown haploinsufficient genes (from Fig. 2B) is shown on the Right.

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population of cells is decreased for genes where small changes in ex-pression had a large impact on fitness (17).

DiscussionWhile changes to gene dosage can lead to imbalances in proteincomplex stoichiometry that adversely affect cellular fitness (11, 12),several lines of evidence indicate that haploinsufficiency cannot beexplained by fitness decrease due to stoichiometric imbalance alone.First, haploinsufficiency and overexpression toxicity are not mutuallydefined, even among genes encoding protein complex subunits.Second, deletion of haploinsufficient genes in a haploid strain ismore detrimental than in a diploid strain even though the number ofuncomplexed protein subunits is the same in both cell types. Third,overexpression of individual members of a complex lead to a differentphenotype than their underexpression, as is showcased by ribosomalproteins and CCT subunits. Finally, at least in the case of eIF2, het-erozygous deletion of all complex member genes does not alleviate thegrowth defect caused by deletion of individual subunit genes.

Our data support a hybrid model for haploinsufficiency, which wecall the dosage-stabilizing hypothesis (Fig. 5B). It builds upon andincorporates core principles from both the insufficient amounts hy-pothesis and the dosage balance hypothesis. The dosage-stabilizinghypothesis posits that HI gene products are limiting for fitness whenunderexpressed (insufficient amount hypothesis), and toxic when over-expressed, most likely due to adverse effects on protein homeostasis andimbalances in protein complex stoichiometry (dosage balance hy-pothesis). In other words, haploinsufficient genes are evolutionarily“stuck,” unable to increase or decrease expression over time to ac-commodate fluctuations in gene dosage because a fitness penalty is as-sociated with both downregulating and upregulating HI gene expression.Thus, haploinsufficient genes are “living on the edge,” representing aunique class of genes that must carefully balance their expression,ensuring that the cost of overproduction does not outweigh thepotential benefit of maximizing growth.

It is worth noting that haploinsufficiency is extremely contextdependent. Deutschbauer et al. (2005) showed that there is littleoverlap between genes that are haploinsufficient under maximalgrowth conditions and those that are limiting when cells are grown inminimal medium (6). Applying more stringent pressures on cell growththrough glucose-, ammonium-, or phosphate-limited continuous cul-ture revealed a set of haploinsufficient genes that is highly conservedacross nutrient-limiting conditions, yet distinct from those definedunder maximal growth conditions (28). Interestingly, under these severegrowth restriction conditions, the frequency of haploinsufficient genes

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LegendLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddd

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YPD benomyl(15 g/ml)

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Fig. 4. Testing the dosage balance and insufficientamount hypotheses. (A) Doubling time of haploid strainscontaining deletions in nonessential top_HI genes, withmeasurements made at 30 °C in YPD. 2N-1 diploidsare included for comparison (paired t test, P < 0.0001).Connecting lines show corresponding genes. (B) Dou-bling time of strains carrying heterozygous deletionsfor genes encoding members of the eIF2 complex, incombination and in isolation. Measurements weremade at 30 °C in YPD. Significance compared with WTis shown above each bar; select other comparisons aremade with brackets (ANOVA with multiple compari-sons, ****P < 0.0001 Bonferroni correction). (C) Volumeof diploid cycling cells with heterozygous deletionsfor all top_HI genes, and a subset of HI genes encodingribosomal proteins (two-tailed t test with Welch cor-rection **P = 0.0076). (D) A fivefold serial dilution ofstrains containing confirmed heterozygous deletionsfor CCT complexmembers, plated on benomyl (15 ug/mL)and YPD control. (E) Haploinsufficient profiles of theheterozygous deletion collection for latrunculin treat-ment (0.9 μM) or benomyl treatment (27 μM). Data arefrom Hoepfner et al. (2014) (33). Genome position of aheterozygous deletion is plotted against normalizeddrug sensitivity score, where a negative score repre-sents impaired proliferation in the presence of thedrug. Purple dots are subunits of the CCT chaperonecomplex.

gene dosage

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Fig. 5. The gene expression range of haploinsufficient genes is narrow. (A)Cell-to cell variability (CV2) in gene expression for HI genes compared withgenes that are highly expressed but are not haploinsufficient (high expres-sion not HI), genes that are haploinsufficient but not highly expressed (HInot high expression) and all non-HI genes in the genome (genome) based onFACS fluorescent measurements of promoter-YFP fusions for 1000 genes (27)(****P < 0.0001, **P = 0.0058, *P = 0.0197). Central line = median. Plotwhiskers span 10–90th percentile. (B) The dosage-stabilizing hypothesis: HIgenes cause a decrease in fitness when underexpressed and overexpressed.This narrowed fitness distribution is driven by the evolutionary pressure toboth increase and decrease expression.

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appears to increase (12–20%) (28) and could be as high as 76%, atleast among essential yeast genes, based on single-cell morphologicalphenotyping (29). Together, these studies indicate that conditionsexist for most if not all genes under which they are haploinsufficient.We speculate that this could account for why all genes are maintainedin two copies over evolutionary time even in organisms that propagateby a predominantly nonsexual lifestyle.

Given the persistence of haploinsufficient genes, how has cellularphysiology been driven by, and perhaps adapted to, their presence?Given the enrichment of transcription and translation factors amongHI genes, which are by nature growth limiting, our data lead us towonder whether haploinsufficient genes might play a role in set-ting the division rate of cells. In other words, the expression levels ofhaploinsufficient genes may be partially responsible for buddingyeast cells having a doubling time of 90 min in YEPD medium at 30°.Changing the expression of any individual gene will have little effecton cell division length, but increasing their expression coordinatelywill, we predict, produce yeast cells with shorter doubling times.Additionally, if increasing gene expression of individual genes is nota solution to the problem of haploinsufficiency, do organisms haveanother way to escape it evolutionarily? Previous work (6, 30) hassuggested that gene duplication, where two copies of the gene nowsplit the overall expression level, may be better buffered against geneexpression fluctuation and loss, providing organisms a way out ofhaploinsufficiency. Future studies should seek to address thesequestions.

MethodsStrains harboring heterozygous deletions of haploinsufficient genes wereobtained from the BY4743 yeast knockout collection; haploid deletion strainsare BY4741 (MATa) (13). Centromeric (CEN) plasmids were obtained from theMolecular barcoded ORF collection of yeast genes (MoBY) (14). Doublingtimes were measured in 96-well format in rich YEP medium +2% glucose at30 °C, taking OD600nm measurements every 15 min for 24 h. MoBY-CENtransformant pools (∼30× genome coverage) were competed in 0.5 L YPDand collected every 8 h (∼5 generations) for 48 h. The relative contributionof strains was captured by amplification of plasmid-specific tag sequences aspreviously described (31), using primers with indices for multiplex sequenc-ing on the Illumina Next-seq platform. For DNA copy number, DNA wasisolated from individual transformants in stationary phase by zymolyasedigestion and phenol extraction, followed by digestion with RNase A. qPCRof DNA was carried out on a Roche LightCycler 480 II using TaKaRa mixfor real-time PCR: SYBR Premix Ex Taq. Cell volume was determined usinga Multisizer 3 Coulter Counter, counting 105 particles per strain at a thresholddiameter of 2 μm. Detailed experimental procedures are provided in SIAppendix.

ACKNOWLEDGMENTS. We thank Andrew Murray, Gene-Wei Li, and mem-bers of the A.A. laboratory for suggestions and critical reading of thismanuscript. This work was supported by NIH grant CA206157 and GM118066to A.A., who is an investigator of the Howard Hughes Medical Institute andthe Glenn Foundation for Medical Research. On behalf of S.A.M., this mate-rial is based upon work supported by the National Science Foundation Grad-uate Research Fellowship under Grant 1122374. Sequencing work at the MITBioMicro Center was supported in part by the Koch Institute Support (core)Grant P30-CA14051 from the National Cancer Institute.

1. R. A. Fisher, The possible modification of the response of the wild type to recurrentmutations. Am. Nat. 62, 115–126 (1928).

2. H. A. Orr, A test of Fisher’s theory of dominance. Proc. Natl. Acad. Sci. U.S.A. 88,11413–11415 (1991).

3. S. Wright, Physiological and evolutionary theories of dominance. Am. Nat. 68, 24–53(1934).

4. F. A. Kondrashov, E. V. Koonin, A common framework for understanding the origin ofgenetic dominance and evolutionary fates of gene duplications. Trends Genet. 20,287–290 (2004).

5. R. A. Veitia, M. C. Potier, Gene dosage imbalances: Action, reaction, and models.Trends Biochem. Sci. 40, 309–317 (2015).

6. A. M. Deutschbauer et al., Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast. Genetics 169, 1915–1925 (2005).

7. V. T. Dang, K. S. Kassahn, A. E. Marcos, M. A. Ragan, Identification of humanhaploinsufficient genes and their genomic proximity to segmental duplications. Eur. J.Hum. Genet. 16, 1350–1357 (2008).

8. N. Huang, I. Lee, E. M. Marcotte, M. E. Hurles, Characterising and predicting hap-loinsufficiency in the human genome. PLoS Genet. 6, e1001154 (2010).

9. J. Steinberg, F. Honti, S. Meader, C. Webber, Haploinsufficiency predictions withoutstudy bias. Nucleic Acids Res. 43, e101 (2015).

10. M. de Clare, P. Pir, S. G. Oliver, Haploinsufficiency and the sex chromosomes fromyeasts to humans. BMC Biol. 9, 15 (2011).

11. B. Papp, C. Pál, L. D. Hurst, Dosage sensitivity and the evolution of gene families inyeast. Nature 424, 194–197 (2003).

12. R. A. Veitia, Exploring the etiology of haploinsufficiency. BioEssays 24, 175–184(2002).

13. G. Giaever et al., Functional profiling of the Saccharomyces cerevisiae genome. Na-ture 418, 387–391 (2002).

14. C. H. Ho et al., A molecular barcoded yeast ORF library enables mode-of-actionanalysis of bioactive compounds. Nat. Biotechnol. 27, 369–377 (2009).

15. A. S. Karim, K. A. Curran, H. S. Alper, Characterization of plasmid burden and copynumber in Saccharomyces cerevisiae for optimization of metabolic engineering ap-plications. FEMS Yeast Res. 13, 107–116 (2013).

16. T. R. Hughes et al., Widespread aneuploidy revealed by DNA microarray expressionprofiling. Nat. Genet. 25, 333–337 (2000).

17. L. Keren et al., Massively parallel interrogation of the effects of gene expression levelson fitness. Cell 166, 1282–1294.e18 (2016).

18. D. Burke, P. Gasdaska, L. Hartwell, Dominant effects of tubulin overexpression in

Saccharomyces cerevisiae. Mol. Cell. Biol. 9, 1049–1059 (1989).19. W. Katz, B. Weinstein, F. Solomon, Regulation of tubulin levels and microtubule as-

sembly in Saccharomyces cerevisiae: Consequences of altered tubulin gene copy

number. Mol. Cell. Biol. 10, 5286–5294 (1990).20. A. Wagner, Energy constraints on the evolution of gene expression. Mol. Biol. Evol.

22, 1365–1374 (2005).21. K. Makanae, R. Kintaka, T. Makino, H. Kitano, H. Moriya, Identification of dosage-

sensitive genes in Saccharomyces cerevisiae using the genetic tug-of-war method.

Genome Res. 23, 300–311 (2013).22. D. M. Gelperin et al., Biochemical and genetic analysis of the yeast proteome with a

movable ORF collection. Genes Dev. 19, 2816–2826 (2005).23. R. Sopko et al., Mapping pathways and phenotypes by systematic gene over-

expression. Mol. Cell 21, 319–330 (2006).24. R. A. Veitia, J. A. Birchler, Models of buffering of dosage imbalances in protein

complexes. Biol. Direct 10, 42 (2015).25. P. Jorgensen, M. Tyers, How cells coordinate growth and division. Curr. Biol. 14,

R1014–R1027 (2004).26. P. Jorgensen, J. L. Nishikawa, B.-J. Breitkreutz, M. Tyers, Systematic identification of

pathways that couple cell growth and division in yeast. Science 297, 395–400 (2002).27. L. Keren et al., Noise in gene expression is coupled to growth rate. Genome Res. 25,

1893–1902 (2015).28. D. Delneri et al., Identification and characterization of high-flux-control genes of

yeast through competition analyses in continuous cultures. Nat. Genet. 40, 113–117

(2008).29. S. Ohnuki, Y. Ohya, High-dimensional single-cell phenotyping reveals extensive

haploinsufficiency. PLoS Biol. 16, e2005130 (2018).30. Y. Guan, M. J. Dunham, O. G. Troyanskaya, Functional analysis of gene duplications in

Saccharomyces cerevisiae. Genetics 175, 933–943 (2007).31. C. Payen et al., High-throughput identification of adaptive mutations in experimen-

tally evolved yeast populations. PLoS Genet. 12, e1006339 (2016).32. N. Dephoure et al., Quantitative proteomic analysis reveals posttranslational re-

sponses to aneuploidy in yeast. eLife 3, e03023 (2014).33. D. Hoepfner et al., High-resolution chemical dissection of a model eukaryote reveals

targets, pathways and gene functions. Microbiol. Res. 169, 107–120 (2014).

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