Gene balance hypothesis: Connecting issues of dosagesensitivity across biological disciplinesJames A. Birchlera,1 and Reiner A. Veitiab,c

aDivision of Biological Sciences, University of Missouri, Columbia, MO 65211; bCentre National de la Recherche Scientifique Unité Mixte de Recherche 7592,Institut Jacques Monod, 75013 Paris, France; and cUnité de Formation et de Recherche Sciences du Vivant, Université Paris Diderot, 75013 Paris, France

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2011.

Contributed by James A. Birchler, May 7, 2012 (sent for review January 30, 2012)

We summarize, in this review, the evidence that genomic balanceinfluences gene expression, quantitative traits, dosage compensa-tion, aneuploid syndromes, population dynamics of copy numbervariants and differential evolutionary fate of genes after partial orwhole-genome duplication. Gene balance effects are hypothesizedto result from stoichiometric differences amongmembers of macro-molecular complexes, the interactome, and signaling pathways. Theimplications of gene balance are discussed.

The concept of gene dosage balance arose in the early days of thefield of genetics, with the work by Blakeslee et al. (1) using the

flowering plantDatura stramonium and thework byBridges (2) usingthe fruit fly Drosophila melanogaster. Both studies found that theaddition of a single chromosome to a genotype was highly detri-mental or lethal, whereas the addition of a complete genome tomake a polyploid was viable and resulted in lesser effects on thephenotype. Thework onDaturawas greatly extended to include fourcopies of each chromosome arm andmultiple whole genomes (3, 4).Subsequently, related principles governing aneuploidy, with somedifferences, were found throughout eukaryotes (5–7).More recently,the removal of chromosomes to produce monosomic genotypesshows that there are quite severe phenotypic effects compared withthe haploid, which has only one copy of every chromosome (8, 9).This principle is illustrated in Fig. 1. Maize genotypes were pro-

duced that were diploid with one, two, or three doses of represen-tative chromosome arms as well as haploids plus the chromosomearm in question all in a highly similar genetic background. For allthree chromosome arms examined, it was clear that addition ofa chromosome to the diploid has much less severe effect than ad-dition of a chromosome to a haploid. However, the removal of oneof two chromosome arms is also more severe than the reduction inploidy fromdiploid to haploid. Thedoubling of amere chromosomearm in a haploid leads to a deformity, whereas doubling the wholegenome leads to a normal diploid. These comparisons illustrate thatthe relative dosage of chromosomal segments is critical for normaldevelopment and phenotypic characteristics.Extending this relationship to the molecular level, the studies

by Birchler (10) and Birchler and Newton (11) found that ex-pression patterns of enzyme and protein levels were modulatedmore in aneuploid genotypes than with changes in ploidy. Thework by Birchler and Newton (11) suggested that a perturbationof stoichiometric relationships of regulatory gene products wasa contributor to the effects of genomic imbalance and playeda role in these transacting dosage effects on gene expression.The modulations are both positive and negative depending on

the gene, tissue, or chromosomal region studied (11, 12). Althoughmost modulations are within direct or inverse limits between theexpression level and the chromosomal dose (Fig. 2), some greatermodulations occur. A common dosage effect on gene expressionfound in aneuploids was an inverse correlation of gene product withthe copy number of an unlinked chromosomal region (i.e., an in-verse dosage effect) (10–12). In other words, monosomics fora particular chromosome arm would modulate the expression ofunlinked genes to an upper limit of approximately twofold, whereasthe corresponding trisomic would reduce the same gene product to

about two-thirds of the amount in the balanced diploid with twocopies of all chromosomal regions (Fig. 2). The amount of any onetarget gene product could be modulated by multiple regions of thegenome. This effect is quite common in the data for trisomic seg-ments in other species. including Datura, barley, Arabidopsis, Dro-sophila, and human trisomic cells (13–32). The ability to recovercorresponding monosomics in maize facilitated the finding that thiseffect can be proportional through one, two, and three doses.Furthermore, the combination of a region of the genome that

produces an inverse dosage effect on a particular gene product to-gether with the gene encoding that product could result in dosagecompensation (Fig. 2). In this case, despite the fact that the genewasvaried in one to three copies, the total output realized was more orless constant in the chromosome arm dosage series (10, 11). By di-viding the varied chromosome arm into smaller segments andassaying gene expression, the basis of this type of dosage compen-sation was shown to involve a combination of an inverse dosageeffect cancelling a structural gene dosage effect (33). Specifically, thestructural gene locus in a smaller segment exhibited a proportionaldosage effect, and another region of the larger aneuploid producedan inverse dosage effect. Similar cases of dosage compensation werefound for chromosome arm trisomics in Drosophila in the work byDevlin et al. (34). The work by Birchler et al. (35) showed in Dro-sophila that the basis of this effect also involved the combination ofa structural gene dosage effect and an inverse effect operating si-multaneously, and it documented the effect on themRNA level (35).Because an inverse effect may regularly occur on variation of manygenomic segments, dosage compensation can also regularly occurwhen a regulator and a target gene are varied together. The inverseeffect can cause compensation of different target genes regardless ofthe level of activity, because the effect involves a relative stoichio-metric relationship of the varied segment to the remainder of thegenome, which will be the same for all target genes.The work by Rabinow et al. (36) tested whether the aneuploid

effects on gene expression could be reduced to the action ofsingle genes. Various leaky alleles of the white eye color gene inDrosophila were used as phenotypic reporters to screen for sec-ond site modifiers that, when heterozygous, would increase ordecrease the expression within a twofold range. The first suchmutation found increased the expression of the white eye colorgene approximately twofold as a heterozygote and thus, wouldmimic a monosomic situation on the single gene level. Whena small aneuploid region around this gene was present in threecopies, the expression of white was reduced to about two-thirds ofthe normal diploid. In contrast, the eye color of diploid andtriploid flies was similar. The introduction of these mutations intoa triploid background causes an ∼50% increase in expression,thus conforming to an inverse relationship for genomic balance.

Author contributions: J.A.B. and R.A.V. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: birchlerj@missouri.edu.

14746–14753 | PNAS | September 11, 2012 | vol. 109 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1207726109

The effect was first found on the phenotypic level and thendocumented in some developmental stages on the mRNA level.The work by Sabl and Birchler (37) surveyed the autosomes of

Drosophila for phenotypic dosage-sensitive modifiers of the whiteeye color gene. Earlier, in studies of X chromosome dosagecompensation, the work by Muller (38) described evidence formultiple X-linked dosage-sensitive modifiers of white. In thesestudies, segments of the genome were found that modulated theexpression of white either positively or negatively and illustratedthat multiple aneuploid regions could affect the phenotype ofa single trait. More regions were found to modulate white nega-tively compared with those that positively affect white. The workby Birchler (39) found that the dosage of the whole X chromo-some produced an inverse dosage effect on the phenotypic ex-pression of white when it was deleted from its normal location onthe X chromosome and was present as a transgene on the auto-somes, although the up-regulation in normal males with one Xchromosome was not a full twofold effect. These phenotypicstudies also illustrate the multigenic nature of these modulatingeffects. However, variation of the whole genome in dosage pro-duces a very similar phenotype in diploid and triploid flies (36),exhibiting little modulation and a proportional expressionwith ploidy.The work by Guo and Birchler (12) documented aneuploid

modulations of gene expression on the RNA level in maize andfound that, in general, themagnitude ofmodulationwas directly orinversely correlated with the degree of genomic imbalance. Thework byGuo andBirchler (12) also noted the parallels between themultigenic additive control of quantitative phenotypic traits andthe impact of multiple aneuploidies on the same quantitativecharacteristics. By comparing the effects in the diploid embryo ofthe maize kernel with the triploid endosperm, it was again foundthat the magnitude of modulations depended on the deviation ofthe chromosomal change from the balanced euploid. RNAmeasurements comparing whole-genome ploidy changes showfewer modulations (40).The work by Birchler et al. (41) summarized the molecular

nature of the known single gene dosage-sensitive modifiers of thewhite eye color gene in Drosophila that had been recovered overthe course of two decades. The collection consisted of tran-scription factors, chromatin components, and members of signaltransduction systems. Several other laboratories noted that tran-scription factors, tumor suppressor genes, and components ofsignal transduction are dosage-sensitive (42–45). This realizationwas consistent with the fact that many transcription factorscontrol developmental decisions in Drosophila in a concentra-tion-dependent manner (46–51), and the myriad of modifiers ofposition effect variegation are dosage-sensitive (52). Dosagesensitivity will operate through a cascade of regulatory steps,allowing many connected genes to affect any one process (41).The gene dosage effects could potentially result simply from

a change in concentration of a gene product in the cell, such asthe change that occurs with changing the dosage of the mostcontrolling step of biochemical pathways (53). However, thereare several theoretical and experimental results that suggest aninvolvement of relative stoichiometric relationships for manydosage effects. The studies by Veitia (43, 44, 54) modeled thekinetics of assembly of macromolecular complexes to explain

Fig. 1. Effect of genomic imbalance on quantitative phenotypic charac-teristics in maize. (A) From left to right, this series of plants is haploid,haploid plus the long arm of chromosome 1 (1L), one copy of 1L, two copiesof 1L, and three copies of 1L in an otherwise diploid background. (B and C)Analogous genotypes for the short arm of chromosomes 5 (5S) and 9 (9S),respectively. A meter stick is included for scale. These examples include oneof the longer (1L) and one of the shorter (9S) chromosome arms in the maizegenome and illustrate that similar imbalance phenomena occur for all tested

arms. The addition of a chromosome arm to a haploid plant producesa much greater impact on the phenotype than adding the same arm to anotherwise diploid plant, illustrating the concept of genomic balance. Dosagemanipulation of chromosome arms is made using translocations with thesupernumerary B chromosome (10, 11), and haploids are produced using aninducer line (12). The dosage and ploidy were determined by metaphasechromosome spreads of root tips, and then, the classified seedlings weregrown into plants in the greenhouse. Photographs by Fangpu Han.

Birchler and Veitia PNAS | September 11, 2012 | vol. 109 | no. 37 | 14747

GEN

ETICS

INAUGURA

LART

ICLE

how changes in stoichiometry of the component members couldproduce a dominant phenotypic effect (43). In the context of amultisubunited complex, changing the amount of one subunit canshift the reaction to unproductive subcomplexes and produce adifferent amount of the complete complex (Fig. 3). The work byPapp et al. (55) examined heterozygous gene KOs in yeast andfound a negative correlation between the involvement in macro-molecular complexes and fitness. Increased gene copy number tothe genome produced a similar effect, and selected co–up-regulationof interactors could correct each other. The work by Papp et al. (55)coined the term balance hypothesis.In molecular terms, dosage imbalance can be illustrated with

the heuristic example of the trimeric factor A-B-A. If its as-sembly follows a random pathway, allowing the formation ofintermediate species AB and BA before assembly of the com-plete A-B-A trimer, a decrease in the concentration of A canlead to a reduction of ABA yield. This result is the case, becauseat some point in the assembly reaction, A will become limitingduring the production of AB and BA that will not yield trimers(Fig. 3). Subunit B will be limiting at low concentration but inexcess, will cause a reduction in the ABA yield (43). Examples ofthis relationship have been noted (56). Thus, both positive andnegative effects on the ABA yield will depend on the relativeamount of B to A (57). This example illustrates that the relativeamount of subunits entering the assembly reaction of a complexcan affect the amount of functional product, which can havedownstream genetic consequences.The work by Liang et al. (58) compared the degree of protein

underwrapping to the occurrence of gene duplicability acrosstaxa. Protein underwrapping is the property of proteins to bepenetrated by water molecules. With increasing protein–proteininteractions, a greater level of underwrapping can be tolerated,

because such interactions stabilize the relevant proteins. There isan overall negative correlation between the degree of under-wrapping and the ability to survive as a gene duplicate in a widerange of taxa from bacteria to humans. This result suggests that,with a greater number of protein–protein interactions involvedwith macromolecular complexes, there are increasing negativefitness consequences of single gene duplication, which manifestsas a stoichiometric imbalance.The work by Schuster-Böckler et al. (59) examined this issue in

a different manner. Using a protein domain database, a negativecorrelation was found between the number of protein–protein in-teraction domains that were present in a protein and its ability to bemaintained as a copy number variant (CNV) in human populations.Also, the dosage-sensitive genes exhibited less expression variationamong tissues and among individuals for the same tissue. Theseresults also support the hypothesis that changing the stoichiometryof components of macromolecular complexes or the interactomewill produce a dominant phenotypic effect that affects fitness.The work by Lemos et al. (60) showed that the number of

interactions of a protein (i.e., its connectivity) constrains geneticvariation of its expression in yeast and fruit fly populations. Namely,they reported a negative correlation between the variation of geneexpression and the number of protein–protein interactions. More-over, the degree of expression variation among genes encodinginteractors was smaller than the degree of expression variation ofrandom gene pairs. Finally, the levels of expression of interactorsdisplayed a positive correlation across strains.

Gene Balance HypothesisThe experimental and theoretical findings involved with gene bal-ance that are described above were formulated from phenotypicdata, gene expression patterns in dosage manipulations, and iden-tification of their underlying basis in dosage-sensitive regulatorygenes. Connections were then made to interacting members ofmultisubunit complexes and their kinetics and mode of assembly.The principle can also be stated in reverse. The stoichiometry ofmembers of multisubunit complexes can affect the amount offunctional complete product, which in turn, affects patterns of geneexpression (if the complex is regulatory) and ultimately, the

Fig. 2. Diagrammatic representation of the types of effects observed forgene expression in aneuploids. The x axis depicts the chromosomal dosage.The y axis depicts the percentage of expression in the aneuploid comparedwith the diploid. (A) A gene dosage effect occurs when the structural geneproduces a proportional amount of product to its copy number. (B) There arealso direct transacting effects, in which a gene is modulated in expression indirect correlation with a different chromosomal dosage. (C) Another trans-acting modulation is the inverse dosage effect, in which the expression ofa target gene is inversely correlated with the dosage of another chromo-somal region. (D) Dosage compensation occurs when the expression ofa structural gene does not change with its dosage. Dosage compensationresults when an inverse dosage effect of an aneuploid region includes,among its target genes, those genes that are also on the varied chromo-some. The two effects, structural gene and inverse, combined togethercancel to produce nearly equal expression in all chromosomal doses. Modi-fied from Birchler (13).

Fig. 3. Heuristic examples of stoichiometric imbalance in the context ofa trimer A-B-A. For simplicity, we consider that the assembly of ABA is ran-dom and irreversible. (A) Normal condition with a particular stoichiometricbalance between the molar amounts of A and B. (B) Halving the amount ofmonomer A leads to a decrease of trimer yield, because there is not enoughof A to complete the reaction. (C) Increasing the relative amount of B leadsto a decrease of ABA for the same reasons. The molar ratio of each conditionis indicated.

14748 | www.pnas.org/cgi/doi/10.1073/pnas.1207726109 Birchler and Veitia

phenotype and evolutionary fitness. Clearly, there are complicatedprocesses involved in these steps that will impact the outcome, butthe bulk of the data suggests this generalization, which has impli-cations as outlined below.

Implications for Evolutionary GenomicsFrom a different perspective, the field of evolutionary genomicscame to the realization that sequenced genomes, such as yeast andArabidopsis, revealed the remnants of ancient whole-genomeduplications (WGD) (61–68). After these tetraploidization events,many genes were lost in a return to diploidy but in a nonrandomfashion depending on function. The classes of genes remaining forlonger periods from the WGD were revealed to be those classesinvolved withmacromolecular complexes in general, which includethe ribosome (69) and proteasome and of note for the discussionhere, transcription factors and components of signal transductionpathways (64, 70–74). These classes of genes were similar to thedosage-sensitivemodifiers of thewhite eye color gene inDrosophila(41). The implication is that deletion of one member of a duplicatepair of these classes would have a negative fitness effect and beselected against. Thus, the duplicate pair would be retained forlonger periods of evolutionary time than other gene classes. Theretained genes, in fact, show evidence of purifying selection in bothduplicates (66, 75), which is consistent with a selection of main-tenance of the stoichiometric relationship. The studies by Birchleret al. (53) and Freeling and Thomas (72) noted the relationship ofthese evolutionary results to classical studies of genomic balance,in that variation of part of the genome is more detrimental thanvariation of the whole genome.A corollary of this concept is that segmental genomic dupli-

cations that include genes encoding members of macromolecularcomplexes would be underrepresented in genomes, because theywould alter the stoichiometry of interacting genes. Studies inyeast, Arabidopsis, rice, poplar, and mammals (64, 67, 76–80) re-veal that this is the case. Furthermore, the study of copy numberpolymorphisms indicates an underrepresentation of CNVs ofgenes that are heavily connected in the interactome (81–83). Thus,there are complementary patterns of classes of genes for thosethat are selected to survive longer after WGD vs. those that cansurvive in populations as segmental or CNVs.For these relationships to occur, there must be a reasonable

correlation between gene copy number and protein level ex-pression. There are little data available on this point in multi-cellular organisms on the whole-genome level, but the work bySpringer et al. (84) examined this point in baker’s yeast. Bystudying a collection of heterozygous gene KOs, the proteinquantities were determined. In this study, only 5% of genes hadlittle to no correlation between functional gene copy number andencoded protein abundance. For 80% of genes, there was a strongcorrelation between copy number and protein quantity. The roleof protein degradation in the context of genomic balance has notbeen investigated.The retention of some classes of genes for longer evolutionary

periods than others is unlikely to be because of divergence offunction or expression in most cases. There are several reasons forthis conclusion. First, after WGDs, most duplicates eventuallybecome reduced back to the singleton state. If the duplicates haddiverged and acquired novel functions (neofunctionalization) orsplit functions (subfunctionalization) as the basis of longer re-tention, the remaining member of the pair would need to back-mutate to regain all functions, which is highly improbable. Second,as noted, the spectrum of genes generally found in segmentalduplications is complementary to the spectrum retained afterWGD. This circumstance is not predicted by the divergence hy-pothesis. Indeed, duplication by whole genome or segments wouldprovide an equal opportunity for divergence, which does not ex-plain the observed pattern (74). Despite these considerations, wedo note that a subset of duplicate genes has certainly changed or

split their functions as an important aspect of evolution whenconsidering all classes of genes (85). The longer retention of du-plicate pairs afterWGD through gene balance might allow greaterperiods of evolutionary time to provide the opportunity for gradualdivergence of dosage-sensitive genes, among them being thosegenes with critical regulatory functions. Also, over evolutionarytime, the relative constraints on duplicate genes can shift to ab-solute constraints (86).The eventual deterioration of the duplicate state of members

of macromolecular complexes can be attributed to several factorsrecognized at present. First, for several cases of allopolyploidy,there is an overall difference in gene expression contributed byone or the other genome present (87–91). When the two genomesare examined for the fraction of genes removed over evolutionarytime, the genome with lesser expression has suffered a greaternumber of deletions (87, 92–94). Deletion of a member of a bal-anced set of duplicates from the lesser expressed genome wouldbe expected to have fewer detrimental effects than deletion forthe more highly expressed genome. If there are insufficient det-rimental effects of the deletion event on reproductive fitness,there will be no selection against it. Second, to the extent thatthere is an imperfect relationship of gene copy number and theencoded protein abundance in the cell, some deletions would beof little consequence. An intertwined consideration is that de-letion of critical downstream target genes of transcription factorsand signal transduction components might eventually modulatehow the quantity of the regulatory complex is effective. Third, therole of microRNAs in affecting genomic balance is unknown butpotentially important in terms of modulating the expression oftranscription factors, because microRNAs can operate in a dos-age-sensitive manner (95). Indeed, microRNAs from duplicategenomes in the grass family are preferentially retained from bothgenomes (96, 97) after WGD events, presumably because of theirimpact on the amount of regulatory proteins. Fourth, differentsubunits of a complex might have different magnitudes of dosagesensitivity, and also, selection on one member of the complexmight affect the others (98). Modeling of the kinetics and modeof complex formation illustrates that changing the concentrationof each subunit can behave differently (57). Also, the penetranceand expressivity of variants of highly connected gene productsmight affect their evolutionary outcome.

Implications for Chromosomal-Level EvolutionGenomic balance phenomena at the chromosomal level havebeen experimentally observed in polyploid plant species. Com-mon wheat is an allopolyploid consisting of three genomes, eachcomposed of seven chromosomes for a total of 21 homolog pairs.The work by Sears (99) generated a series of monosomics withonly one copy of each homolog. In addition, because of themultiple genomes, the work by Sears (99) was also able to pro-duce nullisomics for each homolog (i.e., having no copy ofa chromosome). This condition is possible in wheat, becausethere are two other similar genomes present that provide thegenes that are missing in the nullisomic. Each nullisomic hasa characteristic detrimental phenotype. However, Sears (99)constructed plants that carried four copies of a related (home-ologous) chromosome and was able to partially correct the ab-normality associated with each nullisomic. These constructs werereferred to as compensating nullisomic–tetrasomic lines.This type of effect has been found to rebalance the genome in

newly formed cases of whole-genome duplication (100). Inresynthesized allotetraploid Brassica napus, chromosome segre-gation was not faithful in the first few generations, which pro-duced many lineages exhibiting aneuploidy. With continuationthrough additional generations, the chromosome number re-solved to the number typical of the balanced genome. However,when the chromosomes were examined using a karyotypingmethod that allows one to distinguish all chromosomes, many

Birchler and Veitia PNAS | September 11, 2012 | vol. 109 | no. 37 | 14749

GEN

ETICS

INAUGURA

LART

ICLE

lineages were found in which four chromosomes from one dip-loid progenitor were present but no chromosomes from theother; also, there were equally balanced cases of three chromo-somes of one genome and one chromosome of the other ge-nome. In other words, compensating aneuploids were resolvedthat were similar to the experimentally derived cases in wheat.A natural case of a rebalancing allopolyploid has been described

for the genus Tragopogon (101). Newly formed allotetraploids havebeen documented in recent time because of the introduction of onecontributing species to North America from Europe. Differentcases of allopolyploid formation exhibit varying numbers of chro-mosomes from one diploid progenitor or the other, but they seldomhave more than four of each chromosome, thus maintaining a ge-nomic balance. The shift in the chromosomal complement betweengenomes might have an impact on the resolution of the WGD indivergent lineages because of different genetic variants being fixedon different chromosomes. This finding illustrates how genomicbalance at this level might influence the evolutionary trajectory.

Implications for Human DiseaseAlthough the concept of gene balance has been considered asa basis of aneuploidy syndromes, dosage sensitivity of the re-sponsible genes would also impact other human medical con-ditions. There can be abnormal phenotypes that arise from theabsence of one allele at a given locus (i.e., one-half of the normalamount of gene product is not sufficient to ensure a normalphenotype). This phenomenon is called haploinsufficiency whencompatible with survival of the individual or haplolethality whenit leads to death. There is a myriad of human genetic conditionsinvolving mutations of transcription factors and signal trans-duction components that leads to haploinsufficiency (42, 43, 102).The work by Pessia et al. (103) noted that the expression of

genes encoding components of macromolecular complexes on themammalian X chromosome is similar to the expression of auto-somal genes encoding other members of the same complexes,suggesting selection to maintain their relative stoichiometry de-spite a difference in dosage between the X chromosome and theautosomes. The work by Pessia et al. (103) also suggested that Xinactivation in female mammals evolved as a mechanism tomaintain this similar relative expression. Pessia et al. (103) pos-tulated the potential contribution of dosage-sensitive genes to Xchromosomal aneuploid syndromes.The work by Berger et al. (104) summarized the evidence that

partial inactivation of tumor suppressor genes (very often by so-matic mutation) can contribute to cancer development. Theamount of a particular product of a tumor suppressor gene iscritical. Indeed, null alleles of tumor suppressor genes as hetero-zygotes can condition tumorigenesis in the context of stoichio-metric complexes. Moreover, a particular allele may have differingeffects in different backgrounds, which might be a reflection ofaltered expression of the gene of interest or the relationship of itsgene product levels to other interacting gene products in the cell.Most cancers are associated with a highly aneuploid state in-

volving many changes in the chromosomal constitution of thecells (105). Interestingly, cancerous cells are highly proliferative(106), which contrasts the cellular and organismal detrimentalaneuploid phenotypes described above. No studies have directlyaddressed the issue of stoichiometric effects in cancer cells in thecontext of regulatory balance and whether the aneuploid con-dition optimizes the dosage relationship of a subset of regulatoryfactors in a manner that would relieve the otherwise detrimentaleffects of altered segmental dosage.

Implications for Quantitative TraitsAnother tenet of the gene balance hypothesis is that quantitativetraits will be affected by many loci that exhibit dosage effects(107). The parallels between the genetic control of quantitativetraits and the effect of multiple aneuploidies on any particular

phenotypic characteristic were noted in the work by Guo andBirchler (12), and also, they are illustrated in Fig. 1. Becausemany dosage modifiers affect any one phenotypic characteristic,variation in these genes would be expected to affect quantitativetraits. The identification of the molecular basis of severalquantitative trait loci indicates that transcription factors andsignal transduction components are major contributors (108–111). Transgene-generated dosage series of such candidatesconfirm the dosage sensitivity (112). Human height illustratesthat a quantitative trait can be affected by a large number ofgenes. Genome-wide association studies have documented atleast 180 genes that can affect this trait (113). Nevertheless, thevariation in any one population is a small fraction of the meanheight. A large number of genes has also been found to affectany particular trait in experiments involving inbred lines of maizeand Drosophila that would, however, detect effects of variants inboth dosage-sensitive and -insensitive genes (114, 115).Another consequence of genomic balance is that there will be

highly multigenic subtle variation that can allow selection inmany directions using standing variation. In animal and plantbreeding, when hard selection is applied to a population, traitscan readily be shifted by subtle measures over time. The work byDarwin (116) highlighted the diversity of pigeons and dogs asexamples in which artificial selection had produced a wide vari-ety of forms within one species. An example in which the un-derlying genetics has been examined involves the Illinois highand low oil selection in maize (117). Starting with an open-pol-linated population, selection was applied for increased and de-creased oil content in the kernels. Both types of selectionrespond well and have not shown a plateau over many decades.Reversal of the direction of selection responds equally well.A determination of the genetic differences between high and lowoil lines indicated a multigenic difference of at least 50 geneswith additive effects (117).With subtle standing variation in many regulatory genes that

could impact a particular quantitative trait, the potential exists fornatural selection to change the phenotype to significant extremes,although through gradual steps. If the standing subtle variation indosage-balanced regulatory genes is neutral, the status quo will bemaintained, which as the work by Williams (118) pointed out, isa major aspect of evolution. However, if a selection pressure ona trait arises because of changing conditions and is strong enoughto overcome any detrimental aspects of a potential shift in thestiochiometries of interacting gene products, the potential forextreme changes in the phenotype in a gradual stepwise manner ispresent if the pressure continues over generations. Epistasis, inwhich one gene affects the manifestation of another, is also likelyto impact this process as well as an interaction between dosage-sensitive and qualitative variants.The evolutionary overretention of highly connected genes after

WGD and underrepresentation among CNVs in populationssuggest that a mere 25% change in quantity of gene product isusually selected against for these gene classes. Also, the fact thatCNVs in humans cause recognizable detrimental clinical con-ditions (119) illustrates that changes of gene product quantity inthis range impact the phenotype. These results have implicationsfor the fate of natural variants that do not involve gene copynumber change but alter the expression level in other ways. Ananalogy can be made to genes that mimic aneuploid syndromes,in that changes in quantity of gene products will be detrimental.Thus, mutations that change the quantity of a balanced geneproduct in this range will likely be selected against. Only moresubtle changes can remain neutral or nearly neutral. Thus, thetransacting regulatory variation affecting a particular trait is likelyto be multigenic but with each variant being of small magnitudebecause of these selective dosage constraints.Indeed, results of mutation accumulation studies conducted in

nematodes (120) and fruit flies (121) suggest constraints on

14750 | www.pnas.org/cgi/doi/10.1073/pnas.1207726109 Birchler and Veitia

regulatory modulations. In experiments in which mutations wereallowed to accumulate in lineages for many generations and thenglobal gene expression studies conducted and compared with theprogenitor state or related species, the changes in expression oflargest magnitude involved individual target genes, whereas theglobal transcriptional patterns of gene expression were more orless maintained. These studies suggest that the apparent regula-tory variation, as opposed to individual target gene expression, ismore constrained. In other studies of cis and trans variation, cisvariation for a target gene varies to greater magnitude than transregulatory variation, which is, however, multigenic if the variationis great enough to be detected (122–134). The multigenic low-magnitude transacting effects on gene expression, the mutationaccumulation results, and the finding that members of proteincomplexes that exhibit dosage effects have limited variation forma consistent set of observations suggesting that there is a gener-alized constraint on regulatory variation. These considerationsare also consistent with the phenotypic effects of aneuploidy asillustrated in Fig. 1, in which no genotype differs from the bal-anced state by more than a twofold dosage but nevertheless, cancondition rather detrimental effects. Modulations at this magni-tude, and even below, will likely be selected against because of thenegative effects of altered regulatory balance.In eukaryotic organisms with a primarily diploid phase, new

mutations that arise will be present in a heterozygous condition.In this state, they will not be subject to selection unless they havesome degree of dominance. Strictly recessive mutations are onlyavailable for selection in the homozygous state. This situation forrecessive mutations is usually restricted to small populations inwhich the new alleles can change in frequency by random drift.However, for mutations in genes involved with dosage-sensitiveinteractions, they will produce a semidominance. If the change inamount of gene product caused by the new allele is detrimental,it will be selected against. As noted above for preferentiallyretained genes from WGD, there is, indeed, evidence for puri-fying selection. However, if a change in quantity confers a re-productively adaptive state, it can spread through the populationbecause of the partial dominance.The role of epigenetic variation in dosage balance has not been

explored. Epigenetic effects can change the function of an alleleor gene without changing the nucleotide sequence (135). Epige-netically silenced alleles have been documented, and these allelescan be inherited over generations (136). Parental imprinting of

alleles, in which the history of an allele will determine whether itis expressed or not, creates a critical dosage effect for the enco-ded gene product. If the silencing mechanism is defective and theusually silent allele is expressed, detrimental effects result. Thisfact illustrates that a quantitative change in gene product oftwofold is critical. The driving evolutionary force for uniparentalexpression is likely to be a nonmutational means to modulate theamount of gene product (137).

Concluding RemarksIn the synthesis described in this article, it is postulated thatalterations of the stoichiometric balance of members of macro-molecular complexes will affect the assembly of the whole. Byextension, gene dosage balance also operates in the context ofsignal transduction (54, 111). This stoichiometric principle hasimplications for the control of gene expression and the constraintson variation for various regulatory genes. In turn, these con-sequences will affect developmental processes and thus, modulatequantitative traits, providing at least a partial explanation for theirmultigenic inheritance. The dosage effects will contribute to themolecular basis of aneuploid syndromes and the phenotypicmanifestations of CNV on the single gene level. Within pop-ulations, this principle governs the fate of natural variants that alterthe quantity of regulatory molecules as well as the actual genenumber. The evolutionary consequence of gene dosage balanceimpacts the differential retention of classes of genes depending onwhether they are duplicated by WGD or segmentally. Subtle var-iations can exist for the multitude of regulatory genes, which havethe potential to affect any one trait. With the appropriate strongselection in one direction, gradual accumulation of variants con-tributing to more phenotypic extremes than the progenitor canoccur. With the gene balance hypothesis, an initial synthesis isproposed for findings in the realm of biophysics, gene expression,chromosome biology, quantitative traits, and evolutionary biology.

ACKNOWLEDGMENTS. Maya Benavides, Zhi Gao, and Fangpu Han classifiedthe plants shown in Fig. 1 and produced the photographs. We thank PatrickEdger, Chris Pires, Bernardo Lemos, andKathleenNewton for comments. J.A.B.thanks many former associates who contributed to the cited work. Researchrelated to this topic has been supported by National Institutes of Health GrantR01GM068042. R.A.V. is supported by the Centre National de la RechercheScientifique, the University Paris VII, the Institut Universitaire de France, LaLigue National Contre le Cancer (Comité de Paris), and the Groupement d’en-treprises françaises dans la lutte contre le cancer (GEFLUC).

1. Blakeslee AF, Belling J, FarnhamME (1920) Chromosomal duplication and Mendelianphenomena in Datura mutants. Science 52:388–390.

2. Bridges CB (1925) Sex in relation to chromosomes and genes. Am Nat 59:127–137.3. Blakeslee AF (1934) New Jimson weeds from old chromosomes. J Hered 24:80–108.4. Satina S, Blakeslee AF, Avery AG (1937) Balanced and unbalanced haploids in Datura.

J Hered 28:192–202.5. Bond DJ, Chandley AC (1983) Aneuploidy (Oxford Univ Press, London).6. Lindsley DL, et al. (1972) Segmental aneuploidy and the genetic gross structure of

the Drosophila genome. Genetics 71:157–184.7. Torres EM, et al. (2007) Effects of aneuploidy on cellular physiology and cell division

in haploid yeast. Science 317:916–924.8. Weber DF (1983) Monosomic analysis in diploid crop species. Cytogenetics of Crop

Plants, eds Swaminathan MS, Gupta PK, Sinha U (Macmillan India Limited, NewDelhi), pp 351–378.

9. Lee EA, Darrah LL, Coe EH (1996) Dosage effects on morphological and quantitativetraits in maize aneuploids. Genome 39:898–908.

10. Birchler JA (1979) A study of enzyme activities in a dosage series of the long arm ofchromosome one in maize. Genetics 92:1211–1229.

11. Birchler JA, Newton KJ (1981) Modulation of protein levels in chromosomal dosageseries of maize: The biochemical basis of aneuploid syndromes. Genetics 99:247–266.

12. Guo M, Birchler JA (1994) Trans-acting dosage effects on the expression of modelgene systems in maize aneuploids. Science 266:1999–2002.

13. Birchler JA (2010) Reflections on studies of gene expression in aneuploids. Biochem J426:119–123.

14. McDaniel RG, Ramage RT (1970) Genetics of primary trisomic series in barley: Iden-tification by protein identification. Can J Genet Cytol 12:155–167.

15. Smith HH, Conklin ME (1975) Effects of gene dosage on peroxidase isozymes inDatura stramonium trisomics. Isozymes, Developmental Biology, ed Markert CL(Academic, New York), Vol 3, pp 603-618.

16. O’Brien SJ, Gethmann RC (1973) Segmental aneuploidy as a probe for structural

genes in Drosophila: Mitochondrial membrane enzymes. Genetics 75:155–167.17. Rawls JM, Jr., Lucchesi JC (1974) Regulation of enzyme activities in Drosophila. I. The

detection of regulatory loci by gene dosage responses. Genet Res 24:59–72.18. Hodgetts RB (1975) The response of dopa decarboxylase activity to variations in

gene dosage in Drosophila: A possible location of the structural gene. Genetics 79:

45–54.19. Hall JC, Kankel DR (1976) Genetics of acetylcholinesterase in Drosophila mela-

nogaster. Genetics 83:517–535.20. Pipkin SB, Chakrabartty PK, Bremner TA (1977) Location and regulation of Dro-

sophila fumarase. J Hered 68:245–252.21. Detwiler C, MacIntyre R (1978) A genetic and developmental analysis of an acid

deoxyribonuclease in Drosophila melanogaster. Biochem Genet 16:1113–1134.22. Moore GP, Sullivan DT (1978) Biochemical and genetic characterization of kynur-

enine formamidase from Drosophila melanogaster. Biochem Genet 16:619–634.23. Oliver MJ, Huber RE, Williamson JH (1978) Genetic and biochemical aspects of tre-

halase from Drosophila melanogaster. Biochem Genet 16:927–940.24. Devlin RH, Holm DG, Grigliatti TA (1988) The influence of whole-arm trisomy on

gene expression in Drosophila. Genetics 118:87–101.25. Birchler JA, Hiebert JC, Krietzman M (1989) Gene expression in adult metafemales of

Drosophila melanogaster. Genetics 122:869–879.26. Altug-Teber O, et al. (2007) Specific transcriptional changes in human fetuses with

autosomal trisomies. Cytogenet Genome Res 119:171–184.27. Prandini P, et al. (2007) Natural gene-expression variation in Down syndrome

modulates the outcome of gene-dosage imbalance. Am J Hum Genet 81:252–263.28. Huettel B, Kreil DP, Matzke M, Matzke AJM (2008) Effects of aneuploidy on genome

structure, expression, and interphase organization in Arabidopsis thaliana. PLoS

Genet 4:e1000226.

Birchler and Veitia PNAS | September 11, 2012 | vol. 109 | no. 37 | 14751

GEN

ETICS

INAUGURA

LART

ICLE

29. Aït Yahya-Graison E, et al. (2007) Classification of human chromosome 21 gene-expression variations in Down syndrome: Impact on disease phenotypes. Am J HumGenet 81:475–491.

30. Kahlem P, et al. (2004) Transcript level alterations reflect gene dosage effects acrossmultiple tissues in a mouse model of down syndrome. Genome Res 14:1258–1267.

31. Sun X, Birchler JA (2009) Interaction study of the male specific lethal (MSL) complexand trans-acting dosage effects in metafemales of Drosophila melanogaster. Cyto-genet Genome Res 124:298–311.

32. Nawata H, et al. (2011) Dysregulation of gene expression in the artificial humantrisomy cells of chromosome 8 associated with transformed cell phenotypes. PLoSOne 6:e25319.

33. Birchler JA (1981) The genetic basis of dosage compensation of alcohol de-hydrogenase-1 in maize. Genetics 97:625–637.

34. Devlin RH, Holm DG, Grigliatti TA (1982) Autosomal dosage compensation Dro-sophila melanogaster strains trisomic for the left arm of chromosome 2. Proc NatlAcad Sci USA 79:1200–1204.

35. Birchler JA, Hiebert JC, Paigen K (1990) Analysis of autosomal dosage compensationinvolving the alcohol dehydrogenase locus in Drosophila melanogaster. Genetics124:679–686.

36. Rabinow L, Nguyen-Huynh AT, Birchler JA (1991) A trans-acting regulatory gene thatinversely affects the expression of the white, brown and scarlet loci in Drosophila.Genetics 129:463–480.

37. Sabl JF, Birchler JA (1993) Dosage dependent modifiers ofwhite alleles in Drosophilamelanogaster. Genet Res 62:15–22.

38. Muller HJ (1950) Evidence of the precision of genetic adaptation. Harvey Lect 43:165–229.

39. Birchler JA (1992) Expression of cis-regulatory mutations of the white locus inmetafemales of Drosophila melanogaster. Genet Res 59:11–18.

40. Guo M, Davis D, Birchler JA (1996) Dosage effects on gene expression in a maizeploidy series. Genetics 142:1349–1355.

41. Birchler JA, Bhadra U, Bhadra MP, Auger DL (2001) Dosage-dependent gene regu-lation in multicellular eukaryotes: Implications for dosage compensation, aneuploidsyndromes, and quantitative traits. Dev Biol 234:275–288.

42. Seidman JG, Seidman C (2002) Transcription factor haploinsufficiency: When halfa loaf is not enough. J Clin Invest 109:451–455.

43. Veitia RA (2002) Exploring the etiology of haploinsufficiency. Bioessays 24:175–184.44. Veitia RA (2003) Nonlinear effects in macromolecular assembly and dosage sensi-

tivity. J Theor Biol 220:19–25.45. Kondrashov FA, Koonin EV (2004) A common framework for understanding the

origin of genetic dominance and evolutionary fates of gene duplications. TrendsGenet 20:287–290.

46. Driever W, Nüsslein-Volhard C (1988) The bicoid protein determines position in theDrosophila embryo in a concentration-dependent manner. Cell 54:95–104.

47. Sauer F, Jäckle H (1991) Concentration-dependent transcriptional activation or re-pression by Krüppel from a single binding site. Nature 353:563–566.

48. Cribbs DL, Benassayag C, Randazzo FM, Kaufman TC (1995) Levels of homeoticprotein function can determine developmental identity: Evidence from low-levelexpression of the Drosophila homeotic gene proboscipedia under Hsp70 control.EMBO J 14:767–778.

49. Schulz C, Tautz D (1994) Autonomous concentration-dependent activation and re-pression of Krüppel by hunchback in the Drosophila embryo. Development 120:3043–3049.

50. Struhl G, Struhl K, Macdonald PM (1989) The gradient morphogen bicoid is a con-centration-dependent transcriptional activator. Cell 57:1259–1273.

51. Warrior R, Levine M (1990) Dose-dependent regulation of pair-rule stripes by gapproteins and the initiation of segment polarity. Development 110:759–767.

52. Henikoff S (1996) Dosage-dependent modification of position-effect variegation inDrosophila. Bioessays 18:401–409.

53. Birchler JA, Riddle NC, Auger DL, Veitia RA (2005) Dosage balance in gene regula-tion: Biological implications. Trends Genet 21:219–226.

54. Veitia RA (2004) Gene dosage balance in cellular pathways: Implications for domi-nance and gene duplicability. Genetics 168:569–574.

55. Papp B, Pál C, Hurst LD (2003) Dosage sensitivity and the evolution of gene familiesin yeast. Nature 424:194–197.

56. Veitia RA (2009) On gene dosage balance in protein complexes: A comment onSemple JI, Vavouri T, Lehner B. A simple principle concerning the robustness ofprotein complex activity to changes in gene expression. BMC Syst Biol 3:16.

57. Veitia RA, Bottani S, Birchler JA (2008) Cellular reactions to gene dosage imbalance:Genomic, transcriptomic and proteomic effects. Trends Genet 24:390–397.

58. Liang H, Plazonic KR, Chen J, Li WH, Fernández A (2008) Protein under-wrappingcauses dosage sensitivity and decreases gene duplicability. PLoS Genet 4:e11.

59. Schuster-Böckler B, Conrad D, Bateman A (2010) Dosage sensitivity shapes the evo-lution of copy-number varied regions. PLoS One 5:e9474.

60. Lemos B, Meiklejohn CD, Hartl DL (2004) Regulatory evolution across the proteininteraction network. Nat Genet 36:1059–1060.

61. Wolfe KH, Shields DC (1997) Molecular evidence for an ancient duplication of theentire yeast genome. Nature 387:708–713.

62. Simillion C, Vandepoele K, Van Montagu MC, Zabeau M, Van de Peer Y (2002) Thehidden duplication past of Arabidopsis thaliana. Proc Natl Acad Sci USA 99:13627–13632.

63. Bowers JE, Chapman BA, Rong J, Paterson AH (2003) Unravelling angiosperm ge-nome evolution by phylogenetic analysis of chromosomal duplication events. Nature422:433–438.

64. Maere S, et al. (2005) Modeling gene and genome duplications in eukaryotes. ProcNatl Acad Sci USA 102:5454–5459.

65. Blomme T, et al. (2006) The gain and loss of genes during 600 million years of ver-tebrate evolution. Genome Biol 7:R43.

66. Aury J-M, et al. (2006) Global trends of whole-genome duplications revealed by theciliate Paramecium tetraurelia. Nature 444:171–178.

67. Makino T, McLysaght A (2010) Ohnologs in the human genome are dosage balancedand frequently associated with disease. Proc Natl Acad Sci USA 107:9270–9274.

68. Jiao Y, et al. (2011) Ancestral polyploidy in seed plants and angiosperms. Nature 473:97–100.

69. Rosado A, Raikhel NV (2010) Application of the gene dosage balance hypothesis toauxin-related ribosomal mutants in Arabidopsis. Plant Signal Behav 5:450–452.

70. Blanc G, Wolfe KH (2004) Functional divergence of duplicated genes formed bypolyploidy during Arabidopsis evolution. Plant Cell 16:1679–1691.

71. Seoighe C, Gehring C (2004) Genome duplication led to highly selective expansion ofthe Arabidopsis thaliana proteome. Trends Genet 20:461–464.

72. Freeling M, Thomas BC (2006) Gene-balanced duplications, like tetraploidy, providepredictable drive to increase morphological complexity. Genome Res 16:805–814.

73. Barker MS, et al. (2008) Multiple paleopolyploidizations during the evolution of theCompositae reveal parallel patterns of duplicate gene retention after millions ofyears. Mol Biol Evol 25:2445–2455.

74. Freeling M (2009) Bias in plant gene content following different sorts of duplication:Tandem, whole-genome, segmental, or by transposition. Annu Rev Plant Biol 60:433–453.

75. Yang L, Gaut BS (2011) Factors that contribute to variation in evolutionary rateamong Arabidopsis genes. Mol Biol Evol 28:2359–2369.

76. Davis JC, Petrov DA (2005) Do disparate mechanisms of duplication add similar genesto the genome? Trends Genet 21:548–551.

77. Hakes L, Pinney JW, Lovell SC, Oliver SG, Robertson DL (2007) All duplicates are notequal: The difference between small-scale and genome duplication. Genome Biol 8:R209.

78. Coate JE, Schlueter JA, Whaley AM, Doyle JJ (2011) Comparative evolution of pho-tosynthetic genes in response to polyploid and nonpolyploid duplication. PlantPhysiol 155:2081–2095.

79. Freeling M, et al. (2008) Many or most genes in Arabidopsis transposed after theorigin of the order Brassicales. Genome Res 18:1924–1937.

80. Rodgers-Melnick E, et al. (2012) Contrasting patterns of evolution following wholegenome versus tandem duplication events in Populus. Genome Res 22:95–105.

81. Dopman EB, Hartl DL (2007) A portrait of copy-number polymorphism in Drosophilamelanogaster. Proc Natl Acad Sci USA 104:19920–19925.

82. Korbel JO, et al. (2008) The current excitement about copy-number variation: How itrelates to gene duplications and protein families. Curr Opin Struct Biol 18:366–374.

83. Ionita-Laza I, Rogers AJ, Lange C, Raby BA, Lee C (2009) Genetic association analysisof copy-number variation (CNV) in human disease pathogenesis. Genomics 93:22–26.

84. Springer M, Weissman JS, Kirschner MW (2010) A general lack of compensation forgene dosage in yeast. Mol Syst Biol 6:368.

85. Lynch M, Conery JS (2000) The evolutionary fate and consequences of duplicategenes. Science 290:1151–1155.

86. Bekaert M, Edger PP, Pires JC, Conant GC (2011) Two-phase resolution of polyploidyin the Arabidopsis metabolic network gives rise to relative and absolute dosageconstraints. Plant Cell 23:1719–1728.

87. Thomas BC, Pedersen B, Freeling M (2006) Following tetraploidy in an Arabidopsisancestor, genes were removed preferentially from one homeolog leaving clustersenriched in dose-sensitive genes. Genome Res 16:934–946.

88. Wang J, et al. (2006) Genomewide nonadditive gene regulation in Arabidopsis al-lotetraploids. Genetics 172:507–517.

89. Flagel LE, Wendel JF (2010) Evolutionary rate variation, genomic dominance andduplicate gene expression evolution during allotetraploid cotton speciation. NewPhytol 186:184–193.

90. Buggs RJ, et al. (2010) Characterization of duplicate gene evolution in the recentnatural allopolyploid Tragopogon miscellus by next-generation sequencing andSequenom iPLEX MassARRAY genotyping. Mol Ecol 19(Suppl 1):132–146.

91. Chang PL, Dilkes BP, McMahon M, Comai L, Nuzhdin SV (2010) Homoeolog-specificretention and use in allotetraploid Arabidopsis suecica depends on parent of originand network partners. Genome Biol 11:R125.

92. Woodhouse MR, et al. (2010) Following tetraploidy in maize, a short deletionmechanism removed genes preferentially from one of the two homologs. PLoS Biol8:e1000409.

93. Schnable JC, Springer NM, Freeling M (2011) Differentiation of the maize sub-genomes by genome dominance and both ancient and ongoing gene loss. Proc NatlAcad Sci USA 108:4069–4074.

94. Buggs RJ, et al. (2012) Rapid, repeated, and clustered loss of duplicate genes in al-lopolyploid plant populations of independent origin. Curr Biol 22:248–252.

95. Guo H, Ingolia NT, Weissman JS, Bartel DP (2010) Mammalian microRNAs pre-dominantly act to decrease target mRNA levels. Nature 466:835–840.

96. Zhang L, et al. (2009) A genome-wide characterization of microRNA genes in maize.PLoS Genet 5:e1000716.

97. Abrouk M, et al. (2012) Grass microRNA gene paleohistory unveils new insights intogene dosage balance in subgenome partitioning after whole genome duplication.Plant Cell 24:1776–1792.

98. Veitia RA (2010) A generalized model of gene dosage and dominant negative ef-fects in macromolecular complexes. FASEB J 24:994–1002.

99. Sears ER (1953) Nullisomic analysis in common wheat. Am Nat 87:245–252.100. Xiong Z, Gaeta RT, Pires JC (2011) Homoeologous shuffling and chromosome com-

pensation maintain genome balance in resynthesized allopolyploid Brassica napus.Proc Natl Acad Sci USA 108:7908–7913.

14752 | www.pnas.org/cgi/doi/10.1073/pnas.1207726109 Birchler and Veitia

101. Chester M, et al. (2012) Extensive chromosomal variation generated in a recentlyformed polyploidy species, Tragopogon miscellus (Asteraceae). Proc Natl Acad SciUSA 109:1176–1181.

102. Veitia RA, Birchler JA (2010) Dominance and gene dosage balance in health anddisease: Why levels matter! J Pathol 220:174–185.

103. Pessia E, Makino T, Bailly-Bechet M, McLysaght A, Marais GA (2012) Mammalian Xchromosome inactivation evolved as a dosage-compensation mechanism for dosage-sensitive genes on the X chromosome. Proc Natl Acad Sci USA 109:5346–5351.

104. Berger AH, Knudson AG, Pandolfi PP (2011) A continuum model for tumour sup-pression. Nature 476:163–169.

105. Weaver BA, Cleveland DW (2007) Aneuploidy: Instigator and inhibitor of tumori-genesis. Cancer Res 67:10103–10105.

106. Williams BR, et al. (2008) Aneuploidy affects proliferation and spontaneous im-mortalization in mammalian cells. Science 322:703–709.

107. Tanksley SD (1993) Mapping polygenes. Annu Rev Genet 27:205–233.108. Frary A, et al. (2000) fw2.2: A quantitative trait locus key to the evolution of tomato

fruit size. Science 289:85–88.109. Liu J, Van Eck J, Cong B, Tanksley SD (2002) A new class of regulatory genes un-

derlying the cause of pear-shaped tomato fruit. Proc Natl Acad Sci USA 99:13302–13306.

110. Cong B, Liu J, Tanksley SD (2002) Natural alleles at a tomato fruit size quantitativetrait locus differ by heterochronic regulatory mutations. Proc Natl Acad Sci USA 99:13606–13611.

111. Cong B, Barrero LS, Tanksley SD (2008) Regulatory change in YABBY-like transcrip-tion factor led to evolution of extreme fruit size during tomato domestication. NatGenet 40:800–804.

112. Liu J, Cong B, Tanksley SD (2003) Generation and analysis of an artificial genedosage series in tomato to study the mechanisms by which the cloned quantitativetrait locus fw2.2 controls fruit size. Plant Physiol 132:292–299.

113. Lango Allen H, et al. (2010) Hundreds of variants clustered in genomic loci and bi-ological pathways affect human height. Nature 467:832–838.

114. Buckler ES, et al. (2009) The genetic architecture of maize flowering time. Science325:688–689.

115. Mackay TFC, et al. (2012) The Drosophila melanogaster Genetic Reference Panel.Nature 482:173–178.

116. Darwin C (1859) The Origin of Species by Means of Natural Selection (John Murray,London).

117. Laurie CC, et al. (2004) The genetic architecture of response to long-term artificialselection for oil concentration in the maize kernel. Genetics 168:2141–2155.

118. Williams GC (1966) Adaptation and Natural Selection (Princeton Univ Press,Princeton).

119. Cooper GM, et al. (2011) A copy number variation morbidity map of developmental

delay. Nat Genet 43:838–846.120. Denver DR, et al. (2005) The transcriptional consequences of mutation and natural

selection in Caenorhabditis elegans. Nat Genet 37:544–548.121. Rifkin SA, Houle D, Kim J, White KP (2005) A mutation accumulation assay reveals

a broad capacity for rapid evolution of gene expression. Nature 438:220–223.122. Schadt EE, et al. (2003) Genetics of gene expression surveyed in maize, mouse and

man. Nature 422:297–302.123. Yvert G, et al. (2003) Trans-acting regulatory variation in Saccharomyces cerevisiae

and the role of transcription factors. Nat Genet 35:57–64.124. Wittkopp PJ, Haerum BK, Clark AG (2004) Evolutionary changes in cis and trans gene

regulation. Nature 430:85–88.125. Morley M, et al. (2004) Genetic analysis of genome-wide variation in human gene

expression. Nature 430:743–747.126. Wayne ML, Pan Y-J, Nuzhdin SV, McIntyre LM (2004) Additivity and trans-acting

effects on gene expression in male Drosophila simulans. Genetics 168:1413–1420.127. Landry CR, et al. (2005) Compensatory cis-trans evolution and the dysregulation of

gene expression in interspecific hybrids of Drosophila. Genetics 171:1813–1822.128. Hughes KA, et al. (2006) Segregating variation in the transcriptome: cis regulation

and additivity of effects. Genetics 173:1347–1355.129. West MAL, et al. (2007) Global eQTL mapping reveals the complex genetic archi-

tecture of transcript-level variation in Arabidopsis. Genetics 175:1441–1450.130. Wang D, et al. (2007) Expression evolution in yeast genes of single-input modules is

mainly due to changes in trans-acting factors. Genome Res 17:1161–1169.131. Lemos B, Araripe LO, Fontanillas P, Hartl DL (2008) Dominance and the evolutionary

accumulation of cis- and trans-effects on gene expression. Proc Natl Acad Sci USA

105:14471–14476.132. Wittkopp PJ, Haerum BK, Clark AG (2008) Regulatory changes underlying expression

differences within and between Drosophila species. Nat Genet 40:346–350.133. Petretto E, et al. (2006) Heritability and tissue specificity of expression quantitative

trait loci. PLoS Genet 2:e172.134. Tirosh I, Reikhav S, Levy AA, Barkai N (2009) A yeast hybrid provides insight into the

evolution of gene expression regulation. Science 324:659–662.135. Johannes F, Colot V, Jansen RC (2008) Epigenome dynamics: A quantitative genetics

perspective. Nat Rev Genet 9:883–890.136. Schmitz RJ, et al. (2011) Transgenerational epigenetic instability is a source of novel

methylation variants. Science 334:369–373.137. Beaudet AL, Jiang Y-H (2002) A rheostat model for a rapid and reversible form of

imprinting-dependent evolution. Am J Hum Genet 70:1389–1397.

Birchler and Veitia PNAS | September 11, 2012 | vol. 109 | no. 37 | 14753

GEN

ETICS

INAUGURA

LART

ICLE