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. 2012 Jul 2;109(29):11752–11757. doi: 10.1073/pnas.1201816109

Expression reduction in mammalian X chromosome evolution refutes Ohno’s hypothesis of dosage compensation

Fangqin Lin a, Ke Xing a, Jianzhi Zhang b,1, Xionglei He a,1
PMCID: PMC3406839  PMID: 22753487

Abstract

Susumu Ohno proposed in 1967 that, during the origin of mammalian sex chromosomes from a pair of autosomes, per-allele expression levels of X-linked genes were doubled to compensate for the degeneration of their Y homologs. This conjecture forms the foundation of the current evolutionary model of sex chromosome dosage compensation, but has been tested in mammals only indirectly via a comparison of expression levels between X-linked and autosomal genes in the same genome. The test results have been controversial, because examinations of different gene sets led to different conclusions that either support or refute Ohno’s hypothesis. Here we resolve this uncertainty by directly comparing mammalian X-linked genes with their one-to-one orthologs in species that diverged before the origin of the mammalian sex chromosomes. Analyses of RNA sequencing data and proteomic data provide unambiguous evidence for expression halving (i.e., no change in per-allele expression level) of X-linked genes during evolution, with the exception of only ∼5% of genes that encode members of large protein complexes. We conclude that Ohno’s hypothesis is rejected for the vast majority of genes, reopening the search for the evolutionary force driving the origin of chromosome-wide X inactivation in female mammals.

The X and Y chromosomes of therian (placental and marsupial) mammals originated ∼170 million years ago from a pair of autosomes (1). Evolutionary degeneration of the Y (2) halved the dose of most X-linked genes that had been present in two copies, resulting in chromosome-wide X “monosomy” in males—a phenomenon that is usually fatal when it happens to autosomes. To explain how mammalian cells deal with X monosomy, Susumu Ohno, a pioneer in the study of sex chromosome evolution, suggested that the per-allele expression levels of X-lined genes must have been doubled (3). This presumed twofold up-regulation of all X-linked genes, however, would create the equivalent of X-“tetrasomy” in females, which is believed to have driven the evolution of the inactivation of one X in females, thus equalizing the expression levels between sexes. Ohno’s hypothesis of a twofold up-regulation of X-linked genes forms the basis of the current evolutionary model of sex chromosome dosage compensation and has directed the research in this field for more than four decades (4–8). However, because of the unavailability of the ancestral proto-X chromosome (denoted as X), Ohno’s hypothesis is commonly tested indirectly by estimating the expression ratio between the present-day X and the present-day autosomes (i.e., X:AA) (9–12), under two assumptions (13): (i) gene expressions are comparable between X and proto-autosomes (A), the progenitors of present-day therian autosomes (i.e., XX:AA ∼1); and (ii) gene expressions are comparable between A and A (i.e., AA:AA ∼1). Ohno’s hypothesis of X:XX ∼1 would be supported by an observation of X:AA ∼1.

Several groups have reported similar microarray gene expression levels between one active X and two autosomes in a variety of mammalian tissues (X:AA ∼1) (9–11), supporting Ohno’s hypothesis. However, RNA sequencing (RNA-Seq) revealed an X:AA expression ratio of ∼0.5, refuting Ohno’s hypothesis (12). A number of authors subsequently noted that the fraction of inactive genes is greater in X than in autosomes and that X:AA ∼1 when only active genes are considered (14–17). We suggested that the extra inactive genes in X originated from the most weakly expressed genes in X as a consequence of Y degeneration, and that X:AA ∼0.5 when appropriate sets of active genes are compared (13). Nevertheless, all of the above are indirect tests of Ohno’s hypothesis and are thus inconclusive (13, 18). Fortunately, this problem can be circumvented by directly comparing therian X-linked genes with their orthologs in close outgroup species that diverged from therians before the origin of X (1). Taking advantage of a recently published RNA-Seq dataset (19) that contains six organs from each of chicken, platypus, opossum, mouse, and human, we here trace the evolution of gene expression levels of orthologous genes in these five species. Our analysis provides direct evidence for expression reduction of X-linked genes during mammalian evolution, invalidating Ohno’s hypothesis as a general principle of the evolution of X-linked genes.

Results

Expression Reduction of X-linked Genes in Mammalian Evolution.

The five species analyzed here have the following phylogenetic relationship: (chicken, (platypus, (opossum, (mouse, human)))). The therian X chromosome emerged since therians diverged from the platypus (1). Because of the relatively low quality of the platypus genome assembly (20), we first compared human and chicken. For simplicity, chicken genes that are one-to-one orthologous to human X-linked (or autosomal) genes are denoted as chicken X (or A) genes (Materials and Methods). Although mammals and birds diverged ∼310 million years ago (21), the expression levels of human and chicken one-to-one orthologs are strongly correlated, with a correlation coefficient of ∼0.6 in each tissue examined (Fig. S1). We calculated the human:chicken expression ratio for each orthologous gene pair (Fig. S2 and Materials and Methods). After normalizing the median AA:AA ratio to 1, we found the median X:XX ratio to be ∼0.5 (Fig. 1A), which is in direct contradiction to Ohno’s hypothesis. The rationale of Ohno’s hypothesis is that, provided that expression levels of autosomal genes are unchanged in evolution, expression levels of X-linked genes should not change either (i.e., per-allele expression from X should be doubled due to the degeneration of Y). Hence, Ohno’s hypothesis is equivalent to X:AA = XX:AA. Using the same set of genes, we estimated that the ratio between the median expression level of chicken X genes and that of A genes is ∼1 (i.e., XX:AA∼1; Fig. 1B). By contrast, the X:AA ratio is ∼0.5 in humans (Fig. 1B). Clearly, this result also conflicts with Ohno’s hypothesis. Similar results were obtained when we defined active gene expression by RPKM (reads per kilobases of exonic sequence per million total reads) >1 (15) and considered only those genes that are active in chicken (Fig. 1 C and D). This finding holds even when the chicken RPKM cutoff is raised to 3 (13) (Fig. S3). The results were similar when only those genes that are actively expressed in humans (RPKM >1) were considered (Fig. S4). Qualitatively similar results were obtained in chicken–mouse, platypus–human, and platypus–mouse comparisons (Figs. S5, S6, and S7).

Fig. 1.

Fig. 1.

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Comparison of expression levels of orthologous genes from human and chicken. (A) Human X:chicken XX expression ratios for one-to-one orthologs in six male (M) or four female (F) tissues, when the medians of human AA:chicken AA expression ratios are normalized to 1. The null hypothesis of X:XX = AA:AA is rejected in each tissue (P < 10−9, Mann–Whitney U test). There are 325 gene pairs for calculating X:XX and 10,171 gene pairs for calculating AA:AA. The central bold line shows the median, the box encompasses 50% of genes, and the error bars include 90% of genes. (B) Ratios of chicken XX:AA median expressions and human X:AA median expressions in 10 tissues. Error bars show 95% confidence intervals estimated by bootstrapping genes 1000 times. The null hypothesis of X:AA = XX:AA is rejected in each tissue (P ≤ 0.001, bootstrap test). (C) Same as A, except that only genes actively expressed (RPKM >1) in the indicated chicken tissue are considered. The null hypothesis of X:XX = AA:AA is rejected in each tissue (P < 10−9, Mann–Whitney U test). (D) Same as B, except that only genes actively expressed in the indicated chicken tissue are considered. The null hypothesis of X:AA = XX:AA is rejected in each tissue (P < 0.001, bootstrap test).

Expression Profiles of X-linked Genes That Have One-to-One Chicken Orthologs.

To study the evolution of across-tissue expression profiles for X-linked genes during sex chromosome evolution, we correlated the expression levels of chicken X and human X one-to-one orthologous genes across tissues. As a control, we compared the same set of chicken X genes with their platypus one-to-one orthologs (Fig. 2A). We found the chicken–human expression profile similarity comparable to the chicken–platypus expression profile similarity (Fig. 2B). Use of mouse instead of human data yielded similar results (Fig. S8). Because the chicken–human (or chicken–mouse) divergence time is the same as the chicken–platypus divergence time, and because platypus X genes are autosomal, our observation suggests little impact of sex chromosome–specific selection on the evolution of expression profiles of therian X genes that existed in X.

Fig. 2.

Fig. 2.

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Evolution of across-tissue expression profiles for human X-linked genes that have platypus and chicken one-to-one orthologs. (A) Expression levels, measured by log2(RPKM), of 241 human X-linked genes and their one-to-one orthologous genes in platypus and chicken across 10 tissues. Each row is a gene. (B) Comparable expression profile similarity between chicken and platypus and that between chicken and human for human X-linked genes that have platypus and chicken one-to-one orthologs. Expression profile similarity between species is measured by Pearson’s correlation in expression level across 10 tissues. Each circle is a gene, and the numbers of circles above and below the diagonal are not significantly different (P > 0.09, binomial test).

Testis-biased Expressions of X-linked Young Genes.

Let us define human X-linked genes that have neither one-to-one nor one-to-many orthologs in chicken and platypus as young X genes and the rest as old X genes (Materials and Methods). Many of the young genes belong to multigene families such as various cancer/testis antigen genes (22). Compared with old X genes, a substantially smaller fraction of young X genes are actively expressed in each tissue examined except the testis (Fig. 3 A and B). For each gene, we calculated a testis bias index (TBI) by dividing its testis expression level by the median expression across all tissues. Young X genes have significantly larger TBI than old X genes (P < 10−7; Mann–Whitney U test). For example, 35.8 ± 2.4% of young X genes have TBI >5, whereas the corresponding number is 15.7 ± 1.7% for old X genes. By our definition, human young X genes include (i) those with no identifiable orthologs and (ii) those with many-to-one orthologs in chicken or platypus. Twelve cases belong to the second type in which a young X gene has an autosomal copy that has one chicken ortholog and one platypus ortholog, suggesting that these young X genes originated from duplications of autosomal genes after therians diverged from the platypus (Materials and Methods). In each of these 12 cases, the human X copy has a much lower expression than its autosomal paralog (Fig. 3C). These observations, together with the results of the previous section, suggest that human X-linked genes that existed before the origin of X and those acquired since the origin of X have been subject to distinct evolutionary pressures and play different functional roles. These results are consistent with an earlier study that was based solely on mammalian gene expressions (23).

Fig. 3.

Fig. 3.

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Testis-biased expressions of evolutionarily young X-linked genes in humans. (A) Expression levels of old and young human X-linked genes across 10 tissues. (B) Proportions of human old and young X-linked genes that are actively expressed (RPKM >1) in each tissue. Error bars show one SE of the observed proportion. (C) Expression evolution of 12 young human X-linked genes generated by autosome (A)–derived gene duplication since therians diverged from the platypus. RPKM values relative to the median RPKM of all genes in a transcriptome are shown. In A and C, each row is a gene, and log2(TBI) is presented in the last column. TBI, testis bias index.

Human Proteomic Data Show an X:AA Ratio of ∼0.5.

Ohno’s hypothesis can and should also be tested at the proteome level because dosage compensation must eventually show up at the protein level to have an effect (12). Using a high-throughput proteome dataset of a human cell line (24), we measured the X:AA ratio in protein concentration for human genes with one-to-one orthologs in chicken. Genes are sorted from high to low protein concentrations, and X-linked genes and autosomal genes are divided separately into 100 equal-size bins. Under the current proteomic coverage, most genes have no detectable protein expression; hence, only the first 25 X bins and 25 autosomal bins are compared. The X:AA ratios of median protein concentrations between matched bins are ∼0.5 (Fig. 4A), consistent with the RNA-Seq results as well as our previous results from a small mouse proteomic dataset (12). Genes with active expressions in X are most relevant to Ohno’s hypothesis. We examined genes with RPKM >1 in every chicken tissue examined; they are likely to be house-keeping in chicken. We found the human X:AA protein expression ratio to be ∼0.5 for the one-to-one orthologs of this set of chicken genes (Fig. 4B). These results refute Ohno’s hypothesis at the proteomic level.

Fig. 4.

Fig. 4.

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Comparison between X-linked genes and autosomal genes for protein abundances in a human cell line using (A) human genes that have one-to-one chicken orthologs and (B) human genes whose chicken one-to-one orthologs are likely to be house-keeping. The median protein abundance for each gene bin is shown. The X-linked and autosomal genes are separately divided into 100 equal-size bins, based on the ranks of protein expression levels, and the top 25 bins are compared.

Marsupials and Placentals Show Similar Expression Reductions on X.

Because marsupials and placentals diverged from each other shortly after the origin of the therian X, their X chromosomes have a long history of independent evolution and possess distinct features. For instance, female X inactivation differs between placentals and marsupials (25). Instead of random inactivation of one X in placentals, marsupials preferentially inactivate the paternally derived X. Furthermore, marsupial female X inactivation is incomplete in some cells or tissues. We tested Ohno’s hypothesis in marsupials by comparing opossum genes with their one-to-one orthologs in chicken, and obtained results similar to those from placentals (Fig. 5). The X:XX ratio is 0.66, 0.76, 0.53, 0.50, 0.84, 0.64, 0.65, 0.59, 0.75, 0.75, and 0.46 in brain (Female, F), brain (Male, M), cerebellum (F), cerebellum (M), heart (F), heart (M), kidney (F), kidney (M), liver (F), liver (M), and testis (M), respectively (Fig. 5A), suggesting a general reduction in the expressions of opossum X genes. Note, however, that in a few tissues the X:XX ratio is well above 0.5, which may be due to artifacts resulting from incomplete annotation of the opossum genome (26), the small number (197) of X genes examined, incomplete female X inactivation, and/or a small degree of up-regulation of X genes. Ohno’s hypothesis is most meaningful to active genes on X, and our examination of genes that are active in chicken tissues revealed an X:XX ratio close to 0.5 in all male tissues except the brain (Fig. 5C). Further studies are necessary to understand the complex pattern of the X:XX ratios observed in the opossum. Together, our results from placentals (Fig. 1) and marsupials (Fig. 5) depict an overall picture of expression reduction of X-linked genes that predated the separation between marsupials and placentals, which is consistent with the recent finding that accelerated evolution of expression levels of X-linked genes occurred in an early stage of X evolution (19).

Fig. 5.

Fig. 5.

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Comparison of expression levels of orthologous genes from opossum and chicken. (A) Opossum X:chicken XX expression ratios for one-to-one orthologs in 11 tissues, when the medians of opossum AA:chicken AA expression ratios are normalized to 1. The null hypothesis of X:XX = AA:AA is rejected in each tissue (P < 0.0003, Mann–Whitney U test). There are 197 gene pairs for calculating X:XX and 9,612 gene pairs for calculating AA:AA. The central bold line shows the median, the box encompasses 50% of genes, and the error bars include 90% of genes. (B) Ratios of chicken XX:AA median expressions and the corresponding opossum X:AA ratios. Error bars show 95% confidence intervals estimated by bootstrapping genes 1,000 times. The null hypothesis of X:AA = XX:AA is rejected at P = 0.001, 0.001, <0.001, <0.001, 0.039, 0.001, 0.005, <0.001, 0.003, <0.001, and 0.005, respectively, for the 11 tissues shown from left to right (bootstrap test). (C) Same as A, except that only those genes that are actively expressed in chicken tissues (RPKM >1) are considered. The null hypothesis of X:XX = AA:AA is rejected in each tissue (P < 0.0003, Mann–Whitney U test). (D) Same as B, except that only actively expressed genes in chicken tissues are considered. The null hypothesis of X:AA = XX:AA is rejected at P = 0.001, <0.001, <0.001, <0.001, 0.008, <0.001, 0.016, 0.005, 0.002, <0.001, and <0.001, respectively, for the 11 tissues from left to right (bootstrap test).

Up-regulation of X-linked Genes Encoding Members of Large Protein Complexes.

It was recently reported that human genes encoding components of large protein complexes (≥7 proteins) have a median X:AA expression ratio of ∼1, whereas the corresponding ratio for small protein complexes is ∼0.5 (27). Because dosage balance among components of large protein complexes is believed to be critical to the functionality of these complexes (28, 29), this observation suggests that up-regulation may have occurred to those X-linked genes whose dosages are particularly important. To test this hypothesis directly, we used the expression data of human and chicken orthologous genes to calculate R = (X:XX):(AA:AA). Within each protein complex, R measures the median fold change between chicken and human in the expressions of X-linked genes, relative to that of ausosomal genes. Interestingly, the median R is greater for large protein complexes (seven or more proteins) than for small complexes (fewer than seven proteins) in each of the 10 tissues examined, although their difference is not statistically significant in three of the 10 tissues (Fig. 6). Thus, our result is generally consistent with the suggestion of up-regulation of X-linked genes encoding components of large protein complexes (27). Nonetheless, these genes constitute only ∼5% of all X-linked genes, explaining why there is no chromosome-wide signal of X-up-regulation.

Fig. 6.

Fig. 6.

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Human X-linked genes encoding members of large protein complexes are subject to up-regulation, compared with those encoding members of small complexes. Each open symbol is a protein complex, whereas the solid symbols show median values. Within each complex, the median expression ratio of human X-linked genes to their chicken orthologs (X:XX) is divided by the median expression ratio of autosomal genes (AA:AA). The null hypothesis of equal log2((X:XX):(AA:AA)) values between large and small complexes is true with probabilities of 0.03, 0.17, 0.07, 0.01, 0.05, 0.05, 0.01, 0.03, 0.26, and 0.002, respectively, for the 10 tissues shown from left to right (Mann–Whitney U test).

Discussion

Ohno hypothesized that, compared with their progenitors on X, therian X-linked genes should have doubled their expressions to compensate for the loss of the Y-linked copy (3). Under the assumption that per-allele expression levels are comparable among X, A, and A, Ohno’s hypothesis predicts comparable gene expressions between one active X and two sets of autosomes (X:AA = 1) (13). However, from the rationale of this prediction, it is clear that when the X:AA expression ratio is used to test Ohno’s hypothesis indirectly, one should consider either all genes or all active genes in X and A. Consideration of active genes in X and A (14–17), a strategy that could lead to an inflation of the X:AA ratio (Fig. S9), was rooted in a misunderstanding of Ohno’s hypothesis as the expression balance between X and A (13). To test the expression balance, one may consider only active genes in X and A, because other genes do not contribute. However, to test Ohno’s hypothesis (indirectly), either all genes from X and A or matched fractions of genes from X and A should be included to make a fair comparison (13). In this study, however, we are able to circumvent this problem by directly comparing therian X-linked genes with their X orthologs, and our results conclusively reject Ohno’s hypothesis with the exception of ∼5% of genes encoding large protein complexes.

A natural question is how such chromosome-wide expression halving has been tolerated, given that autosomal monosomy is lethal in humans? Because Y degeneration is stepwise (30), expression reduction happened gradually to more and more X-linked genes during evolution. Thus, a possible explanation is that, at any time in evolution, an organism is faced with the expression halving of only one additional gene, which might have been slightly deleterious and thus can be fixed. This evolutionary process contrasts the sudden loss of an entire chromosome in monosomy that causes a large fitness reduction at once. Consistent with the above explanation is the observation that up to 97% of yeast genes have no detectable fitness effect when one allele is deleted from a diploid cell (31). Although haplosufficiency has not been systematically examined in mammals, it is probable that, for most genes, expression halving has little fitness effect. In this context, it is interesting to note that although the expression levels of human autosomal genes are overall strongly correlated with those of their chicken one-to-one orthologs, at the individual gene level, it is not uncommon to observe substantial expression differences between the orthologs. For instance, 65% of these autosomal one-to-one orthologs exhibit a more than twofold expression difference in kidney between human and chicken, whereas the corresponding number between two individuals of the same species is 41%. Because these genes are subject to neither hemizygosity nor gene duplication, the most likely explanation is that a twofold expression change is acceptable to many genes, especially when the expression changes of many genes do not all occur at once. Furthermore, it is possible that sex chromosomes tend to originate from autosomes that are overall insensitive to dose changes (32). For haploinsufficient genes on X, they may escape the fate of expression halving by relocation to an autosome via either gene duplication or translocation. Indeed, we found a dozen of human autosomal genes that have chicken orthologs residing in the chromosome blocks orthologous to X (Figs. S10 and S11), although gene traffic need not arise from the pressure for dosage balance (33, 34). This said, expression doubling to avoid haploinsufficiency appears to have occurred in ∼5% of X-linked genes that encode members of large protein complexes and was also demonstrated previously in one X-linked gene that was relocated from an autosome (35). Hence, in contrast to Ohno’s hypothesis of a chromosome-wide up-regulation of X-linked genes during sex chromosome evolution, such expression up-regulation is apparently uncommon.

As mentioned, the current evolutionary model of between-sex dosage compensation relies on the occurrence of X up-regulation. Whether the up-regulation of ∼5% of X-linked genes was sufficient to drive the origin of the chromosome-wide X inactivation in female mammals (27) is an open question. Outside mammals, sex chromosomes have evolved independently many times, but chromosome-wide dosage compensation between sexes is not universal (8, 32). For example, in birds, which have ZZ males and ZW females, expression levels of most Z-linked genes are substantially higher in males than in females (36, 37). With our demonstration of a lack of chromosome-wide up-regulation of X-linked genes in mammals and the recent findings of no or incomplete between-sex dosage compensation in many nonmammalian organisms (8, 32), the study of dosage compensation in sex chromosome evolution enters a new era in which new models and theories are urgently needed.

Materials and Methods

RNA-Seq gene expression levels were downloaded from the Supplementary Information of Brawand et al. (19). Protein concentrations were from the Supplementary Information of Beck et al. (24). Genes and their orthology information were retrieved from Ensembl release 57 (www.ensembl.org) using BioMart. Only protein-coding genes were considered in the analyses, and there were 16,736, 17,951, 19,466, 23,060, and 22,229 protein-coding genes with RNA-seq expression information in chicken, platypus, opossum, mouse, and human, respectively. We download 211 human protein complexes, each containing at least one X-linked gene and at least one autosomal gene, from HPRD release 9 (www.hprd.org).

We obtained 11,372 human:chicken one-to-one orthologous gene pairs that correspond to 336 X-linked and 11,024 autosomal genes in humans. Because chicken chromosomes 1 and 4 are orthologous to human X, we considered 325 gene pairs in which the chicken copies are on chromosome 1 or 4 when computing the X:XX ratio, and 10,171 gene pairs in which the chicken copies are not sex chromosome linked when computing the AA:AA ratio. RNA-Seq gene expression levels were measured by RPKM, considering reads mapped to all protein-coding genes of a given species. To facilitate the calculation of expression ratios, genes with RPKM < 0.01 (among which >99% are RPKM = 0) were assigned RPKM = 0.01. The raw RPKM values are suitable for expression comparisons of the same gene among different tissues of a species, but are inappropriate for between-species comparisons. Therefore, a scaling procedure was adopted. Specifically, for any pair of species, we linearly adjusted the expression levels of individual genes of a species by the same factor to make the median expression levels of one-to-one orthologs of the two species identical. This scaling strategy is similar to what was used by Brawand et al. (19), and generates results similar to those of Brawand et al.(19). (Dataset S1 provides both the raw and rescaled RPKM values used in Fig. 1.)

For orthologous genes with one chicken copy, one platypus copy, one human X copy, and one human autosomal copy, it is likely that there was a gene duplication event in human lineage. Synteny information was used to distinguish between the ancestral copy and the duplicate copy. We identified 12 cases with conserved synteny between the chicken copy and the human autosomal copy and 17 cases with conserved synteny between the chicken copy and the human X copy, and the raw RPKM value divided by the median RPKM of the sample was used for comparing the expressions of these genes.

Following Pessia et al. (27), we designated human protein complexes with seven or more members as large and fewer than seven members as small, resulting in a total of 589 autosomal and 41 X-linked genes encoding members of 75 large complexes, and 220 autosomal and 52 X-linked genes encoding members of 136 small complexes. We excluded protein complex members with no one-to-one chicken orthologs, resulting in 42 large complexes and 71 small complexes, each containing at least one X-linked gene and one autosomal gene. A total of 221 autosomal and 20 X-linked genes in large complexes, and 127 autosomal and 43 X-linked genes in small complexes were considered in Fig. 6. We first computed the human to chicken X:XX or AA:AA ratio of an orthologous gene pair. For each protein complex, the median X:XX ratio divided by the median AA:AA ratio was then calculated.

Supplementary Material

Supporting Information
supp_109_29_11752__index.html (1.2KB, html)

Acknowledgments

We thank Xiaoshu Chen for technical assistance. This work was supported by a U.S. National Institutes of Health research grant (to J.Z.) and a grant from the Guangzhou Municipal Government (#11BppZLjj2050035 to X.H.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1201816109/-/DCSupplemental.

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