Tissue- and Stage-Dependent Dosage Compensation on the Neo-X Chromosome in Drosophila pseudoobscura

Molecular Biology and Evolution, Mar 2014

Sex chromosome dosage compensation (DC) is widely accepted in various organisms. This concept is mostly supported by comparisons of gene expression between chromosomes and between sexes. However, genes on the X chromosome and autosomes are mostly not homologous, and the average gene expression level on these chromosomes may not be the same even under DC, which complicates comparisons between chromosomes. Many genes with sex-biased expression also make comparisons between sexes difficult. To overcome these issues, we investigated DC by comparing the expression of neo-X-linked genes in Drosophila pseudoobscura with those of their autosomal orthologs in other Drosophila species. The ratio of the former to the latter in males would be 1 under DC, whereas it becomes 0.5 without DC. We found that the ratio was ∼0.85 for adult whole bodies, indicating that the DC is incomplete on the neo-X chromosome in adults as a whole. The ratio (∼0.90) was also significantly less than 1 for adult bodies without gonads, whereas it was ∼1.0 for adult heads. These results indicate that DC varies among tissues. Our sliding-window analysis of the ratio also revealed that the upregulation of neo-X-linked genes in males occurred chromosome wide in all tissues analyzed, indicating global upregulation mechanisms. However, we found that gene functions also affected the levels of DC. Furthermore, most of the genes recently moved to the X were already under DC at the larval stage but not at the adult stage. These results suggest that DC in Drosophila species operates in a tissue/stage-dependent manner.

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Tissue- and Stage-Dependent Dosage Compensation on the Neo-X Chromosome in Drosophila pseudoobscura

Mol. Biol. Evol. Masafumi Nozawa 1 2 Nana Fukuda 2 Kazuho Ikeo 1 2 Takashi Gojoboriz 0 1 2 0 Department of Life Science, National Cheng Kong University , Tainan , Taiwan 1 Department of Genetics, SOKENDAI, Hayama , Kanagawa , Japan 2 Center for Information Biology, National Institute of Genetics , Mishima, Shizuoka , Japan Sex chromosome dosage compensation (DC) is widely accepted in various organisms. This concept is mostly supported by comparisons of gene expression between chromosomes and between sexes. However, genes on the X chromosome and autosomes are mostly not homologous, and the average gene expression level on these chromosomes may not be the same even under DC, which complicates comparisons between chromosomes. Many genes with sex-biased expression also make comparisons between sexes difficult. To overcome these issues, we investigated DC by comparing the expression of neo-X-linked genes in Drosophila pseudoobscura with those of their autosomal orthologs in other Drosophila species. The ratio of the former to the latter in males would be 1 under DC, whereas it becomes 0.5 without DC. We found that the ratio was ~0.85 for adult whole bodies, indicating that the DC is incomplete on the neo-X chromosome in adults as a whole. The ratio (~0.90) was also significantly less than 1 for adult bodies without gonads, whereas it was ~1.0 for adult heads. These results indicate that DC varies among tissues. Our sliding-window analysis of the ratio also revealed that the upregulation of neo-X-linked genes in males occurred chromosome wide in all tissues analyzed, indicating global upregulation mechanisms. However, we found that gene functions also affected the levels of DC. Furthermore, most of the genes recently moved to the X were already under DC at the larval stage but not at the adult stage. These results suggest that DC in Drosophila species operates in a tissue/stage-dependent manner. dosage compensation; Drosophila; comparative transcriptome; sex chromosome evolution Introduction Heteromorphic sex chromosomes have repeatedly evolved from a pair of autosomes in different groups of organisms (Charlesworth 1978). Recombination between the X and Y chromosomes is then suppressed because the sex-determining genes and the alleles of the genes involved in sexual antagonism should be linked (e.g., Nei 1969; Charlesworth et al. 2005), which in turn leads to the degeneration of the Y chromosome in males (or W in females in the case of the ZW system) because of the inefficacy of natural selection in the absence of recombination. This degeneration causes a massive loss and inactivation of genes as well as the accumulation of repetitive elements on the Y chromosome (Steinemann et al. 1993). Consequently, most of the X-linked genes in males become monoallelic (or hemizygous), whereas the genes in females remain biallelic. This dosage imbalance seems to reduce the fitness of males (Ohno 1967), which may lead to an evolutionary dead end of the species. Based on this idea, Muller (1948) and Ohno (1967) proposed the sex chromosome dosage compensation (DC) hypothesis in which the expression of X-linked genes should be about two times greater in males than in females. After this proposal, many researchers extensively examined this hypothesis (e.g., see Gurbich and Bachtrog 2008; Vicoso and Bachtrog 2009; Kaiser and Bachtrog 2010; Disteche 2012; Johnson and Lachance 2012 for reviews). They have found that DC exists in many species including humans, Drosophila, and nematodes, whereas it is absent or incomplete in other species like chickens (e.g., Gupta et al. 2006; Nguyen and Disteche 2006; Wolf and Bryk 2011). These studies are largely based on the comparison of the gene expression levels between the X (or Z) chromosome and autosomes and/or between females and males. This type of study became so popular when the microarray and next-generation sequencing technologies were widely available. However, these approaches potentially contain serious limitations. First, when comparing expression levels of X-linked genes with those of autosomal genes, there is no guarantee that the expression levels are the same on average even if DC exists, because the genes on the X chromosome and autosomes are mostly not homologous and are totally different with respect to functions. Second, when comparing expression levels of orthologous genes between females and males, it is very difficult to eliminate the genes with sexbiased or sex-specific expression, which may also lead to erroneous conclusions. Indeed, upregulation of X-linked genes in mammals remains controversial because of methodological issues. Xiong et al. (2010) reported that the ratio of expression of the X-linked genes to the autosomal genes is ~0.5 rather than ~1.0, and they concluded that X-linked genes were not upregulated in mammals. The researchers who supported the idea of upregulation immediately criticized Xiong et al.’s approach (Deng et al. 2011; Kharchenko et al. 2011; Lin et al. 2011). They claimed that Xiong et al. used all genes, including transcriptionally inactive genes, which resulted in this biased conclusion. Actually, the proportion of inactive genes was much higher in the X chromosome than in autosomes (Deng et al. 2011; Kharchenko et al. 2011). When they reanalyzed the same RNA-seq data considering only active genes, the ratio was ~1.0. Therefore, different conclusions can be drawn from essentially the same data set. To overcome these controversies, we should ideally compare the X-linked genes in a species with their orthologous genes on autosomes (or proto-X) in other species. Actually, when the expression levels of the X-linked genes in mammals were compared with the levels of their autosomal orthologs in chickens, the median expression ratio of the former to the latter was ~0.5 (Lin et al. 2012). From this result, Lin et al. (2012) finally refuted the hypothesis of upregulation of X-linked genes at least in mammals, although they did not refute DC itself because the gene expression ratio in males to females is still about 1 for the X (see also Julien et al. 2012). In this case, however, some controversy still remains because the divergence time between mammals and chickens was as much as ~300 Ma, and the expression levels of chicken genes would be different from those of mammalian ancestors before forming sex chromosomes. Yet, it is clear that analytical approaches seem to affect the conclusion regarding DC (Jue et al. 2013). In this context, Drosophila species are excellent targets to examine DC using this interspecific transcriptome approach because several chromosomal fusions between autosomes and sex chromosomes recently occurred in several lineages independently and generated so-called neo-X and neo-Y chromosomes (Steinemann and Steinemann 1998; Chang et al. 2008; Schaeffer et al. 2008). Of course, the molecular mechanisms of DC have been studied extensively in D. melanogaster and, to some extent, in other species (e.g., Bone and Kuroda 1996; Park et al. 2007; Alekseyenko et al. 2008; Gelbart and Kuroda 2009; Lott et al. 2011; Conrad and Akhtar 2012; Dunlap et al. 2012; Alekseyenko et al. 2013). Yet, this evolutionary approach based on comparing the expression of orthologous genes between species is quite meaningful to strictly quantify the extent of DC in Drosophila species. This approach will also enable us to discuss the DC not only on the entire X chromosome but also on individual X-linked genes. In this study, we particularly focused on D. pseudoobscura, in which the neo-X chromosome evolved about 13 Ma (Kaiser and Bachtrog 2010). DC in D. pseudoobscura has been studied since the 1970s. Abraham and Lucchesi (1974) compared the enzymatic activities of one gene on the neo-X (XR) chromosome and two genes on the old-X (XL) chromosome between females and males and concluded that DC is operating on both X chromosomes. It was also reported that a DC complex (DCC, which is supposed to be important for initiating DC) certainly bound to many regions of the neo-X of D. pseudoobscura (Bone and Kuroda 1996; Marin et al. 1996). However, there is no large-scale study of gene expression with a special interest in DC on the neo-X. In this study, we therefore report the detailed and global pictures of DC on the neo-X based on our interspecific transcriptome approach. Extent of DC on the Neo-X Chromosome Varies with Tissues In the ancestral lineage leading to D. pseudoobscura and other closely related species, a chromosomal fusion occurred between an autosome (i.e., Muller element D) and an old-X (i.e., Muller element A), which turned the autosome into the neoX (fig. 1A). Therefore, the genes on the Muller element D are X-linked in D. pseudoobscura, whereas their orthologs are located on autosomes in other species with ancestral karyotypes. Because RNA-seq data from adult whole bodies, adult bodies without gonads, and adult heads were available for D. pseudoobscura, D. melanogaster (diverged from D. pseudoobscura ~55 Ma), D. simulans (~55 Ma), and D. mojavensis (~63 Ma) in the Sequence Read Archive (SRA) database (supplementary table S1, Supplementary Material online), we compared the expression levels of the orthologous genes in D. pseudoobscura and these other species. To take account of the possibility that expression levels have changed during evolution even without the chromosomal fusion, we newly defined the following measure, the ratio of male-to-female expression changes to evaluate DC (RDC), as cRPKMM,tar=cRPKMM,com , RDC ¼ cRPKMF,tar=cRPKMF,com where cRPKM is the corrected reads per kilobase of exon per million mapped reads, which would be appropriate for comparing gene expression level between species (see Materials and Methods). Subscripts F and M denote females and males, respectively, and the subscripts tar and com are for target species (D. pseudoobscura in this study) and comparing species (D. melanogaster, D. simulans, or D. mojavensis), respectively. The essence of this equation is that we can detect changes in male expression between species due to the chromosomal fusion using female expression change as a control. If there is no DC, the RDC value will be 0.5 because D. pseudoobscura males have only one copy of the Muller element D, whereas males in other species carry two copies. In contrast, the value is expected to be 1 if DC is perfect. As controls, we also computed the RDC values for the genes located on the X or autosomes in both species. In adult whole bodies, all species comparisons showed that the RDC values in the category of X-Au (i.e., genes are located on the neo-X in D. pseudoobscura but located on autosomes in other species; gray bars in fig. 1B) are significantly lower than the values in other categories where the gene copy numbers are balanced (i.e., black and white bars for X-X and Au-Au, respectively, in fig. 1B). Yet, the median RDC value (~0.85) of the X-Au category was significantly greater than 0.5. Therefore, DC on the neo-X of D. pseudoobscura certainly exists, but it seems to be incomplete in adult whole bodies. This would be natural because the adult male bodies contain a substantial fraction of male germline tissues in which no DC has been suggested (Meiklejohn et al. 2011). Yet, even in the data of adult bodies without gonads, the median RDC value (~0.90) of the X-Au category is significantly lower than 1 and lower than the values of the X-X and Au-Au categories (fig. 1B). In contrast, when we analyzed the adult head data, the median values of the X-Au category were ~1.0 in both cases where D. melanogaster and D. mojavensis were used as the comparing species. We also examined the distributions of RDC values (fig. 1C). In the whole body data, a majority of genes had RDC values that were less than 1 with a mode of ~0.7. The distribution was slightly shifted to the right in the data of adult bodies without gonads, but the mode was still ~0.8. In contrast, the mode of RDC values was very close to 1 in the adult head data. These results indicate that the DC on the neo-X is largely regulated in a global manner irrespective of the data, but the level of DC varies even among somatic tissues. It should be mentioned that the RDC values on the neoX vary with tissues, but the values in a specific tissue are highly correlated irrespective of the comparing species (supplementary table S2, Supplementary Material online). In the following, we therefore used D. melanogaster as a comparing species to analyze adult whole bodies and adult heads unless otherwise stated. For the data of adult bodies without gonads, we could only use D. simulans because of the data availability. We next conducted a sliding-window analysis of RDC with the window size of 1 Mb and the sliding size of 100 kb along the neo-X (fig. 2). In adult whole bodies, the values were always greater than 0.5 but mostly less than 1 except for some subcentromeric and telomeric regions. This observation also indicates the incomplete but widespread DC on the neoX in adult whole bodies. The variance of RDC among regions was much smaller in adult bodies without gonads, but the RDC values were mostly less than 1. In contrast, the adult head data showed that the RDC values were mostly between 0.9 and 1.1. This result also indicates that the level of DC and the genes under DC varies with tissues, but there must be mechanisms of the chromosome-wide upregulation of gene expression. Gene Functions Also Affect the Level of DC of Each Neo-X-Linked Gene The above analyses suggest that a global upregulation mechanism of DC exists on the neo-X as previously indicated (Abraham and Lucchesi 1974; Bone and Kuroda 1996; Marin et al. 1996). Yet, the extent of the DC of each gene may be partly under gene-by-gene regulation, as well, because the RDC values considerably vary with genes (fig. 1). To clarify the factors that affect the levels of DC of each gene, we classified the genes on the neo-X into DC genes and non-DC genes based on the RDC. To strictly define the DC and non-DC genes, a gene was regarded as a DC gene if the gene showed 0.9 RDC < 1.1, whereas a gene with 0.4 RDC < 0.6 was classified as a non-DC gene. All other genes were regarded as unclassified genes and eliminated from the analyses. The ratio of the numbers of DC to non-DC genes was 0.79 (218/276) in adult whole bodies, 1.78 (315/177) in adult bodies without gonads, and 2.71 (398/147) in adult heads (see also supplementary table S3, Supplementary Material online, for details). This is also consistent with the idea that the DC on the neo-X varies among tissues and is more complete in adult heads. In adult whole bodies, we found that the genes under DC evolve significantly slower than the non-DC genes with respect to nonsynonymous substitution rates (fig. 3A). Because the synonymous substitution rates were similar between the categories (data not shown), the DC genes are likely to be more conserved than non-DC genes because of stronger functional constraints. Consistent with this observation, the proportion of essential genes (i.e., gene knockdown by RNAi causes lethality) was higher for DC genes than for non-DC genes (fig. 3B). In addition, expression levels of DC genes were higher than those of non-DC genes in both females and males (fig. 3C), implying that genes with high expression tend to be under DC, probably to maintain a large amount of gene products. Moreover, the DC gene products interact with a significantly greater number of proteins than the non-DC gene products (fig. 3D). These results suggest that the genes with low evolutionary rates, essential functions, high expression levels, and large numbers of interacting genes tend to be under DC. A similar trend was also observed in the data of adult bodies without gonads, although there was no significant difference in gene expression levels. In contrast, in adult FIG. 2. Median RDC values along the neo-X of Drosophila pseudoobscura. Each dot is a median RDC value for the genes in a 1-Mbp window size along the neo-X with a 100-kb sliding size. The RDC value of 1 is shown by a broken line. The actual neo-X starts at the ~6.8 Mb region on the XR because of an inversion (Schaeffer et al. 2008). For the data of adult whole bodies and adult heads, D. melanogaster was used as a comparing species. For the data of adult bodies without gonads, D. simulans was used as a comparing species. heads, DC and non-DC genes were not statistically different with respect to the evolutionary rate, essentiality, expression level, and number of interacting genes. It is possible that the functional constraints on heads may not necessarily reflect the constraints on the whole bodies. For example, even if a gene is a non-DC gene in heads due to a dispensable function in heads, the gene may show a slow evolutionary rate because of its functional importance in other tissues. We also examined the types of genes that tend to be under DC using gene ontology (GO) with the GOrilla software (Eden et al. 2009) (supplementary table S4, Supplementary Material online). The genes whose products often form large protein complexes such as macromolecular complexes tend to be overrepresented relative to the non-DC genes in adult whole bodies, although the trend is not so conspicuous. This result is consistent with the finding that the genes under DC tend to have greater numbers of protein–protein interactions (PPIs) (fig. 3D). This is probably because the genes forming large complexes are expected to interact with many other genes and should maintain balanced expression with many genes (Lin et al. 2012; Pessia et al. 2012). Yet, the enriched GO terms were mostly different in the data set of adult bodies without gonads. Furthermore, there was no GO term that was enriched in the adult head data. These results again indicate that the genes under DC are different among tissues. Paralogs on Autosomes May Weakly Affect the Level of DC of Neo-X-Linked Genes The above observations indicated that the levels of DC are partly dependent on gene functions. However, if a neoX-linked gene has at least one paralog with high sequence similarity on an autosome, DC may not be necessary for the gene because the paralog may reduce the effect of the dosage imbalance of the neo-X-linked gene. If this is the case, genes that are not under DC are expected to have paralogs on autosomes with a higher proportion compared with genes under DC. Indeed, non-DC genes tend to have paralogs on autosomes with slightly higher proportions compared with DC genes irrespective of the data sets and the cutoff E-values for detecting paralogs, although the difference is not statistically significant (fig. 4). This observation suggests that the existence of paralogs on autosomes may weakly affect the level of DC of neo-X-linked genes. DC Appears to be More Essential at the Larval Stage Than the Adult Stage In the above analyses, we considered the evolution of DC when a pair of autosomes became sex chromosomes. However, it is also possible that a single gene or a small number of genes are individually transposed from autosomes to the X after the formation of sex chromosomes. Therefore, we examined whether the genes recently moved to the X are under DC. For this analysis, we used two pairs of closely related species, D. simulans–D. sechellia and D. pseudoobscura– D. persimilis, both of which diverged ~1 Ma (Tamura et al. 2004). First, we downloaded the orthologous gene data of 12 Drosophila species (gene_orthologs_fb_2012_01.tsv) and identified the genes that were transposed from autosomes to the X after the speciation of D. simulans and D. sechellia or D. pseudoobscura and D. persimilis under the parsimony principle. To eliminate spurious candidates as much as possible, we then removed the genes that had paralogs using TBlastN against each genome and against all annotated coding sequences with the cutoff E-value of 10 5. In this way, we finally identified six and three genes that were moved from autosomes to the X in the D. simulans and D. pseudoobscura lineages after the divergence from D. sechellia and D. FIG. 4. Proportion of DC and non-DC genes that have paralogs on autosomes. Different cutoff E-values were used to detect paralogs on autosomes. Error bars indicate the 95% confidence interval based on the 1,000 bootstrap resampling. persimilis, respectively. Using quantitative polymerase chain reaction (qPCR), we estimated the RDC values for these genes and examined whether these genes that were recently transposed to the X are under DC. Total RNA from whole bodies of larva, pupa, and adults was used for the qPCR experiments. At the larval stage, most of the genes had RDC values that were close to 1 (fig. 5), and three of them were between 0.9 and 1.1 (supplementary table S5, Supplementary Material online). At the pupal stage, the RDC values varied from gene to gene, but there was only one gene with an RDC value that was less than 0.5, and three genes had RDC values that were greater than 1.5. One might think that the DC is more complete at the pupal stage than the larval stage because the RDC values tended to be larger in the former than the latter. However, the expression level of a gene is unlikely to be upregulated very much when the DCC binds to the surrounding regions of the gene to initiate DC (Bachtrog et al. 2010). Therefore, DC appears to be more complete at the larval stage than the pupal stage. At the adult stage, in contrast, five of nine genes had values that were less than 0.4, indicating that the DC for these genes is quite incomplete. In addition, no gene had an RDC value between 0.9 and 1.1 (supplementary table S5, Supplementary Material online). Therefore, although the number of genes analyzed is only nine, it is likely that DC seems to be necessary at the larval stage immediately after the transpositions. At the adult stage, however, DC might not be essential. Yet, we cannot completely deny the possibility that DC is required in some of the adult tissues. This stage-dependent DC on the neo-X was also observed at the genomic level. When the expression level of each of the transcripts was compared between females and males at larval, pupal, and adult stages of D. pseudoobscura by using our own RNA-seq data, the deviation from the slope of 1 under perfect DC increased with development (supplementary figs. S1 and S2, Supplementary Material online). As the slope values decrease smaller with development, the level of DC appears to be more complete at the larval stage than the FIG. 5. Distributions of RDC for the genes that were recently moved to the X chromosome. adult stage. However, when the data of adult bodies without gonads were analyzed, the deviation from the diagonal was smaller. The deviation was even smaller when the adult head data were analyzed. Of course, we should not depend on this type of comparison too much because of the effects of male-biased and female-biased genes (Meiklejohn et al. 2003; Parisi et al. 2003; Ranz et al. 2003; Sturgill et al. 2007). Yet, it is clear that DC is also stage dependent as well as tissue dependent. In this study, we investigated the evolutionary aspects of DC in Drosophila species by taking advantage of the massive available data of genomes and transcriptomes in combination with our own experimental data. Our interspecies approach, where the expression levels of orthologous genes on the X in one species but on autosomes in other species were compared, clearly demonstrated a chromosome-wide DC on the neo-X of D. pseudoobscura. This finding is consistent with previous studies based on the comparison of gene expression between females and males or between the X and autosomes (Abraham and Lucchesi 1974; Strobel et al. 1978; Gupta et al. 2006; Nguyen and Disteche 2006; Deng et al. 2011; Lott et al. 2011; Zhou and Bachtrog 2012a). Our results also agree with those of previous reports regarding the molecular basis of DC in D. melanogaster (Lucchesi et al. 2005; Gelbart and Kuroda 2009; Larschan et al. 2011; Conrad and Akhtar 2012). Yet, our study for the first time found that the level of DC varies even among somatic tissues. It has been proposed that DCC recognizes high-affinity target sites (HAS), which initiates the global upregulation of male X-linked genes in D. melanogaster (Conrad and Akhtar 2012 for review). Our study implies that this type of initiation of DC also occurs in the D. pseudoobscura neo-X as well. We clearly showed that the RDC value is greater than 0.5 throughout the neo-X (fig. 2) in all adult tissues examined, which is consistent with the idea of the global upregulation of the neoX-linked genes in males. Indeed, the GA-rich motif seems to be commonly used as the HAS that is recognized by the DCC in D. melanogaster (Alekseyenko et al. 2008) and D. miranda (Alekseyenko et al. 2013). Therefore, it is possible that the same sequence motif is used for the neo-X of D. pseudoobscura as well. We indeed found that the number of the (GA)4 repeat, a core part of the HAS in D. melanogaster and D. miranda, is slightly greater in the regions surrounding the DC genes than those surrounding the non-DC genes on the neo-X of D. pseudoobscura, although no statistical significance was detected (supplementary fig. S3, Supplementary Material online). This may indicate that the same type of HAS also plays a role in initiating the global upregulation of the neo-X-linked genes in D. pseudoobscura. However, this trend disappeared when the data of adult bodies without gonads and adult heads were analyzed (supplementary fig. S3, Supplementary Material online). Therefore, the usage of this motif as the HAS and/or the motif sequence itself might be different among tissues. Further studies are necessary to resolve this issue. We also found that the DC on the neo-X is stage dependent. Most of the genes that were transposed to the X chromosome within 1 Ma are already under DC at the larval stage, whereas most of these genes are not under DC at the adult stage (fig. 5). In addition, the expression levels of genes between sexes are more balanced at the larval stage than the adult stage, when whole body data were analyzed (supplementary figs. S1 and S2, Supplementary Material online). These observations suggest that DC is particularly essential at the larval stage, possibly for pupation and metamorphosis, but it may be less important once flies complete developmental processes and become adults. This could be one of the reasons why a certain group of genes with less important functions are not under DC at the adult stage (fig. 3). Indeed, several mutants such as msl1 and msl2 mutants (both of the genes are components of DCC) mostly show male lethality at the larval stage (Belote and Lucchesi 1980). Yet, it should be mentioned that the fraction of germline tissues in bodies may also affect the extent of DC as a whole. This is because male germline tissues apparently lack the DC (Meiklejohn et al. 2011) and the fraction of germline tissues is much greater in adult bodies than in larval bodies. In addition to the global upregulation, we also discovered the gene-by-gene regulation of DC. We clearly showed that at least the evolutionary rate, essentiality, expression level, GO, and number of PPIs of each gene are correlated with DC to some extent (fig. 3 and supplementary table S4, Supplementary Material online). This type of DC is consistent with the original idea of Ohno (1967), who stated “During the course of evolution of this insect, the dosage compensation mechanism must have developed one by one for each individual X-linked gene.” We may be able to strengthen this hypothesis if we compare the gene expression level between D. melanogaster and D. pseudoobscura not only at the adult stage but also at the larval and pupal stages using our interspecies evolutionary approach, which will also be examined in future work. It is known that the expression ratio of the X-linked genes in the Drosophila SL2 cells with msl2 knocked down to those in the normal SL2 cells is not 0.5 but ~0.75 (Meiklejohn et al. 2011). This is also true for the aneuploid Drosophila S2 cells (Zhang et al. 2010) and the autosomal deficiency hemizygotes of D. melanogaster (Stenberg et al. 2009). These results indicated that there must be some mechanism of DCC-independent DC in addition to DCC-dependent DC (Zhang et al. 2010), but the details of the DCC-independent DC mechanism have not been clarified yet. The gene-by-gene regulation of DC, which we described in this study, might be responsible for at least a part of the DCC-independent DC, although we still do not know how the level of DC of each gene is determined and it may be too speculative at this moment. Our findings regarding DC in Drosophila species are quite different from those in mammalian species, where the expression ratio of X-linked genes in mammals to their autosomal orthologs in chickens rejected the existence of widespread upregulation of X-linked genes (Julien et al. 2012; Lin et al. 2012). Why the DC is quite contrasting between these two groups of organisms is still a mystery, but we would like to discuss several possibilities below. First, in the case of Drosophila species, the upregulation of the X-linked genes is likely to occur only in males, which can compensate the dosage imbalance between females and males as well as between the X and autosomes simultaneously (Kaiser and Bachtrog 2010). In the case of mammals, in contrast, the upregulation of the X-linked genes has been supposed to occur in both females and males (Deng et al. 2013), so that one of two X chromosomes in females has to be inactivated (Lyon 1961; Adler et al. 1997; Disteche 2012). This complicated process may make the interpretation of the results difficult. Second, mammalian genomes generally contain a lot of duplicated genes that provide functional redundancies. Therefore, it is possible that acquiring DC may not be so essential in the evolution of heteromorphic sex chromosomes, although a certain number of genes with specific functions seem to be compensated (Lin et al. 2012; Pessia et al. 2012). Indeed, in yeast, where a whole-genome duplication occurred (Wolfe and Shields 1997) and there is great functional redundancy by duplicated genes (Gu et al. 2003), 97% of genes have no detectable fitness effect when one allele is deleted from a diploid cell (Deutschbauer et al. 2005). In Drosophila species, in contrast, the number of duplicated genes and resultant functional redundancy seem to be much less than in mammals. For example, the number of genes per gene family is 1.9–2.3 in mammalian species (Demuth et al. 2006) but only 1.1–1.2 in Drosophila species (Hahn et al. 2007). In this situation, acquiring DC appears to be more essential in Drosophila species. Indeed, there seems to be some effect of duplicated genes on DC in D. pseudoobscura (fig. 4). Third, Lin et al.’s (2012) study of DC in mammals is based on the comparison of mammals with chickens, which diverged as much as ~300 Ma (Kumar and Hedges 2011). In this case, gene expression levels in chickens may not be the same as those in the mammalian ancestor before having the sex chromosomes. Therefore, their approach to evaluate DC might potentially be biased. In this sense, our study is mainly based on the two Drosophila species, D. pseudoobscura and D. melanogaster (i.e., within the same genus). Indeed, the divergence time of the two species is ~55 Ma which is much more recent than the divergence of mammals and chickens. Of course, gene expression levels may have changed during this time period, but we have taken care of this effect as much as possible by defining a new measure, RDC, where the female expression change during evolution was used as a control. Of course, our approach to study DC has some limitations. First, our approach requires an orthologous autosomal chromosome as a reference, and it may be difficult to find such a chromosome in reality. For example, the Muller element A is the X chromosome (old-X) in all known Drosophila species; therefore, it was difficult to examine the DC on this X chromosome. However, because the cost for genome and transcriptome sequencing has drastically become less expensive, it is now possible to sequence the genome of other species in which the Muller element A is an autosome. Indeed, the Muller element A was found to be an autosome in some other Dipteran species such as the gray fleshfly (Vicoso and Bachtrog 2013). Therefore, we believe that this limitation will be overcome in the near future. Second, our approach assumes that gene expression changes occur equally in both sexes during evolution if there is no chromosomal fusion. However, this assumption may not always be the case. For example, if an expression level of a neo-X-linked gene becomes half only in females of D. pseudoobscura after splitting from D. melanogaster, the RDC value for the gene would be 1 even if there is no DC (i.e., no upregulation in males). How to deal with this issue is also a critical challenge for future studies. In this study, we clearly showed that the DC is quite widespread on the neo-X chromosome of D. pseudoobscura in a stage/tissue-dependent manner. However, the major issue still remains: how was the DC mechanism established in relation to the formation of sex chromosomes, in particular the degeneration of the Y chromosome. The Y chromosome of D. pseudoobscura has almost completely been degenerated (Carvalho and Clark 2005), so that we cannot test whether the degeneration of the Y triggers the development of DC on the X. To clarify this point, we should ideally investigate a species where sex chromosomes were generated so recently that the degeneration of the Y is still ongoing. Several Drosophila species such as D. miranda, D. albomicans, and D. americana have recently (~1 Ma or less) experienced fusions between autosomes and sex chromosomes independently (Chang et al. 2008; Kaiser and Bachtrog 2010) and are potentially good targets to test this hypothesis. Although Bachtrog and her colleagues have vigorously studied the evolution of sex chromosomes using D. miranda (e.g., Bachtrog 2004; Bachtrog 2006; Kaiser et al. 2011; Zhou and Bachtrog 2012a, 2012b; Alekseyenko et al. 2013; Ellison and Bachtrog 2013) and D. albomicans (Zhou et al. 2012), our interspecies approach to examine DC has great potential to clarify the detailed evolutionary processes of DC in these Drosophila species as well as in other groups of organisms in future. Materials and Methods Drosophila Species In this study, we used D. pseudoobscura (Stock no. 140110121.94), D. persimilis (14011-0111.49), D. simulans (140210251.194), and D. sechellia (14021-0248.25). All of them are the strains that were used in genome sequencing projects (Clark et al. 2007). The fly stocks were obtained from KYORIN-Fly Drosophila Stock Center (http://kyotofly.kit.jp/ cgi-bin/kyorin/index.cgi, last accessed December 17, 2013). RNA Extraction In this study, RNA-seq and qPCR methods were used to examine DC in Drosophila species. For both experiments, total RNA was extracted from five females or males of third instar larvae (before wandering), pupae (48–60 h after pupation), or virgin adults (72–96 h after eclosion) using a standard acid phenol-guanidinium thiocyanate-chloroform extraction method with slight modifications (Sambrook and Russell 2001). Total RNA was then treated with DNase I to digest potentially contaminating genomic DNA following the manufacturer’s instructions (TaKaRa, Ohtsu, Shiga, Japan). RNA-seq To exhaustively determine the expression level of X-linked and autosomal genes in D. pseudoobscura, we first purified mRNA from total RNA using Dynabeads mRNA Purification Kit (Life Technologies, Carlsbad, CA, USA). cDNA libraries were then constructed using TruSeq RNA Sample Preparation Kit ver. 2 (Illumina, San Diego, CA, USA) for the HiSeq 2000 sequencer or Ion Total RNA-seq Kit ver. 2 (Life Technologies) for the Ion PGM sequencer. Single-end sequencing was performed by these two sequencers. All reads from the HiSeq 2000 were 101 bp in length, whereas the read length varies with reads in the data from Ion PGM. Library construction and sequencing for Hiseq 2000 were done by Hokkaido System Science. Analyses of RNA-seq Data Sequence data from HiSeq 2000 were processed using CASAVA ver. 1.8.1 (Illumina) to remove low-quality sequences and adaptor sequences. Internal adaptor sequences were further removed by cutadapt ver. 1.0 (Martin 2011). For the data from Ion PGM, those processes were conducted with Torrent Suite ver. 2.2 (Life Technologies), and the reads with <50 nucleotides were eliminated. All remaining sequences were used as queries for BlastN search (MEGABLAST option, Altschul et al. 1997) against the mRNA sequences of D. pseudoobscura (dpse-all-transcriptr2.27.fasta downloaded from FlyBase [http://flybase.org/, last accessed December 17, 2013]) to map those reads to the known transcripts. The number of reads mapped to each transcript was then counted. Here, we only considered the best-hit transcript showing the lowest E-value for each read. When a read was mapped to more than one transcript with the same E-value, the count was equally divided into these best-hit transcripts without any weighting (e.g., when there were three tie best-hit transcripts, each transcript was counted as one-third). Detailed information of each run is provided in supplementary table S1, Supplementary Material online. Note that we also used TopHat 2.0.4 (Trapnell et al. 2009) and BWA 0.5.9 (Li and Durbin 2010) with default options for mapping, but the mapping rate by these tools is similar to or slightly lower than that by BlastN. We also downloaded the RNA-seq data of D. melanogaster, D. simulans, D. mojavensis, and D. pseudoobscura from the SRA database (http://www.ncbi.nlm.nih.gov/Traces/sra, last accessed December 17, 2013). The procedures for counting the number of reads for each transcript were the same as those for our own experimental data except that we only used 6–80 nt for 101-bp sequences and 6–75 nt for 75- or 76-bp sequences to remove nucleotides with low quality using the FASTX-Toolkit ver. 0.0.13 (http://hannonlab.cshl. edu/fastx_toolkit/index.html, last accessed December 17, 2013). (We used dmel-all-transcript-r5.45.fasta, dsim-alltranscript-r1.4.fasta, and dmoj-all-transcript-r1.3.fasta from FlyBase for the mRNA sequences.) For paired-end sequencing data, only forward sequences were used for the analysis without considering reverse sequences to eliminate the bias that can be caused by the sequencing layout. Summary information for the data is given in supplementary table S1, Supplementary Material online. All the tag count data were then converted to RPKM for normalization (Mortazavi et al. 2008). For the data that were retrieved from the database, we used the average RPKM of two or more replicates for the analysis. Regarding our own RNA-seq data, however, the number of reads from HiSeq 2000 and Ion PGM is considerably different (at most two orders of magnitude). Therefore, we separately analyzed these two biological replicates. Yet, the results were essentially the same regardless of the data. When gene expression level was compared between species, the RPKM values for all alternative transcripts of a gene were summed. However, the RPKM values cannot directly be compared between species because lengths of orthologous genes may be different between species. Therefore, whenever comparing the gene expression level between species, we linearly adjusted an individual RPKM to make the median expression identical to 1 following Lin et al. (2012). Note that many transcripts (or genes) in all species but D. melanogaster are not assigned to the chromosomes; rather, they are just assigned to contigs or scaffolds. Therefore, we assigned these transcripts to the chromosomes as much as possible using Schaeffer et al.’s (2008) chromosomal maps based on genetic and physical map information. qPCR To compare the expression level of genes that are located on the X in one species and autosomes in the closely related species, we used qPCR. cDNA was reverse transcribed from total RNA using the PrimeScript RT reagent Kit (TaKaRa). SYBR Premix Ex Taq (TaKaRa) was then used for qPCR with the Chromo4 thermal cycler (Bio-Rad, Hercules, CA, USA). For this experiment, we made two biological replicates for each sample and three technical replicates for each biological replicate. To compare gene expression levels between species, we used the comparative threshold cycle method (Schefe et al. 2006) with a reference gene, nrv2, which shows stable expression with developmental stages (Ling and Salvaterra 2011). After qPCR, the products were electrophoresed and sequenced to confirm that they were truly derived from the desired genes. PCR conditions and primers used are provided in supplementary table S6, Supplementary Material online. Other Data Analyses To examine the essentiality (or lethality) of DC and non-DC genes, we downloaded the data of RNAi knock-down experiments of D. melanogaster from NIG-FLY (downloaded 2012 Aug 16, http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp, last accessed December 17, 2013). To examine the number of PPIs for the DC and non-DC genes, we used the Drosophila Interaction Database (version 2012_04, http://www.droidb. org/DBdescription.jsp, last accessed December 17, 2013). 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Masafumi Nozawa, Nana Fukuda, Kazuho Ikeo, Takashi Gojobori. Tissue- and Stage-Dependent Dosage Compensation on the Neo-X Chromosome in Drosophila pseudoobscura, Molecular Biology and Evolution, 2014, 614-624, DOI: 10.1093/molbev/mst239