Promoter generation for the chimeric sex-determining gene dm-W in Xenopus frogs.

Many sex-determining genes (SDGs) were generated as neofunctionalized genes through duplication and/or mutation of gonadal formation-related genes. We previously identified dm-W as an SDG in the African clawed frog Xenopus laevis and found that a partial duplication of the masculinization gene dmrt1 created the neofunctionalized dm-W after allotetraploidization by interspecific hybridization. The allotetraploid Xenopus species have two dmrt1 genes, dmrt1.L and dmrt1.S. Xenopus laevis dm-W has four exons: two dmrt1.S-derived exons (exons 2 and 3) and two other exons (noncoding exon 1 and exon 4). Our recent work revealed that exon 4 originated from a DNA transposon, hAT-10. Here, to clarify when and how the noncoding exon 1 and its coexisting promoter evolved during the establishment of dm-W after allotetraploidization, we newly determined nucleotide sequences of the dm-W promoter region from two other allotetraploid species, X. largeni and X. petersii, and performed an evolutionary analysis. We found that dm-W acquired a new exon 1 and TATA-type promoter in the common ancestor of the three allotetraploid Xenopus species, resulting in the deletion of the dmrt1.S-derived TATA-less promoter. In addition, we demonstrated that the TATA box contributes to dm-W promoter activity in cultured cells. Collectively, these findings suggest that this novel TATA-type promoter was important for the establishment of dm-W as a sex-determining gene, followed by the degeneration of the preexisting promoter.

Heat shock factor 1 induces a short burst of transcription of the clock gene Per2 during interbout arousal in mammalian hibernation.

During winter hibernation, a diverse range of small mammals can enter prolonged torpor. They spend the nonhibernation season as a homeotherm but the hibernation season as a heterotherm. In the hibernation season, chipmunks (Tamias asiaticus) cycle regularly between 5 and 6 days-long deep torpor with a body temperature (Tb) of 5 to 7 °C and interbout arousal of ∼20 h, during which, their Tb returns to the normothermic level. Here, we investigated Per2 expression in the liver to elucidate the regulation of the peripheral circadian clock in a mammalian hibernator. In the nonhibernation season, as in mice, heat shock factor 1, activated by elevated Tb during the wake period, activated Per2 transcription in the liver, which contributed to synchronizing the peripheral circadian clock to the Tb rhythm. In the hibernation season, we determined that the Per2 mRNA was at low levels during deep torpor, but Per2 transcription was transiently activated by heat shock factor 1, which was activated by elevated Tb during interbout arousal. Nevertheless, we found that the mRNA from the core clock gene Bmal1 exhibited arrhythmic expression during interbout arousal. Since circadian rhythmicity is dependent on negative feedback loops involving the clock genes, these results suggest that the peripheral circadian clock in the liver is nonfunctional in the hibernation season.

Parallel Evolution of Sex-Linked Genes across XX/XY and ZZ/ZW Sex Chromosome Systems in the Frog Glandirana rugosa.

Genetic sex-determination features male (XX/XY) or female heterogamety (ZZ/ZW). To identify similarities and differences in the molecular evolution of sex-linked genes between these systems, we directly compared the sex chromosome systems existing in the frog Glandirana rugosa. The heteromorphic X/Y and Z/W sex chromosomes were derived from chromosomes 7 (2n = 26). RNA-Seq, de novo assembly, and BLASTP analyses identified 766 sex-linked genes. These genes were classified into three different clusters (XW/YZ, XY/ZW, and XZ/YW) based on sequence identities between the chromosomes, probably reflecting each step of the sex chromosome evolutionary history. The nucleotide substitution per site was significantly higher in the Y- and Z-genes than in the X- and W- genes, indicating male-driven mutation. The ratio of nonsynonymous to synonymous nucleotide substitution rates was higher in the X- and W-genes than in the Y- and Z-genes, with a female bias. Allelic expression in gonad, brain, and muscle was significantly higher in the Y- and W-genes than in the X- and Z-genes, favoring heterogametic sex. The same set of sex-linked genes showed parallel evolution across the two distinct systems. In contrast, the unique genomic region of the sex chromosomes demonstrated a difference between the two systems, with even and extremely high expression ratios of W/Z and Y/X, respectively.

Evolutionary features of ligands and their receptors via protein-protein interactions and essentiality in primates.

Protein evolution rate is negatively correlated with several effectors, such as expression level, expression distribution, protein-protein interactions (PPIs), and essentiality for survival. These effectors can characterize the signaling pathways mediated by ligand-receptor binding. However, it is unclear whether these effectors are constraining factors on the pathway-specific evolution of ligands and receptors. To clarify the relation between the effectors and protein evolution (dN /dS ratio) in ligands and their receptors considering each signaling pathway, we investigated 377 proteins in 20 peptide/protein ligand groups and their receptor groups using 15 primate sequences. The dN /dS ratios between peptide/protein ligand groups and their receptor groups were positively correlated, suggesting the protein evolution under the influence of signaling pathway to which they belong. Comparing each signaling pathway, ligands and receptors mainly related to development and growth (FGF/Hedgehog/Notch/WNT groups) showed lower dN /dS ratios, higher PPI numbers, and higher essentiality, whereas those mainly related to immune process (CSF/IFN/IL/TNF groups) showed higher dN /dS ratios, lower PPI numbers, and lower essentiality. Most ligands and receptors were poorly expressed, and expression level was not a constraining factor on the protein evolution. These findings indicate that PPI and essentiality are constraining factors that characterize the pathway-specific evolution of ligands and receptors.

Identification of ancestral sex chromosomes in the frog Glandirana rugosa bearing XX-XY and ZZ-ZW sex-determining systems.

Sex chromosomes constantly exist in a dynamic state of evolution: rapid turnover and change of heterogametic sex during homomorphic state, and often stepping out to a heteromorphic state followed by chromosomal decaying. However, the forces driving these different trajectories of sex chromosome evolution are still unclear. The Japanese frog Glandirana rugosa is one taxon well suited to the study on these driving forces. The species has two different heteromorphic sex chromosome systems, XX-XY and ZZ-ZW, which are separated in different geographic populations. Both XX-XY and ZZ-ZW sex chromosomes are represented by chromosome 7 (2n = 26). Phylogenetically, these two systems arose via hybridization between two ancestral lineages of West Japan and East Japan populations, of which sex chromosomes are homomorphic in both sexes and to date have not yet been identified. Identification of the sex chromosomes will give us important insight into the mechanisms of sex chromosome evolution in this species. Here, we used a high-throughput genomic approach to identify the homomorphic XX-XY sex chromosomes in both ancestral populations. Sex-linked DNA markers of West Japan were aligned to chromosome 1, whereas those of East Japan were aligned to chromosome 3. These results reveal that at least two turnovers across three different sex chromosomes 1, 3 and 7 occurred during evolution of this species. This finding raises the possibility that cohabitation of the two different sex chromosomes from ancestral lineages induced turnover to another new one in their hybrids, involving transition of heterogametic sex and evolution from homomorphy to heteromorphy.

Neofunctionalization of a Noncoding Portion of a DNA Transposon in the Coding Region of the Chimerical Sex-Determining Gene dm-W in Xenopus Frogs.

Most vertebrate sex-determining genes (SDGs) emerge as neofunctionalized genes through duplication and/or mutation of ancestral genes that are involved with sexual differentiation. We previously demonstrated dm-W to be the SDG in the African clawed frog Xenopus laevis and found that a portion of this gene emerged from the masculinization gene dmrt1 after allotetraploidization by interspecific hybridization between two ancestral species around 17-18 Ma. dm-W has four exons consisting of a noncoding exon 1, dmrt1-derived exons 2 and 3, and an orphan exon 4 (Ex4) of unknown origin that includes coding sequence (CDS). In this study, we searched for the origin of Ex4 and investigated the function of the CDS of this exon. We found that the Ex4-CDS is derived from a noncoding portion of the hAT-10 family of DNA transposon. Evolutionary analysis of transposons and determination of the Ex4 sequences from three other species indicated that Ex4 was generated before the diversification of most or all extant allotetraploid species in subgenus Xenopus, during which time we hypothesize that transposase activity of this hAT superfamily was active. Using DNA-protein binding and transfection assays, we further demonstrate that the Ex4-encoded amino acid sequence increases the DNA-binding ability and transrepression activity of DM-W. These findings suggest that the conversion of the noncoding transposon sequence to the CDS of dm-W contributed to neofunctionalization of a new chimeric SDG in the ancestor of the allotetraploid Xenopus species, offering new insights into de novo origin and functional evolution of chimerical genes.

Activation of DNA Transposons and Evolution of piRNA Genes Through Interspecific Hybridization in Xenopus Frogs.

Interspecific hybridization between two closely related species sometimes resulted in a new species with allotetraploid genomes. Many clawed frog species belonging to the Xenopus genus have diverged from the allotetraploid ancestor created by the hybridization of two closely related species with the predicted L and S genomes. There are species-specific repeated sequences including transposable elements in each genome of organisms that reproduce sexually. To understand what happened on and after the hybridization of the two distinct systems consisting of repeated sequences and their corresponding piRNAs, we isolated small RNAs from ovaries and testes of three Xenopus species consisting of allotetraploid X. laevis and X. borealis and diploid X. tropicalis as controls. After a comprehensive sequencing and selection of piRNAs, comparison of their sequences showed that most piRNA sequences were different between the ovaries and testes in all three species. We compared piRNA and genome sequences and specified gene clusters for piRNA expression in each genome. The synteny and homology analyses showed many distinct piRNA clusters among the three species and even between the two L and/or S subgenomes, indicating that most clusters of the two allotetraploid species changed after hybridization. Moreover, evolutionary analysis showed that DNA transposons including Kolobok superfamily might get activated just after hybridization and then gradually inactivated. These findings suggest that some DNA transposons and their piRNAs might greatly influence allotetraploid genome evolution after hybridization.

Independent pseudogenizations and losses of sox15 during amniote diversification following asymmetric ohnolog evolution.

BACKGROUND: Four ohnologous genes (sox1, sox2, sox3, and sox15) were generated by two rounds of whole-genome duplication in a vertebrate ancestor. In eutherian mammals, Sox1, Sox2, and Sox3 participate in central nervous system (CNS) development. Sox15 has a function in skeletal muscle regeneration and has little functional overlap with the other three ohnologs. In contrast, the frog Xenopus laevis and zebrafish orthologs of sox15 as well as sox1-3 function in CNS development. We previously reported that Sox15 is involved in mouse placental development as neofunctionalization, but is pseudogenized in the marsupial opossum. These findings suggest that sox15 might have evolved with divergent gene fates during vertebrate evolution. However, knowledge concerning sox15 in other vertebrate lineages than therian mammals, anuran amphibians, and teleost fish is scarce. Our purpose in this study was to clarify the fate and molecular evolution of sox15 during vertebrate evolution. RESULTS: We searched for sox15 orthologs in all vertebrate classes from agnathans to mammals by significant sequence similarity and synteny analyses using vertebrate genome databases. Interestingly, sox15 was independently pseudogenized at least twice during diversification of the marsupial mammals. Moreover, we observed independent gene loss of sox15 at least twice during reptile evolution in squamates and crocodile-bird diversification. Codon-based phylogenetic tree and selective analyses revealed an increased dN/dS ratio for sox15 compared to the other three ohnologs during jawed vertebrate evolution. CONCLUSIONS: The findings revealed an asymmetric evolution of sox15 among the four ohnologs during vertebrate evolution, which was supported by the increased dN/dS values in cartilaginous fishes, anuran amphibians, and amniotes. The increased dN/dS value of sox15 may have been caused mainly by relaxed selection. Notably, independent pseudogenizations and losses of sox15 were observed during marsupial and reptile evolution, respectively. Both might have been caused by strong relaxed selection. The drastic gene fates of sox15, including neofunctionalization and pseudogenizations/losses during amniote diversification, might be caused by a release from evolutionary constraints.

Hybridogenesis in the Water Frogs from Western Russian Territory: Intrapopulation Variation in Genome Elimination.

Hybridogenesis in an interspecific hybrid frog is a coupling mechanism in the gametogenic cell line that eliminates the genome of one parental species with endoduplication of the remaining genome of the other parental species. It has been intensively investigated in the edible frog Pelophylax kl. esculentus (RL), a natural hybrid between the marsh frog P. ridibundus (RR) and the pool frog P. lessonae (LL). However, the genetic mechanisms involved remain unclear. Here, we investigated the water frogs in the western Russian territory. In three of the four populations, we genetically identified 16 RL frogs living sympatrically with the parental LL species, or with both parental species. In addition, two populations contained genome introgression with another species, P. bedriagae (BB) (a close relative of RR). In the gonads of 13 RL frogs, the L genome was eliminated, producing gametes of R (or R combined with the B genome). In sharp contrast, one RL male eliminated the L or R genome, producing both R and L sperm. We detected a variation in genome elimination within a population. Based on the genetic backgrounds of RL frogs, we hypothesize that the introgression of the B genome resulted in the change in choosing a genome to be eliminated.

PACT/PRKRA and p53 regulate transcriptional activity of DMRT1.

The transcription factor DMRT1 (doublesex and mab-3 related transcription factor) has two distinct functions, somatic-cell masculinization and germ-cell development in some vertebrate species, including mouse and the African clawed frog Xenopus laevis. However, its transcriptional regulation remains unclear. We tried to identify DMRT1-interacting proteins from X. laevis testes by immunoprecipitation with an anti-DMRT1 antibody and MS/MS analysis, and selected three proteins, including PACT/PRKRA (Interferon-inducible double-stranded RNA dependent protein kinase activator A) derived from testes. Next, we examined the effects of PACT/PRKRA and/or p53 on the transcriptional activity of DMRT1. In transfected 293T cells, PACT/PRKRA and p53 significantly enhanced and repressed DMRT1-driven luciferase activity, respectively. We also observed that the enhanced activity by PACT/PRKRA was strongly attenuated by p53. Moreover, in situ hybridization analysis of Pact/Prkra mRNA in tadpole gonads indicated high expression in female and male germline stem cells. Taken together, these findings suggest that PACT/PRKRA and p53 might positively and negatively regulate the activity of DMRT1, respectively, for germline stem cell fate.

Parallel Evolution of Two dmrt1-Derived Genes, dmy and dm-W, for Vertebrate Sex Determination.

Animal sex-determining genes, which bifurcate for female and male development, are diversified even among closely related species. Most of these genes emerged independently from various sex-related genes during species diversity as neofunctionalization-type genes. However, the common mechanisms of this divergent evolution remain poorly understood. Here, we compared the molecular evolution of two sex-determining genes, the medaka dmy and the clawed frog dm-W, which independently evolved from the duplication of the transcription factor-encoding masculinization gene dmrt1. Interestingly, we detected parallel amino acid substitutions, from serine (S) to threonine (T), on the DNA-binding domains of both ancestral DMY and DM-W, resulting from positive selection. Two types of DNA-protein binding experiments and a luciferase reporter assay demonstrated that these S-T substitutions could strengthen the DNA-binding abilities and enhance the transcriptional regulation function. These findings suggest that the parallel S-T substitutions may have contributed to the establishment of dmy and dm-W as sex-determining genes.

Independent evolution for sex determination and differentiation in the DMRT family in animals.

Some DMRT family genes including arthropod dsx, nematode mab-3, and vertebrate dmrt1 are involved in sex determination and/or differentiation in bilaterian animals. Although there have been some reports about evolutionary analyses of the family by using its phylogenetic trees, it is still undecided as to whether these three sex determination-related genes share orthologous relationships or not. To clarify this question, we analyzed evolutional relationships among the family members in various bilaterians by using not only phylogenetic tree analysis, but also synteny analysis. We found that only four genes, dmrt2a/2b, dmrt3, dmrt4/ 5 and dmrt93B were commonly present in invertebrate bilateria. The syntenies of dmrt2a/2b-dmrt3 and dmrt4/5-dmrt93B are conserved before and after two rounds of whole genome duplication in the ancestral vertebrate. Importantly, this indicates that dmrt1 must have appeared in the common vertebrate ancestor. In addition, dmrt1, dsx, or mab-3 formed each different cluster at a distance in our phylogenetic tree. From these findings, we concluded that the three sex determination-related genes, dmrt1, dsx, and mab-3 have no orthologous relationships, and suggested independent evolution for sex determination and differentiation in the DMRT gene family. Our results may supply clues about why sex-determining systems have diverged during animal evolution.

Circadian transcription factor HSF1 regulates differential HSP70 gene transcription during the arousal-torpor cycle in mammalian hibernation.

Mammalian hibernation is a seasonal phenomenon. The hibernation season consists of torpor periods with a reduced body temperature (Tb), interrupted by euthermic arousal periods (interbout arousal, IBA). The physiological changes associated with hibernation are assumed to be under genetic control. However, the molecular mechanisms that govern hibernation-associated gene regulation are still unclear. We found that HSP70 transcription is upregulated in the liver of nonhibernating (summer-active) chipmunks compared with hibernating (winter-torpid) ones. In parallel, HSF1, the major transcription factor for HSP70 expression, is abundant in the liver-cell nuclei of nonhibernating chipmunks, and disappears from the nuclei of hibernating ones. Moreover, during IBA, HSF1 reappears in the nuclei and drives HSP70 transcription. In mouse liver, HSF1 is regulated by the daily Tb rhythm, and acts as a circadian transcription factor. Taken together, chipmunks similarly use the Tb rhythm to regulate gene expression via HSF1 during the torpor-arousal cycle in the hibernation season.

Epigenetic regulation of hibernation-associated HP-20 and HP-27 gene transcription in chipmunk liver.

The chipmunk hibernation-related proteins (HPs) HP-20 and HP-27 are components of a 140-kDa complex that dramatically decreases in the blood during hibernation. The HP-20 and HP-27 genes are expressed specifically in the liver and are downregulated in hibernating chipmunks. Hibernation-associated physiological changes are assumed to be under genetic control. Therefore, to elucidate the molecular mechanisms of hibernation, here we examined the mechanisms behind the altered HP-20 and HP-27 gene expression in nonhibernating versus hibernating chipmunks. Chromatin immunoprecipitation (ChIP) analyses revealed that histone H3 on the HP-20 and HP-27 gene promoters was highly acetylated at lysine (K) 9 and K14 and highly trimethylated at K4 in the liver of nonhibernating chipmunks, while these active histone modifications were nearly absent in hibernating chipmunks. Furthermore, histone acetyltransferases and a histone methyltransferase were associated with the HP-20 and HP-27 gene promoters primarily in nonhibernating chipmunks. Consistent with a previous finding that HNF-1 and USF can activate HP-20 and HP-27 gene transcription by binding to the proximal promoter region, ChIP-quantitative PCR (qPCR) analyses revealed that significantly less HNF-1 and USF were bound to these gene promoters in hibernating than in nonhibernating chipmunks. These findings collectively indicated that the hibernation-associated HP-20 and HP-27 gene expression is epigenetically regulated at the transcriptional level by the binding of HNF-1 and USF to their proximal promoters, and that histone modification has a key role in hibernation-associated transcriptional regulation.

Masculinization-Related Genes and Cell-Mass Structures During Early Gonadal Differentiation in the African Clawed Frog Xenopus laevis.

The African clawed frog Xenopus laevis has a female heterogametic ZZ/ZW-type sex-determining system. We previously discovered a W-linked female sex-determining gene dm-W that is involved in ovary formation, probably through the up-regulation of the estrogen synthesis genes cyp19a1 and foxl2. We also reported that a unique "mass-in-line structure", which disappears from ZZ gonads during early testicular development, might serve as the basis for ovary differentiation in ZW gonads. However, the molecular mechanisms underlying early masculinization are poorly understood. To elucidate the development of bipotential gonads into testes after sex determination in this species, we focused on the orthologs of five mammalian sex-related genes: three nuclear factor genes, dax1, sf1 (also known as ad4bp), and sox9, and two genes encoding members of the tumor growth factor-ß (TGF-ß) family, anti-Müllerian hormone (amh) and inhibin ßb (inhbb). Quantitative RT-PCR analysis revealed that the expression of dax1, sox9, amh, and inhbb or sf1 was greatly or slightly higher in ZZ than in ZW gonads during early sex development. In situ hybridization analysis revealed that amh and inhbb mRNAs were expressed in somatic cells on the inner and outer sides of cell masses in the mass-in-line structure, respectively, in the developing ZZ gonads. Interestingly, estrogen exposure prevented the disappearance of the mass-in-line structure in early developing ZZ tadpoles. These findings suggest that TGF-ß signaling is involved in the destruction of the mass-in-line structure, which may be maintained by estrogen.

HNF-4 participates in the hibernation-associated transcriptional regulation of the chipmunk hibernation-related protein gene.

The chipmunk hibernation-related protein 25 (HP-25) is involved in the circannual control of hibernation in the brain. The liver-specific expression of the HP-25 gene is repressed in hibernating chipmunks under the control of endogenous circannual rhythms. However, the molecular mechanisms that differentially regulate the HP-25 gene during the nonhibernation and hibernation seasons are unknown. Here, we show that the hibernation-associated HP-25 expression is regulated epigenetically. Chromatin immunoprecipitation analyses revealed that significantly less hepatocyte nuclear receptor HNF-4 bound to the HP-25 gene promoter in the liver of hibernating chipmunks compared to nonhibernating chipmunks. Concurrently in the hibernating chipmunks, coactivators were dissociated from the promoter, and active transcription histone marks on the HP-25 gene promoter were lost. On the other hand, small heterodimer partner (SHP) expression was upregulated in the liver of hibernating chipmunks. Overexpressing SHP in primary hepatocytes prepared from nonhibernating chipmunks caused HNF-4 to dissociate from the HP-25 gene promoter, and reduced the HP-25 mRNA level. These results suggest that hibernation-related HP-25 expression is epigenetically regulated by the binding of HNF-4 to the HP-25 promoter, and that this binding might be modulated by SHP in hibernating chipmunks.

Sex chromosome differentiation and the W- and Z-specific loci in Xenopus laevis.

Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes.

Genome organization of the vg1 and nodal3 gene clusters in the allotetraploid frog Xenopus laevis.

Extracellular factors belonging to the TGF-ß family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-ß family genes in response to allotetraploidization.

Molecular Evolution of Two Distinct dmrt1 Promoters for Germ and Somatic Cells in Vertebrate Gonads.

The transcription factor DMRT1 has important functions in two distinct processes, somatic-cell masculinization and germ-cell development in mammals. However, it is unknown whether the functions are conserved during evolution, and what mechanism underlies its expression in the two cell lineages. Our analysis of the Xenopus laevis and Silurana tropicalis dmrt1 genes indicated the presence of two distinct promoters: one upstream of the noncoding first exon (ncEx1), and one within the first intron. In contrast, only the ncEx1-upstream promoter was detected in the dmrt1 gene of the agnathan sand lamprey, which expressed dmrt1 exclusively in the germ cells. In X. laevis, the ncEx1- and exon 2-upstream promoters were predominantly used for germ-cell and somatic-cell transcription, respectively. Importantly, knockdown of the ncEx1-containing transcript led to reduced germ-cell numbers in X. laevis gonads. Intriguingly, two genetically female individuals carrying the knockdown construct developed testicles. Analysis of the reptilian leopard gecko dmrt1 revealed the absence of ncEx1. We propose that dmrt1 regulated germ-cell development in the vertebrate ancestor, then acquired another promoter in its first intron to regulate somatic-cell masculinization during gnathostome evolution. In the common ancestor of reptiles and mammals, only one promoter got function for both the two cell lineages, accompanied with the loss of ncEx1. In addition, we found a conserved noncoding sequence (CNS) in the dmrt1 5'-flanking regions only among amniote species, and two CNSs in the introns among most vertebrates except for agnathans. Finally, we discuss relationships between these CNSs and the promoters of dmrt1 during vertebrate evolution.

Conservatism and variability of gene expression profiles among homeologous transcription factors in Xenopus laevis.

Xenopus laevis has an allotetraploid genome of 3.1Gb, in contrast to the diploid genome of a closely related species, Xenopus tropicalis. Here, we identified 412 genes (189 homeolog pairs, one homeologous gene cluster pair, and 28 singletons) encoding transcription factors (TFs) in the X. laevis genome by comparing them with their orthologs from X. tropicalis. Those genes include the homeobox gene family (Mix/Bix, Lhx, Nkx, Paired, POU, and Vent), Sox, Fox, Pax, Dmrt, Hes, GATA, T-box, and some clock genes. Most homeolog pairs for TFs are retained in two X. laevis subgenomes, named L and S, at higher than average rates (87.1% vs 60.2%). Among the 28 singletons, 82.1% were deleted from chromosomes of the S subgenome, a rate similar to the genome-wide average (82.1% vs 74.6%). Interestingly, nkx2-1, nkx2-8, and pax9, which reside consecutively in a postulated functional gene cluster, were deleted from the S chromosome, suggesting cluster-level gene regulation. Transcriptome correlation analysis demonstrated that TF homeolog pairs tend to have more conservative developmental expression profiles than most other types of genes. In some cases, however, either of the homeologs may show strongly different spatio-temporal expression patterns, suggesting neofunctionalization, subfunctionalization, or nonfunctionalization after allotetraploidization. Analyses of otx1 suggests that homeologs with much lower expression levels have undergone greater amino acid sequence diversification. Our comprehensive study implies that TF homeologs are highly conservative after allotetraploidization, possibly because the DNA sequences that they bind were also duplicated, but in some cases, they differed in expression levels or became singletons due to dosage-sensitive regulation of their target genes.