Gene Balance Hypothesis: Connecting Issues of Dosage Sensitivity Across Biological Disciplines

Gene balance hypothesis: Connecting issues of dosage sensitivity across biological disciplines

James 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 balance about two-thirds of the amount in the balanced diploid with two influences gene expression, quantitative traits, dosage compensa- copies of all chromosomal regions (Fig. 2). The amount of any one tion, aneuploid syndromes, population dynamics of copy number target gene product could be modulated by multiple regions of the variants and differential evolutionary fate of genes after partial or genome. This effect is quite common in the data for trisomic seg- whole-genome duplication. Gene balance effects are hypothesized ments in other species. including Datura, barley, Arabidopsis, Dro- to result from stoichiometric differences among members of macro- sophila, and human trisomic cells (13–32). The ability to recover molecular complexes, the interactome, and signaling pathways. The corresponding monosomics in maize facilitated the finding that this implications of gene balance are discussed. effect can be proportional through one, two, and three doses. Furthermore, the combination of a region of the genome that he concept of gene dosage balance arose in the early days of the produces an inverse dosage effect on a particular gene product to- Tfield of genetics, with the work by Blakeslee et al. (1) using the gether with the gene encoding that product could result in dosage flowering plant Datura stramonium and the work by Bridges (2) using compensation (Fig. 2). In this case, despite the fact that the gene was the fruit fly Drosophila melanogaster. Both studies found that the varied in one to three copies, the total output realized was more or addition of a single chromosome to a genotype was highly detri- less constant in the chromosome arm dosage series (10, 11). By di- mental or lethal, whereas the addition of a complete genome to viding the varied chromosome arm into smaller segments and make a polyploid was viable and resulted in lesser effects on the assaying gene expression, the basis of this type of dosage compen- phenotype. The work on Datura was greatly extended to include four sation was shown to involve a combination of an inverse dosage copies of each chromosome arm and multiple whole genomes (3, 4). effect cancelling a structural gene dosage effect (33). Specifically, the Subsequently, related principles governing aneuploidy, with some structural gene locus in a smaller segment exhibited a proportional differences, were found throughout eukaryotes (5–7). More recently, dosage effect, and another region of the larger aneuploid produced the removal of chromosomes to produce monosomic genotypes an inverse dosage effect. Similar cases of dosage compensation were shows that there are quite severe phenotypic effects compared with found for chromosome arm trisomics in Drosophila in the work by the haploid, which has only one copy of every chromosome (8, 9). Devlin et al. (34). The work by Birchler et al. (35) showed in Dro- This principle is illustrated in Fig. 1. Maize genotypes were pro- sophila that the basis of this effect also involved the combination of duced that were diploid with one, two, or three doses of represen- a structural gene dosage effect and an inverse effect operating si- tative chromosome arms as well as haploids plus the chromosome multaneously, and it documented the effect on the mRNA level (35). arm in question all in a highly similar genetic background. For all Because an inverse effect may regularly occur on variation of many three chromosome arms examined, it was clear that addition of genomic segments, dosage compensation can also regularly occur a chromosome to the diploid has much less severe effect than ad- when a regulator and a target gene are varied together. The inverse dition of a chromosome to a haploid. However, the removal of one effect can cause compensation of different target genes regardless of of two chromosome arms is also more severe than the reduction in the level of activity, because the effect involves a relative stoichio- ploidy from diploid to haploid. The doubling of a mere chromosome metric relationship of the varied segment to the remainder of the arm in a haploid leads to a deformity, whereas doubling the whole genome, which will be the same for all target genes. genome leads to a normal diploid. These comparisons illustrate that The work by Rabinow et al. (36) tested whether the aneuploid the relative dosage of chromosomal segments is critical for normal effects on gene expression could be reduced to the action of development and phenotypic characteristics. single genes. Various leaky alleles of the white eye color gene in Extending this relationship to the molecular level, the studies Drosophila were used as phenotypic reporters to screen for sec- by Birchler (10) and Birchler and Newton (11) found that ex- ond site modifiers that, when heterozygous, would increase or pression patterns of enzyme and protein levels were modulated decrease the expression within a twofold range. The first such more in aneuploid genotypes than with changes in ploidy. The mutation found increased the expression of the white eye color work by Birchler and Newton (11) suggested that a perturbation gene approximately twofold as a heterozygote and thus, would of stoichiometric relationships of regulatory gene products was mimic a monosomic situation on the single gene level. When a contributor to the effects of genomic imbalance and played a small aneuploid region around this gene was present in three a role in these transacting dosage effects on gene expression. The modulations are both positive and negative depending on copies, the expression of white was reduced to about two-thirds of the gene, tissue, or chromosomal region studied (11, 12). Although the normal diploid. In contrast, the eye color of diploid and triploid flies was similar. The introduction of these mutations into most modulations are within direct or inverse limits between the ∼ expression level and the chromosomal dose (Fig. 2), some greater a triploid background causes an 50% increase in expression, modulations occur. A common dosage effect on gene expression thus conforming to an inverse relationship for genomic balance. found in aneuploids was an inverse correlation of gene product with the copy number of an unlinked chromosomal region (i.e., an inverse dosage effect) (10–12). In other words, monosomics for Author contributions: J.A.B. and R.A.V. wrote the paper. a particular chromosome arm would modulate the expression of The authors declare no conflict of interest. unlinked genes to an upper limit of approximately twofold, whereas Freely available online through the PNAS open access option. the corresponding trisomic would reduce the same gene product to 1To whom correspondence should be addressed. E-mail: [email protected].

14746–14753 | PNAS | September 11, 2012 | vol. 109 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1207726109 Downloaded by guest on September 28, 2021 The effect was ﬁrst found on the phenotypic level and then

documented in some developmental stages on the mRNA level. INAUGURAL ARTICLE The work by Sabl and Birchler (37) surveyed the autosomes of Drosophila for phenotypic dosage-sensitive modifiers of the white eye color gene. Earlier, in studies of X chromosome dosage compensation, the work by Muller (38) described evidence for multiple X-linked dosage-sensitive modifiers of white. In these studies, segments of the genome were found that modulated the expression of white either positively or negatively and illustrated that multiple aneuploid regions could affect the phenotype of a single trait. More regions were found to modulate white negatively compared with those that positively affect white. The work by Birchler (39) found that the dosage of the whole X chromosome produced an inverse dosage effect on the phenotypic expression of white when it was deleted from its normal location on the X chromosome and was present as a transgene on the autosomes, although the up-regulation in normal males with one X chromosome was not a full twofold effect. These phenotypic studies also illustrate the multigenic nature of these modulating effects. However, variation of the whole genome in dosage produces a very similar phenotype in diploid and triploid flies (36), exhibiting little modulation and a proportional expression with ploidy. The work by Guo and Birchler (12) documented aneuploid modulations of gene expression on the RNA level in maize and found that, in general, the magnitude of modulation was directly or

inversely correlated with the degree of genomic imbalance. The GENETICS work by Guo and Birchler (12) also noted the parallels between the multigenic additive control of quantitative phenotypic traits and the impact of multiple aneuploidies on the same quantitative characteristics. By comparing the effects in the diploid embryo of the maize kernel with the triploid endosperm, it was again found that the magnitude of modulations depended on the deviation of the chromosomal change from the balanced euploid. RNA measurements comparing whole-genome ploidy changes show fewer modulations (40). The work by Birchler et al. (41) summarized the molecular nature of the known single gene dosage-sensitive modiﬁers of the white eye color gene in Drosophila that had been recovered over the course of two decades. The collection consisted of transcription factors, chromatin components, and members of signal transduction systems. Several other laboratories noted that transcription factors, tumor suppressor genes, and components of signal transduction are dosage-sensitive (42–45). This realization was consistent with the fact that many transcription factors control developmental decisions in Drosophila in a concentration-dependent manner (46–51), and the myriad of modiﬁers of position effect variegation are dosage-sensitive (52). Dosage sensitivity 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 as the change that occurs with changing the dosage of the most controlling step of biochemical pathways (53). However, there are several theoretical and experimental results that suggest an involvement of relative stoichiometric relationships for many dosage effects. The studies by Veitia (43, 44, 54) modeled the kinetics of assembly of macromolecular complexes to explain

Fig. 1. Effect of genomic imbalance on quantitative phenotypic charac- arms. The addition of a chromosome arm to a haploid plant produces teristics in maize. (A) From left to right, this series of plants is haploid, a much greater impact on the phenotype than adding the same arm to an haploid plus the long arm of chromosome 1 (1L), one copy of 1L, two copies otherwise diploid plant, illustrating the concept of genomic balance. Dosage of 1L, and three copies of 1L in an otherwise diploid background. (B and C) manipulation of chromosome arms is made using translocations with the Analogous genotypes for the short arm of chromosomes 5 (5S) and 9 (9S), supernumerary B chromosome (10, 11), and haploids are produced using an respectively. A meter stick is included for scale. These examples include one inducer line (12). The dosage and ploidy were determined by metaphase of the longer (1L) and one of the shorter (9S) chromosome arms in the maize chromosome spreads of root tips, and then, the classiﬁed seedlings were genome and illustrate that similar imbalance phenomena occur for all tested grown into plants in the greenhouse. Photographs by Fangpu Han.

Birchler and Veitia PNAS | September 11, 2012 | vol. 109 | no. 37 | 14747 Downloaded by guest on September 28, 2021 because such interactions stabilize the relevant proteins. There is an overall negative correlation between the degree of underwrapping and the ability to survive as a gene duplicate in a wide range of taxa from bacteria to humans. This result suggests that, with a greater number of protein–protein interactions involved with macromolecular complexes, there are increasing negative fitness consequences of single gene duplication, which manifests as a stoichiometric imbalance. The work by Schuster-Böckler et al. (59) examined this issue in a different manner. Using a protein domain database, a negative correlation was found between the number of protein–protein interaction domains that were present in a protein and its ability to be maintained as a copy number variant (CNV) in human populations. Also, the dosage-sensitive genes exhibited less expression variation among tissues and among individuals for the same tissue. These results also support the hypothesis that changing the stoichiometry of components of macromolecular complexes or the interactome will produce a dominant phenotypic effect that affects fitness. The work by Lemos et al. (60) showed that the number of Fig. 2. Diagrammatic representation of the types of effects observed for interactions of a protein (i.e., its connectivity) constrains genetic gene expression in aneuploids. The x axis depicts the chromosomal dosage. variation of its expression in yeast and fruit fly populations. Namely, The y axis depicts the percentage of expression in the aneuploid compared they reported a negative correlation between the variation of gene with the diploid. (A) A gene dosage effect occurs when the structural gene – produces a proportional amount of product to its copy number. (B) There are expression and the number of protein protein interactions. More- also direct transacting effects, in which a gene is modulated in expression in over, the degree of expression variation among genes encoding direct correlation with a different chromosomal dosage. (C) Another trans- interactors was smaller than the degree of expression variation of acting modulation is the inverse dosage effect, in which the expression of random gene pairs. Finally, the levels of expression of interactors a target gene is inversely correlated with the dosage of another chromo- displayed a positive correlation across strains. somal region. (D) Dosage compensation occurs when the expression of a structural gene does not change with its dosage. Dosage compensation Gene Balance Hypothesis results when an inverse dosage effect of an aneuploid region includes, The experimental and theoretical findings involved with gene bal- among its target genes, those genes that are also on the varied chromosome. The two effects, structural gene and inverse, combined together ance that are described above were formulated from phenotypic cancel to produce nearly equal expression in all chromosomal doses. Modi- data, gene expression patterns in dosage manipulations, and iden- fied from Birchler (13). tification of their underlying basis in dosage-sensitive regulatory genes. Connections were then made to interacting members of multisubunit complexes and their kinetics and mode of assembly. how changes in stoichiometry of the component members could The principle can also be stated in reverse. The stoichiometry of produce a dominant phenotypic effect (43). In the context of a members of multisubunit complexes can affect the amount of multisubunited complex, changing the amount of one subunit can functional complete product, which in turn, affects patterns of gene shift the reaction to unproductive subcomplexes and produce a expression (if the complex is regulatory) and ultimately, the different amount of the complete complex (Fig. 3). The work by Papp et al. (55) examined heterozygous gene KOs in yeast and found a negative correlation between the involvement in macromolecular complexes and fitness. Increased gene copy number to the genome produced a similar effect, and selected co–up-regulation of 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 assembly follows a random pathway, allowing the formation of intermediate species AB and BA before assembly of the complete A-B-A trimer, a decrease in the concentration of A can lead to a reduction of ABA yield. This result is the case, because at some point in the assembly reaction, A will become limiting during the production of AB and BA that will not yield trimers (Fig. 3). Subunit B will be limiting at low concentration but in excess, will cause a reduction in the ABA yield (43). Examples of this relationship have been noted (56). Thus, both positive and negative effects on the ABA yield will depend on the relative amount of B to A (57). This example illustrates that the relative amount of subunits entering the assembly reaction of a complex can affect the amount of functional product, which can have Fig. 3. Heuristic examples of stoichiometric imbalance in the context of downstream genetic consequences. a trimer A-B-A. For simplicity, we consider that the assembly of ABA is ran- The work by Liang et al. (58) compared the degree of protein dom and irreversible. (A) Normal condition with a particular stoichiometric balance between the molar amounts of A and B. (B) Halving the amount of underwrapping to the occurrence of gene duplicability across monomer A leads to a decrease of trimer yield, because there is not enough taxa. Protein underwrapping is the property of proteins to be of A to complete the reaction. (C) Increasing the relative amount of B leads penetrated by water molecules. With increasing protein–protein to a decrease of ABA for the same reasons. The molar ratio of each condition interactions, a greater level of underwrapping can be tolerated, is indicated.

14748 | www.pnas.org/cgi/doi/10.1073/pnas.1207726109 Birchler and Veitia Downloaded by guest on September 28, 2021 phenotype and evolutionary ﬁtness. Clearly, there are complicated split their functions as an important aspect of evolution when

processes involved in these steps that will impact the outcome, but considering all classes of genes (85). The longer retention of du- INAUGURAL ARTICLE the bulk of the data suggests this generalization, which has impli- plicate pairs after WGD through gene balance might allow greater cations as outlined below. periods of evolutionary time to provide the opportunity for gradual divergence of dosage-sensitive genes, among them being those Implications for Evolutionary Genomics genes with critical regulatory functions. Also, over evolutionary From a different perspective, the field of evolutionary genomics time, the relative constraints on duplicate genes can shift to ab- came to the realization that sequenced genomes, such as yeast and solute constraints (86). Arabidopsis, revealed the remnants of ancient whole-genome The eventual deterioration of the duplicate state of members duplications (WGD) (61–68). After these tetraploidization events, of macromolecular complexes can be attributed to several factors many genes were lost in a return to diploidy but in a nonrandom recognized at present. First, for several cases of allopolyploidy, fashion depending on function. The classes of genes remaining for there is an overall difference in gene expression contributed by longer periods from the WGD were revealed to be those classes one or the other genome present (87–91). When the two genomes involved with macromolecular complexes in general, which include are examined for the fraction of genes removed over evolutionary the ribosome (69) and proteasome and of note for the discussion time, the genome with lesser expression has suffered a greater here, transcription factors and components of signal transduction number of deletions (87, 92–94). Deletion of a member of a bal- pathways (64, 70–74). These classes of genes were similar to the anced set of duplicates from the lesser expressed genome would dosage-sensitive modifiers of the white eye color gene in Drosophila be expected to have fewer detrimental effects than deletion for (41). The implication is that deletion of one member of a duplicate the more highly expressed genome. If there are insufficient det- pair of these classes would have a negative fitness effect and be rimental effects of the deletion event on reproductive fitness, selected against. Thus, the duplicate pair would be retained for there will be no selection against it. Second, to the extent that longer periods of evolutionary time than other gene classes. The there is an imperfect relationship of gene copy number and the retained genes, in fact, show evidence of purifying selection in both encoded protein abundance in the cell, some deletions would be duplicates (66, 75), which is consistent with a selection of main- of little consequence. An intertwined consideration is that de- tenance of the stoichiometric relationship. The studies by Birchler letion of critical downstream target genes of transcription factors et al. (53) and Freeling and Thomas (72) noted the relationship of and signal transduction components might eventually modulate

these evolutionary results to classical studies of genomic balance, how the quantity of the regulatory complex is effective. Third, the GENETICS in that variation of part of the genome is more detrimental than role of microRNAs in affecting genomic balance is unknown but variation of the whole genome. potentially important in terms of modulating the expression of A corollary of this concept is that segmental genomic dupli- transcription factors, because microRNAs can operate in a dos- cations that include genes encoding members of macromolecular age-sensitive manner (95). Indeed, microRNAs from duplicate complexes would be underrepresented in genomes, because they genomes in the grass family are preferentially retained from both would alter the stoichiometry of interacting genes. Studies in genomes (96, 97) after WGD events, presumably because of their yeast, Arabidopsis, rice, poplar, and mammals (64, 67, 76–80) re- impact on the amount of regulatory proteins. Fourth, different veal that this is the case. Furthermore, the study of copy number subunits of a complex might have different magnitudes of dosage polymorphisms indicates an underrepresentation of CNVs of sensitivity, and also, selection on one member of the complex genes that are heavily connected in the interactome (81–83). Thus, might affect the others (98). Modeling of the kinetics and mode there are complementary patterns of classes of genes for those of complex formation illustrates that changing the concentration that are selected to survive longer after WGD vs. those that can of each subunit can behave differently (57). Also, the penetrance survive in populations as segmental or CNVs. and expressivity of variants of highly connected gene products For these relationships to occur, there must be a reasonable might affect their evolutionary outcome. correlation between gene copy number and protein level expression. There are little data available on this point in multi- Implications for Chromosomal-Level Evolution cellular organisms on the whole-genome level, but the work by Genomic balance phenomena at the chromosomal level have Springer et al. (84) examined this point in baker’s yeast. By been experimentally observed in polyploid plant species. Com- studying a collection of heterozygous gene KOs, the protein mon wheat is an allopolyploid consisting of three genomes, each quantities were determined. In this study, only 5% of genes had composed of seven chromosomes for a total of 21 homolog pairs. little to no correlation between functional gene copy number and The work by Sears (99) generated a series of monosomics with encoded protein abundance. For 80% of genes, there was a strong only one copy of each homolog. In addition, because of the correlation between copy number and protein quantity. The role multiple genomes, the work by Sears (99) was also able to pro- of protein degradation in the context of genomic balance has not duce nullisomics for each homolog (i.e., having no copy of been investigated. a chromosome). This condition is possible in wheat, because The retention of some classes of genes for longer evolutionary there are two other similar genomes present that provide the periods than others is unlikely to be because of divergence of genes that are missing in the nullisomic. Each nullisomic has function or expression in most cases. There are several reasons for a characteristic detrimental phenotype. However, Sears (99) this conclusion. First, after WGDs, most duplicates eventually constructed plants that carried four copies of a related (home- become reduced back to the singleton state. If the duplicates had ologous) chromosome and was able to partially correct the ab- diverged and acquired novel functions (neofunctionalization) or normality associated with each nullisomic. These constructs were split functions (subfunctionalization) as the basis of longer re- referred to as compensating nullisomic–tetrasomic lines. tention, the remaining member of the pair would need to back- This type of effect has been found to rebalance the genome in mutate to regain all functions, which is highly improbable. Second, newly formed cases of whole-genome duplication (100). In as noted, the spectrum of genes generally found in segmental resynthesized allotetraploid Brassica napus, chromosome segre- duplications is complementary to the spectrum retained after gation was not faithful in the ﬁrst few generations, which pro- WGD. This circumstance is not predicted by the divergence hy- duced many lineages exhibiting aneuploidy. With continuation pothesis. Indeed, duplication by whole genome or segments would through additional generations, the chromosome number re- provide an equal opportunity for divergence, which does not ex- solved to the number typical of the balanced genome. However, plain the observed pattern (74). Despite these considerations, we when the chromosomes were examined using a karyotyping do note that a subset of duplicate genes has certainly changed or method that allows one to distinguish all chromosomes, many

Birchler and Veitia PNAS | September 11, 2012 | vol. 109 | no. 37 | 14749 Downloaded by guest on September 28, 2021 lineages were found in which four chromosomes from one dip- phenotypic characteristic were noted in the work by Guo and loid progenitor were present but no chromosomes from the Birchler (12), and also, they are illustrated in Fig. 1. Because other; also, there were equally balanced cases of three chromo- many dosage modifiers affect any one phenotypic characteristic, somes of one genome and one chromosome of the other ge- variation in these genes would be expected to affect quantitative nome. In other words, compensating aneuploids were resolved traits. The identification of the molecular basis of several that were similar to the experimentally derived cases in wheat. quantitative trait loci indicates that transcription factors and A natural case of a rebalancing allopolyploid has been described signal transduction components are major contributors (108– for the genus Tragopogon (101). Newly formed allotetraploids have 111). Transgene-generated dosage series of such candidates been documented in recent time because of the introduction of one confirm the dosage sensitivity (112). Human height illustrates contributing species to North America from Europe. Different that a quantitative trait can be affected by a large number of cases of allopolyploid formation exhibit varying numbers of chro- genes. Genome-wide association studies have documented at mosomes from one diploid progenitor or the other, but they seldom least 180 genes that can affect this trait (113). Nevertheless, the have more than four of each chromosome, thus maintaining a ge- variation in any one population is a small fraction of the mean nomic balance. The shift in the chromosomal complement between height. A large number of genes has also been found to affect genomes might have an impact on the resolution of the WGD in any particular trait in experiments involving inbred lines of maize divergent lineages because of different genetic variants being fixed and Drosophila that would, however, detect effects of variants in on different chromosomes. This finding illustrates how genomic both dosage-sensitive and -insensitive genes (114, 115). balance at this level might influence the evolutionary trajectory. Another consequence of genomic balance is that there will be highly multigenic subtle variation that can allow selection in Implications for Human Disease many directions using standing variation. In animal and plant Although the concept of gene balance has been considered as breeding, when hard selection is applied to a population, traits a basis of aneuploidy syndromes, dosage sensitivity of the re- can readily be shifted by subtle measures over time. The work by sponsible genes would also impact other human medical con- Darwin (116) highlighted the diversity of pigeons and dogs as ditions. There can be abnormal phenotypes that arise from the examples in which artificial selection had produced a wide vari- absence of one allele at a given locus (i.e., one-half of the normal ety of forms within one species. An example in which the un- amount of gene product is not sufficient to ensure a normal derlying genetics has been examined involves the Illinois high phenotype). This phenomenon is called haploinsufficiency when and low oil selection in maize (117). Starting with an open-pol- compatible with survival of the individual or haplolethality when linated population, selection was applied for increased and de- it leads to death. There is a myriad of human genetic conditions creased oil content in the kernels. Both types of selection involving mutations of transcription factors and signal trans- respond well and have not shown a plateau over many decades. duction components that leads to haploinsufficiency (42, 43, 102). Reversal of the direction of selection responds equally well. The work by Pessia et al. (103) noted that the expression of A determination of the genetic differences between high and low genes encoding components of macromolecular complexes on the oil lines indicated a multigenic difference of at least 50 genes mammalian X chromosome is similar to the expression of auto- with additive effects (117). somal genes encoding other members of the same complexes, With subtle standing variation in many regulatory genes that suggesting selection to maintain their relative stoichiometry de- could impact a particular quantitative trait, the potential exists for spite a difference in dosage between the X chromosome and the natural selection to change the phenotype to significant extremes, autosomes. The work by Pessia et al. (103) also suggested that X although through gradual steps. If the standing subtle variation in inactivation in female mammals evolved as a mechanism to dosage-balanced regulatory genes is neutral, the status quo will be maintain this similar relative expression. Pessia et al. (103) pos- maintained, which as the work by Williams (118) pointed out, is tulated the potential contribution of dosage-sensitive genes to X a major aspect of evolution. However, if a selection pressure on chromosomal aneuploid syndromes. a trait arises because of changing conditions and is strong enough The work by Berger et al. (104) summarized the evidence that to overcome any detrimental aspects of a potential shift in the partial inactivation of tumor suppressor genes (very often by so- stiochiometries of interacting gene products, the potential for matic mutation) can contribute to cancer development. The extreme changes in the phenotype in a gradual stepwise manner is amount of a particular product of a tumor suppressor gene is present if the pressure continues over generations. Epistasis, in critical. Indeed, null alleles of tumor suppressor genes as hetero- which one gene affects the manifestation of another, is also likely zygotes can condition tumorigenesis in the context of stoichio- to impact this process as well as an interaction between dosage- metric complexes. Moreover, a particular allele may have differing sensitive and qualitative variants. effects in different backgrounds, which might be a reflection of The evolutionary overretention of highly connected genes after altered expression of the gene of interest or the relationship of its WGD and underrepresentation among CNVs in populations gene product levels to other interacting gene products in the cell. suggest that a mere 25% change in quantity of gene product is Most cancers are associated with a highly aneuploid state in- usually selected against for these gene classes. Also, the fact that volving many changes in the chromosomal constitution of the CNVs in humans cause recognizable detrimental clinical con- cells (105). Interestingly, cancerous cells are highly proliferative ditions (119) illustrates that changes of gene product quantity in (106), which contrasts the cellular and organismal detrimental this range impact the phenotype. These results have implications aneuploid phenotypes described above. No studies have directly for the fate of natural variants that do not involve gene copy addressed the issue of stoichiometric effects in cancer cells in the number change but alter the expression level in other ways. An context of regulatory balance and whether the aneuploid con- analogy can be made to genes that mimic aneuploid syndromes, dition optimizes the dosage relationship of a subset of regulatory in that changes in quantity of gene products will be detrimental. factors in a manner that would relieve the otherwise detrimental Thus, mutations that change the quantity of a balanced gene effects of altered segmental dosage. product in this range will likely be selected against. Only more subtle changes can remain neutral or nearly neutral. Thus, the Implications for Quantitative Traits transacting regulatory variation affecting a particular trait is likely Another tenet of the gene balance hypothesis is that quantitative to be multigenic but with each variant being of small magnitude traits will be affected by many loci that exhibit dosage effects because of these selective dosage constraints. (107). The parallels between the genetic control of quantitative Indeed, results of mutation accumulation studies conducted in traits and the effect of multiple aneuploidies on any particular nematodes (120) and fruit flies (121) suggest constraints on

14750 | www.pnas.org/cgi/doi/10.1073/pnas.1207726109 Birchler and Veitia Downloaded by guest on September 28, 2021 regulatory modulations. In experiments in which mutations were alleles, in which the history of an allele will determine whether it

allowed to accumulate in lineages for many generations and then is expressed or not, creates a critical dosage effect for the enco- INAUGURAL ARTICLE global gene expression studies conducted and compared with the ded gene product. If the silencing mechanism is defective and the progenitor state or related species, the changes in expression of usually silent allele is expressed, detrimental effects result. This largest magnitude involved individual target genes, whereas the fact illustrates that a quantitative change in gene product of global transcriptional patterns of gene expression were more or twofold is critical. The driving evolutionary force for uniparental less maintained. These studies suggest that the apparent regula- expression is likely to be a nonmutational means to modulate the tory variation, as opposed to individual target gene expression, is amount of gene product (137). more constrained. In other studies of cis and trans variation, cis variation for a target gene varies to greater magnitude than trans Concluding Remarks regulatory variation, which is, however, multigenic if the variation In the synthesis described in this article, it is postulated that is great enough to be detected (122–134). The multigenic low- alterations of the stoichiometric balance of members of macro- magnitude transacting effects on gene expression, the mutation molecular complexes will affect the assembly of the whole. By accumulation results, and the ﬁnding that members of protein extension, gene dosage balance also operates in the context of complexes that exhibit dosage effects have limited variation form signal transduction (54, 111). This stoichiometric principle has a consistent set of observations suggesting that there is a gener- implications for the control of gene expression and the constraints alized constraint on regulatory variation. These considerations on variation for various regulatory genes. In turn, these con- are also consistent with the phenotypic effects of aneuploidy as sequences will affect developmental processes and thus, modulate illustrated in Fig. 1, in which no genotype differs from the bal- quantitative traits, providing at least a partial explanation for their anced state by more than a twofold dosage but nevertheless, can multigenic inheritance. The dosage effects will contribute to the condition rather detrimental effects. Modulations at this magni- molecular basis of aneuploid syndromes and the phenotypic tude, and even below, will likely be selected against because of the manifestations of CNV on the single gene level. Within pop- negative effects of altered regulatory balance. ulations, this principle governs the fate of natural variants that alter In eukaryotic organisms with a primarily diploid phase, new the quantity of regulatory molecules as well as the actual gene mutations that arise will be present in a heterozygous condition. number. The evolutionary consequence of gene dosage balance In this state, they will not be subject to selection unless they have impacts the differential retention of classes of genes depending on

some degree of dominance. Strictly recessive mutations are only GENETICS whether they are duplicated by WGD or segmentally. Subtle var- available for selection in the homozygous state. This situation for recessive mutations is usually restricted to small populations in iations can exist for the multitude of regulatory genes, which have which the new alleles can change in frequency by random drift. the potential to affect any one trait. With the appropriate strong However, for mutations in genes involved with dosage-sensitive selection in one direction, gradual accumulation of variants con- interactions, they will produce a semidominance. If the change in tributing to more phenotypic extremes than the progenitor can amount of gene product caused by the new allele is detrimental, occur. With the gene balance hypothesis, an initial synthesis is fi it will be selected against. As noted above for preferentially proposed for ndings in the realm of biophysics, gene expression, retained genes from WGD, there is, indeed, evidence for puri- chromosome biology, quantitative traits, and evolutionary biology. fying selection. However, if a change in quantity confers a re- ACKNOWLEDGMENTS. Maya Benavides, Zhi Gao, and Fangpu Han classified productively adaptive state, it can spread through the population the plants shown in Fig. 1 and produced the photographs. We thank Patrick because of the partial dominance. Edger, Chris Pires, Bernardo Lemos, and Kathleen Newton for comments. J.A.B. The role of epigenetic variation in dosage balance has not been thanks many former associates who contributed to the cited work. Research explored. Epigenetic effects can change the function of an allele related to this topic has been supported by National Institutes of Health Grant R01GM068042. R.A.V. is supported by the Centre National de la Recherche or gene without changing the nucleotide sequence (135). Epige- Scientifique, the University Paris VII, the Institut Universitaire de France, La netically silenced alleles have been documented, and these alleles Ligue National Contre le Cancer (Comité de Paris), and the Groupement d’en- can be inherited over generations (136). Parental imprinting of treprises françaises dans la lutte contre le cancer (GEFLUC).

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