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
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
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
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
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
Gene Functions Also Affect the Level of DC of Each
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
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
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
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
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
Materials and Methods
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).
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).
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.
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
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).
Supplementary figures S1–S3 and tables S1–S6 are available at
Molecular Biology and Evolution online (http://www.mbe.
The authors express their gratitude to Muneo Matsuda for
providing the flies that were used in this study. They also
thank Mai Fujimi and Chie Iwamoto for their help in their
experiments; Shu Kondo and Ryu Ueda for their technical
advice about Drosophila experiments; Hiroshi Akashi,
Masafumi Muraoka, Naobumi Sasaki, and Kazuhiro
Satomura for their useful advice about the analysis; and
three anonymous reviewers for their constructive comments
on early versions of the manuscript. This work was supported
by the National Institute of Genetics and JSPS KAKENHI
Grant Number 25711023 to M.N.
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