Reshaping of global gene expression networks and sex-biased gene expression by integration of a young gene
2012; Springer Nature; Volume: 31; Issue: 12 Linguagem: Inglês
10.1038/emboj.2012.108
ISSN1460-2075
AutoresSidi Chen, Xiaochun Ni, Benjamin H. Krinsky, Yong E. Zhang, Maria D. Vibranovski, K White, Manyuan Long,
Tópico(s)Chromosomal and Genetic Variations
ResumoArticle27 April 2012free access Reshaping of global gene expression networks and sex-biased gene expression by integration of a young gene Sidi Chen Sidi Chen Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USAPresent address: Department of Biology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02139, USA Search for more papers by this author Xiaochun Ni Xiaochun Ni Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USA Institute for Genomics and Systems Biology, The University of Chicago and Argonne National Laboratory, Chicago, IL, USA Search for more papers by this author Benjamin H Krinsky Benjamin H Krinsky Committee on Evolutionary Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Yong E Zhang Yong E Zhang Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USAPresent address: Key Laboratory of the Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, PR China. Search for more papers by this author Maria D Vibranovski Maria D Vibranovski Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USA Search for more papers by this author Kevin P White Corresponding Author Kevin P White Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USA Institute for Genomics and Systems Biology, The University of Chicago and Argonne National Laboratory, Chicago, IL, USA Department of Human Genetics, The University of Chicago, Chicago, IL, USA Search for more papers by this author Manyuan Long Corresponding Author Manyuan Long Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USA Search for more papers by this author Sidi Chen Sidi Chen Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USAPresent address: Department of Biology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02139, USA Search for more papers by this author Xiaochun Ni Xiaochun Ni Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USA Institute for Genomics and Systems Biology, The University of Chicago and Argonne National Laboratory, Chicago, IL, USA Search for more papers by this author Benjamin H Krinsky Benjamin H Krinsky Committee on Evolutionary Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Yong E Zhang Yong E Zhang Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USAPresent address: Key Laboratory of the Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, PR China. Search for more papers by this author Maria D Vibranovski Maria D Vibranovski Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USA Search for more papers by this author Kevin P White Corresponding Author Kevin P White Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USA Institute for Genomics and Systems Biology, The University of Chicago and Argonne National Laboratory, Chicago, IL, USA Department of Human Genetics, The University of Chicago, Chicago, IL, USA Search for more papers by this author Manyuan Long Corresponding Author Manyuan Long Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USA Search for more papers by this author Author Information Sidi Chen1,‡, Xiaochun Ni1,2,‡, Benjamin H Krinsky3, Yong E Zhang1, Maria D Vibranovski1, Kevin P White 1,2,4 and Manyuan Long 1 1Department of Ecology and Evolution, The University of Chicago, Chicago, IL, USA 2Institute for Genomics and Systems Biology, The University of Chicago and Argonne National Laboratory, Chicago, IL, USA 3Committee on Evolutionary Biology, The University of Chicago, Chicago, IL, USA 4Department of Human Genetics, The University of Chicago, Chicago, IL, USA ‡These authors contributed equally to this work *Corresponding authors. Department of Ecology and Evolution, The University of Chicago, 1101 E 57th Street, Chicago, IL 60637, USA. Tel.:+1 773 834 3913; Fax:+1 773 834 2877; E-mail: [email protected] or Tel.:+1 773 702 0557; Fax:+1 773 702 9740; E-mail: [email protected] The EMBO Journal (2012)31:2798-2809https://doi.org/10.1038/emboj.2012.108 Present address: Department of Biology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02139, USA PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info New genes originate frequently across diverse taxa. Given that genetic networks are typically comprised of robust, co-evolved interactions, the emergence of new genes raises an intriguing question: how do new genes interact with pre-existing genes? Here, we show that a recently originated gene rapidly evolved new gene networks and impacted sex-biased gene expression in Drosophila. This 4–6 million-year-old factor, named Zeus for its role in male fecundity, originated through retroposition of a highly conserved housekeeping gene, Caf40. Zeus acquired male reproductive organ expression patterns and phenotypes. Comparative expression profiling of mutants and closely related species revealed that Zeus has recruited a new set of downstream genes, and shaped the evolution of gene expression in germline. Comparative ChIP-chip revealed that the genomic binding profile of Zeus diverged rapidly from Caf40. These data demonstrate, for the first time, how a new gene quickly evolved novel networks governing essential biological processes at the genomic level. Introduction As major contributors to adaptation, genes with novel biological functions often emerge during evolution (Long et al, 2003; Kaessmann et al, 2009), acting as new catalytic enzymes (Burki and Kaessmann, 2004; Zhang et al, 2004; Matsuno et al, 2009), new cellular components (Marques et al, 2008; Rosso et al, 2008) or acquiring antiviral immunity functions (Stremlau et al, 2004). These genes frequently recruited regulatory elements from different sources and acquired expression in various tissues (Kaessmann, 2010), especially in male reproductive organs (Vibranovski et al, 2009a; Kaessmann, 2010). Recently, the evolutionary roles of several new genes have been characterized. They can contribute to substrate specificity alteration (Zhang et al, 2004), diet shift (Zhang, 2006), resolving intralocus sexual antagonistic conflict (Gallach et al, 2010), or modulating male-courtship behaviour (Dai et al, 2008). Furthermore, although genetic networks are typically viewed to be robust (Kitano, 2004; Wagner, 2005), evidence shows that some small and local structures in certain pathways are evolutionarily mutable (Matsuno et al, 2009; Ding et al, 2010). However, it is unclear if gene networks can undergo extensive changes on short evolutionary time scales, or just evolve slowly under constraint by the robustness. It is tempting to ask if new genes could integrate into complex biological networks and bring evolutionary innovation to the global network structure. Specifically, how and why new genes interact with pre-existing genes are poorly understood. Here, we describe how a young gene rapidly evolved extensive new gene–gene interactions in the genome and reshaped global sex-biased gene expression. The Zeus (CG9573, Drcd-1-r) gene was initially identified as a young retrogene that is linked to a male-fertility locus (Bai et al, 2007; Quezada-Diaz et al, 2010). We functionally and phenotypically characterized Zeus as well as Caf40, the protein encoded by its parental locus (Caf40, CG14213, or Drcd-1), and examined the role of Zeus in the evolution of the male reproductive network. The interactions of Zeus with the genome provided an opportunity to understand the evolutionary targets of a new gene that has integrated into pre-existing global gene networks. Results Origin and rapid evolution of a nascent retrogene, Zeus Zeus originated 4–6 million years (Myr) ago, from a parental gene Caf40 (CG14213) by an out-of-X chromosome retroposition event in Drosophila (Bai et al, 2007; Figure 1A) The parental gene Caf40 is ancient, shared by a broad range of taxa including yeasts, worms, flies, and mammals (Chen et al, 2001; Garces et al, 2007). While Caf40 is highly conserved in all eukaryotes, the new duplicate, Zeus, evolved rapidly (Quezada-Diaz et al, 2010). We mapped the nucleotide substitutions of Zeus and Caf40 along all major lineages of the D. melanogaster group, and detected an initial burst of 81 replacement substitutions in 1–2 Myr (Figure 1B; Supplementary Figure S1). We sequenced 13 African and cosmopolitan D. melanogaster lines and observed a significant excess of between-species non-synonymous substitutions compared with within-species polymorphisms (Supplementary Table S1; McDonald–Kreitman (MK) test, P=0.00007). These data suggest that strong positive selection has dominated the evolution of Zeus. Figure 1.Origin and adaptive evolution of Zeus. Schematic representation of the origin and subsequent evolution of Zeus. (A) Retroposition event that led to the origin and gene structure evolution of Zeus. Green boxes represent exons of the parental gene Caf40; orange boxes represent exons of the new gene Zeus; grey boxes represent adjacent genes; boxes were not drawn to scale. (B) Sequence evolution of Zeus and Caf40 in the Drosophila phylogeny; replacement (R)/silent (S) substitutions are shown near major internal branches; R/S polymorphism data are shown at external branches where applicable. (C) Substitutions in binding domain and binding evolution: the left panel shows the homology models of the 3D structures of Zeus, Caf40 and the inferred ancestral protein; the numbers on tree branches indicate amino-acid substitutions in those particular lineages, '2 indels' stands for a 9 amino-acid (aa) deletion at the N-terminus and a 6-aa insertion at the C-terminus; the right panel shows the zoom-in view for the amino-acid (aa) substitutions in the nucleic acid binding groove of the Zeus protein; aa substitutions fixed by positive Darwinian selection are represented as spheres and colour coded according to charge/polarity properties. Download figure Download PowerPoint Gene expression pattern of Zeus We then examined the expression pattern of Zeus using enhancer trap line, in which the Gal4 or LacZ element were inserted at the promoter region of 5′-UTR of the gene (Materials and methods). We found that Zeus is mainly expressed in the male reproductive system, including the testes and accessory glands (Figure 2A; Supplementary Figure S2). High levels of Zeus expression were detected during the entire process of spermatogenesis from mitotic spermatogonia, primary spermatocytes, differentiating spermatids, to mature sperm (Figure 2A; Supplementary Figure S2). We confirmed the expression patterns with RT–PCR (Supplementary Figure S2). This expression pattern is consistent with public microarray data (Chintapalli et al, 2007; Supplementary Figure S2) and high-throughput in-situ data from the literature (Tomancak et al, 2002). The male gonad expression of Zeus implies a specific role in male reproduction, and we went on to further test this hypothesis. Figure 2.Characterization of expression and phenotype of Zeus. (A) Expression pattern of Zeus in adult testis, blue arrows point at primary spermatocytes (left), early spermatids (middle) and mature sperms (right). (B) Testis phenotype of Zeus: top panel, wild-type testis overall morphology (left panel, phase contrast) and nuclei (right panel, DAPI staining); bottom panel, constitutive (Act5C-Gal4 driven) Zeus-RNAi testis overall morphology showing ectopic misgrowth at the tip (left panel, phase contrast) and irregular sperm bundles (right panel, DAPI staining). Red arrowheads point to representative abnormalities. (C) Sperm development phenotype shows misorganized sperm bundles (yellow arrows) in a Zeus-RNAi testis. (D) RT–PCR showing in-vivo transcription levels of Zeus, Caf40, CG13102 and gapdh1 in constitutive Zeus-RNAi and control animals. (E) Fecundity phenotype of constitutive Zeus-RNAi animals showing increased male sterility (left), reduced male fertility (middle), and normal female fertility (right); genotypes are shown below each data column; error bars on the column represent standard errors of the mean (s.e.m.); brackets denote statistical comparisons (ANOVA, *P<0.01). (F) Fecundity phenotype of tissue-specific Zeus-RNAi (left), RNAi-rescue (middle) and Zeus EMS mutants (right): nanos-Gal4-Zeus-RNAi; Acp26Aa(X)-Gal4-Zeus-RNAi; nos-Zeus-RNAi; Caf40-Overexpress, nanos-Gal4-Zeus-RNAi with Caf40 overexpression; Q94*, P50L, L182F, and Q110* are mutants with respective mutations in the Zeus protein sequences. Download figure Download PowerPoint Phenotypic characterization of Zeus in male reproduction To investigate the phenotype of Zeus, we used RNA interference (RNAi) to knockdown its expression (Dietzl et al, 2007). Constitutive RNAi against Zeus caused a 70% fertility reduction in male flies compared with wild-type and related controls (ANOVA, P<0.001 in each comparison; Figure 2E), with no detectable effect in females (Figure 2E). Germline-specific (nos-Gal4) Zeus RNAi also produced strong and significant male fertility defect, while accessory gland-specific (Acp26Aa-Gal4) RNAi did not (Figure 2F), suggesting that the primary fecundity function of Zeus lies in the testes. We carried out RT–PCR and verified that in Zeus-RNAi animals, the mRNA levels of Zeus were significantly reduced, while neither the parental gene Caf40 nor its overlapping gene (CG13102) was affected (Figure 2D). Moreover, the Zeus reproductive phenotype was recapitulated by presumptive Zeus null mutants, including two premature stop codons (Q94*, Q110*) and two missense mutations (P50L, L182F) that changed the polarity of amino acids (Figure 2F; Supplementary Figure S2). These point mutation alleles failed to complement each other, or previously isolated P-element insertion at the 5′-UTR of Zeus, which also caused male sterility (Castrion, 1993; Bai et al, 2007) as we confirmed independently (Supplementary Figure S2). Furthermore, testes from Zeus knockdown male flies showed specific defects in structure, including the disorganized cysts, tumour formation, and/or misoriented sperm bundles (Figure 2B and C). Both Zeus knockdown and mutant males can produce motile sperm, but they either fail to fertilize wild-type female eggs, or the fertilized eggs fail to develop (Supplementary Figure S2). These data suggest that Zeus plays an important role in male reproduction, probably functioning in the late stages of spermatogenesis or fertilization. Thus, despite of its recent origin, Zeus is essential for male fitness. Expression pattern and phenotype of the parental gene, Caf40 As a comparison, we also investigated the function and phenotype of the parental gene Caf40, which is highly conserved, and thus possibly performs the ancestral function. The expression patterns and phenotypes of Caf40 are dramatically different from Zeus. First, Caf40 is expressed throughout the life cycle in most animal tissues, particularly at high levels in the musculature, digestive system, appendages, and central and peripheral nervous systems but only weakly in the reproductive tract (Figure 3A). Second, constitutive knockdown of Caf40 led to lethality at the onset of pupation (Figure 3B; Supplementary Table S2), and tissue-specific knockdown in the adult eye lead to missing sensory bristles and aberrant ommatidia development (Supplementary Figure S3). Third, germline knockdown of Caf40 did not produce significant male fertility defect (Supplementary Figure S3). Finally, ectopic expression of Caf40 failed to rescue Zeus male reproduction defects (Figure 2F). These data suggest that, instead of being a simple and redundant copy of Caf40, the new gene Zeus has evolved the capability to influence a distinct phenotype and established a unique role in the organism. Figure 3.Characterization of expression and phenotype of the parental gene Caf40. (A) Caf40 expression during several major stages of the life cycle, including early larvae (a1), late larvae (a2), pupae (a3) and adult (a4), and in several adult tissues nervous system (a5, MB, mushroom body; OL, optic lobe), digestive system (a6), musculature (a7), and weakly in reproductive organs (a8); (a4) note the Caf40::GFP fly showing strong constitutive GFP signal (red arrowhead) and the control fly showing no fluorescence (blue arrow). (B) Prepupae lethal phenotype of Caf40: top panel, normal pupae development of wild-type Can-S (top left) and non-induced control animals (in the same cross as Caf40-RNAi, top right); black arrow heads point to developing head/eye structures; bottom panel, disrupted pupae development of Caf40-RNAi animals under a constitutive driver (Act5C-Gal4), black arrow heads point to necrotic larval head structures (without formation of adult-like head/eye structures); blue arrows point to the empty pupal cuticle of control flies, which fully developed and hatched 7 days after pupation in the same cross; d AP, days after pupation. Download figure Download PowerPoint Perturbation of global gene expression pattern in Zeus animals The new phenotype of the young gene Zeus suggested that the genetic network has changed since its origin, because obviously the ancestral organisms were able to reproduce without the gene. This allowed us to test the concept of network evolution driven by new gene origination. To understand how the global gene expression network was affected when Zeus was perturbed, we carried out expression profiling of the transcriptome of Zeus-RNAi testes, where Zeus is primarily expressed (Materials and methods). In the set of genes differentially expressed between Zeus knockdown and controls (Supplementary Table S5), we found that female-biased genes were highly enriched among the RNAi upregulated genes, whereas male-biased genes were highly enriched in the RNAi downregulated genes (Supplementary Figure S4; Supplementary Table S6, χ2 tests, P=0 for both female- and male-biased gene tests). Moreover, the upregulated, female-biased genes were enriched on the X chromosome, whereas the downregulated, male-biased genes were overrepresented on autosomes (Supplementary Table S6, χ2 test, P=0.0001), consistent with previously reported chromosomal distribution patterns of sex-biased genes (Vibranovski et al, 2009b). These data suggest that Zeus tends to repress female-biased genes and activate male-biased genes in male reproductive organs. To investigate the evolution of gene regulation after retroposition, we knocked down Zeus and Caf40 in parallel with a germline-specific driver and profiled the global gene expression in testes samples (Materials and methods). Compared with controls, Zeus-RNAi reduced 80% of Zeus mRNA, but did not affect Caf40 expression level; and vice versa for Caf40-RNAi. These data sets enabled us to identify distinct downstream genes for each factor. We identified a large set of genes that were significantly differentially expressed (multiple testing corrected P-value of 1). We also identified a set of downstream genes for Caf40. We found that 49.4% (444/899) of Zeus' downstream genes are distinct from Caf40's (Figure 4A), indicating that half of the Zeus downstream genes are not regulated by Caf40. For the genes co-regulated by both factors, the magnitudes, or even the directions of downstream gene expression changes may differ (i.e., upregulation in Zeus and downregulation in Caf40; Figure 4A). These data suggest that Zeus evolved to regulate a largely distinct gene set, implying its new role related to gene regulation in the male germline. Figure 4.Evolution of global gene regulation of Zeus in male germline. (A) Heatmap of male-germline RNA-seq expression profiling showing evolution of globally target genes and altered gene expression between Zeus and Caf40; top panel, expression profile of Zeus-specific target genes; middle panel, Zeus/Caf40 shared target genes; bottom panel, Caf40-specific target genes; left columns, nanosGal4»Zeus-RNAi; middle column, control; right column, nanosGal4»Caf40-RNAi; colour key and histogram with respect to the expression distribution were shown at bottom-right corner; microarray data (not shown) recapitulate consistent patterns; (B) heatmap of male-germline RNA-seq expression profiling of closely related species showing the influence of Zeus on its target genes after gene origination and speciation; top panel, expression profile of Zeus-suppressed orthologous target genes; bottom panel, expression profile of Zeus-activated orthologous target genes; left columns, nanosGal4»Zeus-RNAi; middle column, D. melanogaster wild type; right column, D. simulans wild type; rightmost column, D. yakuba wild type; colour key and histogram with respect to the expression distribution were shown at bottom-right corner; microarray data (not shown) recapitulate consistent patterns. (heatmaps represent difference between samples, gene number not drawn to scale) Download figure Download PowerPoint Genome-wide binding profile of Zeus To understand how Zeus integrated into the biological network, we then investigated how Zeus physically interacts with existing genes in the genome. We first examined where the Zeus protein is localized in the cell. We generated transgenic fly lines expressing GFP-tagged Zeus recombinant protein and found that the majority of Zeus protein is localized in the nucleus (Figure 5A). Being descended from Caf40, the Zeus protein has an Rcd1-like domain (Rcd, Retinoid acid-induced Cell Differentiation). Rcd1 homologues function as transcriptional regulators of cellular differentiation (Liu et al, 1998; Okazaki et al, 1998; Hiroi et al, 2002; Garces et al, 2007). Previous structural analysis revealed that an Rcd1 domain has six Armadillo-like repeats, forming a nucleic acid binding groove (Garces et al, 2007). Gel-shift assays demonstrated that Rcd1 domain physically binds to DNA (Chen et al, 2001; Garces et al, 2007). The putative nucleic acid binding groove of Zeus has experienced excessive amino-acid substitutions fixed by positive selection (Figure 1C; Supplementary Figure S1). Most (91%) of these novel amino acids were either charged or polar residues on the surface of the groove, forming a distinct surface conformation of Zeus (Figure 1C). We then set out to determine where the Zeus protein associates with chromosomes in the cell, and, ultimately how these adaptive amino-acid changes might have led to evolution in binding. We generated transgenic fly lines that expressed 3 × FLAG-tagged Zeus protein in vivo, under the control of an upstream activating sequence (UAS) promoter. We expressed the recombinant proteins either constitutively (Act5C-Gal4 driver) or in a tissue-specific manner (germline driver or accessory gland driver), and confirmed the detection of the recombinant protein (Figure 5B). Figure 5.Chromosomal binding profiles of Zeus and Caf40. (A) Confocal images of Zeus–GFP (green, GFP) localization with respect to the nucleus (blue, DAPI), scale bars are 10 μm. (B) purification and detection of FLAG-tagged Zeus protein in transgenic Drosophila male adults. (C) Overview of genome-wide binding profiles of Zeus (red) and Caf40 (blue) on all four chromosomes in D. melanogaster; black scale bar represents 5 Mb of chromosome. (D) Representative conserved (left), Caf40-specific (middle) and Zeus-specific (right) peaks/binding sites; track colour coding: (from top to bottom) Zeus-IP track (red), Zeus binding profile; Caf40-IP track (blue), Caf40 binding profile coordinate (black), base positions in the chromosome; Ref-seq(+/−) of the annotated D. melanogaster genome (green). (E) Venn diagrams showing the number and overlap of binding target genes of Zeus and Caf40. Download figure Download PowerPoint We performed ChIP-chip (chromatin immunoprecipitation followed by microarray hybridization) to identify the global binding profile of Zeus, using an anti-FLAG antibody. This avoids cross-detection of endogenous Caf40 binding due to protein sequence similarity. We identified 363 peaks (putative binding sites/regions) for Zeus at a cutoff P-value of 1 × 10−3 (Figure 5C–E; Supplementary Figure S5; Supplementary Tables S7 and S8). Almost all of them (362/363, 99.7%) can be found within or near annotated genes (Figure 5C–E; Supplementary Figure S5). With this criterion, we identified 322 genes with Zeus binding sites as Zeus-binding genes (Figure 5E). Unexpectedly, although these binding sites/genes were distributed across all major chromosomal arms, they were highly overrepresented on the X chromosome (Supplementary Table S9) (120/363, 77% excess over random expectation, χ2 test, P=4.0 × 10−11). Because the X chromosome is enriched with female-biased genes (Sturgill et al, 2007; Vibranovski et al, 2009b), this binding pattern is in concordance with the sex-biased regulation of Zeus downstream genes. Evolution of Zeus downstream targets To study the evolution of Zeus downstream targets, we carried out transgenic experiments and ChIP-chip for Caf40 in parallel. By comparing the genome-wide binding profiles of Zeus and Caf40, we found many clear examples of conservation and divergence (Figure 5C and D; Supplementary Figure S5; Supplementary Tables S7 and S8). We found that Zeus' putative targets, the genes bound by Zeus protein as revealed from ChIP, are largely different from those of Caf40's, diverging by 60%. These data revealed that, after origination, Zeus quickly recruited many pre-existing genes as its targets in the genome and established novel functional gene–gene interactions. By intersecting the ChIP-chip and expression profiling data sets, we sought to discover putative Zeus direct targets, which were physically associated with Zeus protein and differentially expressed upon Zeus knockdown (Supplementary Tables S10 and S11). Interestingly, we found that Zeus direct targets were predominantly upregulated (51 out of 63 genes) in Zeus-RNAi samples (Fisher's exact test, P=0.0003; Supplementary Figure S6), suggesting that the direct action of Zeus tends to repress. We analysed Zeus and its downstream genes during spermatogenesis. Zeus expression decreases from the meiotic stage to the post-meiotic stage. Zeus downstream genes were enriched in the increasing expression category at the stage transition compared with randomly simulated genes (37.9% versus 28.7%, χ2 test, P=0.0003), consistent with putatative, repressive action. Role of Zeus in the evolution of male germline gene expression Structural modelling with evolutionary inference revealed that the putative nucleic acid binding groove of Zeus experienced excessive amino-acid substitutions that have been fixed by positive selection. Most (91%) of these novel amino acids are either charged or polar residues on the surface of the groove, forming a distinct surface conformation of Zeus (Figure 1C). In contrast, the Caf40 binding domain did not change in 25 Myr, and the whole protein has remained evolutionarily stagnant. Caf40 downstream genes are also much more conserved than Zeus downstream genes or randomly simulated genes (Supplementary Figure S5). These data imply that Zeus gained a large set of distinct gene–gene interactions in the last several million years. We also analysed the Zeus and Caf40 target gene sets with the stage-specific spermatogenesis expression data (Vibranovski et al, 2009a). During spermatogenesis, the relative expression level of Zeus compared with Caf40 (Zeus/Caf40) decreases during meiosis/post-meiosis stage (Supplementary Table S12). Accordingly, Zeus' target genes'/downstream genes' expression tend to increase compared with Caf40's (Supplementary Table S12), in concordance with the fact that Zeus primarily acts as a repressor. We then sought to detect evolutionary signatures of Zeus in the evolution of gene regulation across multiple Drosophila species. Using RNA-seq, we performed expression profiling with testes samples from four representative species with or without Zeus, which are, D. melanogaster, D. simulans, and D. yakuba. We analysed the orthologues of Zeus downstream genes in these species. Among genes activated by Zeus in D. melanogaster, most (73.6%, or 167/227) are expressed at significantly lower levels in D. yakuba (Figure 4B; Supplementary Figure S4; Supplementary Table S13). On the other hand, among genes repressed by Zeus in D. melanogaster, the majority (58.4%, or 115/197) has higher expression in D. yakuba (Figure 4B; Supplementary Figure S4; Supplementary Table S13). These results revealed that the evolution of the gene expression of Zeus targets is correlated with the presence/absence of Zeus in closely related species (Fisher's exact test, P=4.0 × 10–12). These results were confirmed by expression profiling with custom-designed microarrays for these species. These data suggested that, although the divergence b
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