Sox3: A transcription factor for Cyp19 expression in the frog Rana rugosa

Gene 445 (2009) 38–48

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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e

Sox3: A transcription factor for Cyp19 expression in the frog Rana rugosa Yuki Oshima a, Kiyoshi Naruse b, Yoriko Nakamura c, Masahisa Nakamura a,⁎ a b c

Department of Biology, Faculty of Education and Integrated Arts and Sciences, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan Laboratory of Bioresource, National Institute for Basic Biology, 38 Saigo-naka, Myodaiji, Okazaki, Aichi 444-8585, Japan Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

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Article history: Received 28 March 2009 Received in revised form 11 May 2009 Accepted 21 May 2009 Available online 27 May 2009 Received by D. Mager Keywords: Sox3 Cyp19 Promoter assay ChIP assay Sex determination Rana rugosa Amphibians

a b s t r a c t Cyp19 is expressed at a high level in the gonad of the female tadpole of the frog Rana rugosa during sex determination. To identify sequence elements important for expression of Cyp19, we isolated a genomic clone (∼ 40 kbp) carrying R. rugosa Cyp19 and analyzed the nucleotide sequence of the 5′-flanking region to search for potential transcription factor binding sites. Sox (SRY-related HMG box) protein and Sf1 binding sites were found in the ovary-specific promoter region of Cyp19. Because Sox3 is located on the sex chromosome in R. rugosa, we conducted the luciferase reporter assay in Xenopus A6 cells using the promoter region. Sox3 drove the reporter gene in the cells, but Sf1 did not. When sequential deletion of the 2.7 kbp Cyp19-promoter region was undertaken, a fragment spanning nucleotides − 191 to + 48 was sufficient to drive the transcription of the reporter gene. In site-directed mutagenesis, the binding site at − 57 in the region was critical for Sox3 responsiveness. Sox3 lacking the HMG box had no ability to promote Cyp19 transcription. In addition, a chromatin immunoprecipitation (ChIP) assay showed that DNA fragments were enriched 8-fold, as determined by real-time PCR, when chromatin was immunoprecipitated with the anti-His antibody against His-tagged Sox3. The results, taken together, suggest that Sox3 activates Cyp19 transcription by its direct binding to the binding site of the Cyp19 promoter region. Sox3 appears to be a factor that directs indifferent gonads to develop into an ovary in R. rugosa. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The SRY gene on the Y chromosome has been found in mammals, but not in other vertebrates (Sinclair et al.,1990). This gene is responsible for initiating male sexual differentiation in mammals (Koopman et al., 1991). In amphibians, a gene responsible for primary ovary development, designated DM-W has been found on the W chromosome in Xenopus laevis (Yoshimoto et al., 2008). However, DM-W does not exist in other amphibians such as X. tropicalis and R. rugosa (Uno et al., 2008b). Abbreviations: Sox, SRY-related HMG box; Cyp19, cytochrome P450 family 19; SRY, sex determining region Y; HMG box, high-mobility group box; ChIP, cromatin immunoprecipitation; DNA, deoxyribonucleic acid; PCR, polymerase chain reaction; DM-W, W-linked DM-domain gene; FISH, fluorescence in situ hybridization; Sf1, steroidogenic factor 1; NR5A1, nuclear receptor subfamily 5 group A member 1; HEK293 cell, human embryonic kidney 293 cell; TM3 cell, mouse Leydig cell line; RNA, ribonucleic acid; rRNA, ribosomal RNA; cDNA, complementary DNA; GA3PDH, glyceraldehyde 3 phosphate dehydrogenase; DIG, digoxigenin; RACE, rapid amplification of cDNA ends; SP, specific primer; LB medium, Luria–Bertani medium; Luc, luciferase; PBS, phosphate-buffered saline; FITC, fluoresceinisothiocyanate; IgG, immunoglobulin G; His, histidine; NaBu, sodium butyrate; mRNA, messenger RNA; anti-sense S, sense; DNP, 2,4-dinitrophenol; AP, alkaline phosphatase; HNPP, 2hydroxy-3-naphthoic acid-2'-phenyl-anilide phosphate; CMV, cytomegalovirus A6 cell, Xenopus laevis cell line derived from kidney; NTC, no template control; SEM, standard error of the mean; SE, standard error; st, stage; TK, thymidine kinase; UTR, untranslated region; BS, binding site. ⁎ Corresponding author. Fax: +81 3 5369 7307. E-mail address: [email protected] (M. Nakamura). 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.05.011

SRY encodes a transcription factor containing a 79-amino acid DNAbinding domain known as the HMG box. The HMG box is present in some chromatin-associated proteins of the high-mobility group family and in some transcription factors (Jantzen et al., 1990; Gimelli et al., 2007). After the discovery of SRY, a number of the SRY-related HMG box (Sox) genes were isolated from a wide variety of organisms and were classified into ten subgroups, A–J (Wright et al., 1993; Bowles et al., 2000). Members in the Sox family are involved in a diverse range of developmental processes. For instance, Sox1–3 in the B group are expressed during lens development (Kamachi et al., 1999), and Sox11 in the C group is found in the nervous system in mice (Hargrave et al., 1997). SOX3, which is located on the X chromosome in humans and marsupials (Stevanović et al., 1993; Foster and Graves, 1994), has been highly conserved during mammalian evolution. Based on sequence information from SOX3 homologues in mammals, SOX3 is most closely related to SRY among any members of the SOX family. Gonadal differentiation has been found to be very sensitive to sex steroids in some species of amphibians (Burns, 1961; Schmid et al., 1991), fish (Yamamoto and Kajishima, 1968; Lee et al., 2001), reptiles (Bull et al., 1998), and birds (Shimada, 1998; Villalpando et al., 2000); thus, sex steroids can bring about the masculinization and/or feminization of gonads in many species of vertebrates. Treatment with sex steroids induces the sex-reversal from male to female or the reverse (Adkins-Regan, 1987; Hayes, 1998). It is well known that estradiol-17β, which is converted from testosterone by the Cyp19

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enzyme, can feminize the gonad of many species of vertebrates, e.g., fish (Matthiessen and Sumpter, 1998), amphibians (Mackenzie et al., 2003), reptiles (Crews et al., 1991), birds (Elbrecht and Smith, 1992), and mammals (Coveney et al., 2001). Moreover, Cyp19 encoding a P450 aromatase enzyme is highly conserved in all phyla of vertebrates (Tong and Chung, 2003). In mammals, Cyp19 is expressed in various tissues such as the ovary, testis, brain, placenta, and skin (Mahendroo et al., 1991); this is also true in reptiles (Endo and Park, 2005). How is the tissue-specific expression of Cyp19 achieved in these species? In humans, a single-copy Cyp19 gene with six promoters and first exons is responsible for the tissue-specific expression and multifactorial regulation of a single Cyp19 (Tchoudakova et al., 2001). As it does in humans, a single Cyp19 gene exists in many species of vertebrates, except pigs (Corbin et al., 1995) and fish (Tchoudakova et al., 2001; Chiang et al., 2001). A single copy of Cyp19, which is located on chromosome 3, has been found in R. rugosa by fluorescence in situ hybridization (FISH) analysis (Uno et al., 2008a). This is also true in X. laevis and X. tropicalis (Uno et al., 2008b). At the present time, the existence of multiple Cyp19 genes has not been reported in amphibians. Thus, it is conceivable that these amphibian species carry a single Cyp19 gene. Of interest, the expression of Cyp19 is upregulated significantly in the gonad of R. rugosa female tadpoles (Maruo et al., 2008). The question, then, is which factors up-regulate Cyp19 transcription during gonadal feminization in amphibians? Steroidogenic factor 1 (Sf1), also termed NR5A1 (Biason-Lauber and Schoenle, 2000), is a member of the nuclear hormone receptor superfamily of transcription factors. Sf1 is expressed in all steroidogenic tissue (e.g., adrenal gland, testis, ovary) and is thought to be a key regulator of steroid hormone biosynthesis (Morohashi et al.,1992; Lynch et al., 1993). In the fish Nile tilapia Oreochromis niloticus, Sf1 activates Cyp19a transcription in the luciferase reporter assay using human embryonic kidney 293 (HEK293) and mouse Leydig cell line TM3 (TM3) cells (Wang et al., 2007). However, Sf1 expression does not show any sexual dimorphism during early sex differentiation in tilapia (Ijiri et al., 2008). In amphibians, on the other hand, putative Sf1 binding sites are present in the promoter region of R. rugosa Cyp19 (Oshima et al., 2006). Sf1 from R. rugosa can promote tilapia, but not R. rugosa, Cyp19 transcription in the reporter assay in HEK293 and TM3 cells (Oshima et al., 2006). Thus, there must be another factor that up-regulates Cyp19 transcription in R. rugosa. Such a factor must be identified to understand the molecular mechanism of Cyp19 transcription in this frog. Recently, we found that Sox3 is located on the sex (XY/ZW) chromosomes in R. rugosa (Uno et al., 2008a,b). In addition, we found a few Sox protein binding sites [5′-AACAAAG-3′; Harley et al. (1992)] in the promoter region of R. rugosa Cyp19 by sequence analysis. These results, taken together, led us to examine whether Sox3 activated Cyp19 transcription in this species. Here we report that Sox3 may be a factor to direct indifferent gonads to develop into an ovary in R. rugosa. 2. Materials and methods 2.1. Animals Unfertilized eggs of the ZZ/ZW type R. rugosa were obtained and artificially inseminated as reported elsewhere (Iwade et al., 2008). They were allowed to develop to tadpoles of various stages for use in the experiments. Embryos and tadpoles were staged according to Shumway (1940) and Taylor and Kollros (1946). The sex of tadpoles was determined at a molecular level according to Iwade et al. (2008). Manipulations of embryos, tadpoles, and frogs were approved by the Ethics Committee of Waseda University. 2.2. Sox3 expression in various adult tissues Total RNA was prepared from various tissues of adult R. rugosa and used as the initial templates for real-time PCR. The PCR sequence

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consisted of 4 min at 94 °C, followed by 30 or 25 cycles of 30 s at 95 °C, 30 s at 65 °C, and 1 min at 72 °C, ending with 7 min of extension at 72 °C. DNA fragments for Sox3 (425 bp), Cyp19 (308 bp), and 18S rRNA (534 bp) cDNAs were amplified by PCR using three sets of primers (Sox3, forward primer 5′-AGAGCCGTCCACATGAAGGAA-3′ and backward 5′-CTCTCTGGGTGTGGGAAGTG-3′; Cyp19, forward 5′ACATTGGCCGCATGCATAAA-3′ and backward 5′-CGGGGCTGTGTGCAGAGAAA-3′; 18S rRNA, forward 5′-GCCTGAGAAACGGCTACCACATC-3′ and backward 5′-GCCGGTCCAAGAATTTCACCTC-3′). PCR products were electrophoresed on 1% agarose gel and the gels were stained in ethidium bromide to visualize bands. 2.3. Sox3 expression during sex determination Total RNA was prepared from R. rugosa tadpoles at different stages. Sox3 (GenBank Accession No. AB295441) and Cyp19 (AB178482) cDNAs were amplified by real-time PCR as described elsewhere (Maruo et al., 2008). The reaction consisted of 10 min at 95 °C, followed by 55 cycles of 10 s at 95 °C, 10 s at 65 °C, and 10 s at 72 °C, and then 1 s at 95 °C and 15 s at 65 °C to 95 °C (0.1 °C/s), ending with a step at 40 °C. The increase in the fluorescence signal of SYBR Green I was detected automatically during the 72 °C phase of the reaction by a Light Cycler 1.5 (Roche Diagnostics). For the analysis, primers for Sox3 (forward, 5′-GGCTGGACTAATGGGGCTTATTCTCT-3′ and backward, 5′CTAGGCTCATGACGGTGGAGGT-3′), for Cyp19 (forward, 5′-ACATTGGCCGCATGCATAAA-3′ and backward, 5′-CGGGGCTGTGTGCAGAGAAA3′), and GA3PDH (forward, 5′-GAAGTGAAGGCTGACGGAGGA-3′ and backward, 5′-CGCCTTGTCATAGCTTTCATGGT-3′) were used. 2.4. In situ hybridization In situ hybridization was performed as described elsewhere (Iwade et al., 2008). For this analysis, DIG-labeled RNA fragments were used: 425 bp for Sox3 and 308 bp for Cyp19 corresponding to bp regions of 553–977 for Sox3 and bp 1238–1545 for Cyp19 cDNAs, respectively. After hybridization had been carried out at 50 °C for 16 h, positive signals were detected according to the manufacturer's protocol (Roche Diagnostics). 2.5. Molecular cloning of exon 1 of Cyp19 A single Cyp19 exists in many species of vertebrates except for pigs (Corbin et al., 1995) and some teleost fish (Tchoudakova et al., 2001; Chiang et al., 2001). In mammals, Cyp19 consists of exons 1 to 10. Exon 1 of Cyp19 is not translated in humans and varies in a manner dependent on the tissues in which it is expressed (Harada et al., 1993). In mice, ovary-derived Cyp19 mRNA does not involve untranslated exon 1 (Honda et al., 1996). In humans, the ovaryspecific promoter gives rise to a 5′-untranslated region contiguous with the first coding exon (exon 2) (Bulun et al., 2003). Thus, it was necessary to clarify whether exon 1 of Cyp19 is transcribed in the brain, but not in the ovary. If this is the case in R. rugosa, as in humans and mice, ovary-derived Cyp19 mRNA would be transcribed from exon 2 using the ovary-specific promoter directly upstream of exon 2. Thus, we isolated brain-derived Cyp19 cDNA and determined its nucleotide sequence. For this purpose, we employed the 5′-RACE technique. Total RNA was prepared from the brain of adult R. rugosa. To determine the nucleotide sequence of brain-derived untranslated exon 1, we designed SP1, SP2, SP3, and oligo dG anchor primers for 5′RACE, based on the nucleotide sequence of R. rugosa Cyp19 cDNA (AB178482) isolated from the ovary (Kato et al., 2004). By using the SP1 (5′-ACTCTGACAAATTCCCCATAC-3′), SP2 (5′-CCTAACCCCAGACAGTAAGA-3′), SP3 (5′-TCCAGATGATGATCAGCAGAAATACGA-3′) and oligo dG anchor (5′-GACCACGCGTATGGATGTAGGAAAGGGGGGGGGGGGGH-3′) primers, the extra 77 nucleotides were extended

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2.8. Construction of ovary-specific promoter region/luciferase genes

upstream of the ovary-derived Cyp19 mRNA. The DNA fragment obtained was inserted into a pCR2.1 vector (Invitrogen) and sequenced using an automated DNA sequencer (model 400L, ALOKA). To confirm that we did not simply select exon 2 carrying a shorter 5′-untranslated region adjacent to its coding region, five sets of specific primers were used to amplify Cyp19 cDNA derived from the ovary of R. rugosa. They were designed based on the nucleotide sequence of untranscribed and transcribed regions of R. rugosa Cyp19 (AB379847). Nucleotide sequences of these primers were as follows: forward (1), 5′-GTCTTCCCTTCTGCACGGATACCTA-3′ at nucleotide positions + 1 to +25; (2), 5′-TTTAAAGGTCTTCCCTTCTGCACGGAT-3′ at −7 to +20, (3) 5′-CACCTGGGTTTTTAAAGGTCTTCCCTT-3′ at − 17 to +10; (4), 5′-AATGTCACCTGGGTTTTTAAAGGTCTT-3′ at − 22 to + 5; and (5), 5′-TGTCTAATGTCACCTGGGTTTTTAAAG-3′ at − 27 to −1; and backward (1), 5′-TCCAGATGATGATCAGCAGAAATACGA-3′ at + 140 to +166. The first nucleotide of the 5′ end of the ovary-derived Cyp19 cDNA was mapped as + 1 on Cyp19. Amplification of Cyp19 cDNA fragments was performed by PCR. The reaction consisted of 4 min at 94 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 65 °C, and 1 min at 72 °C, ending with 7 min of extension at 72 °C. PCR products were electrophoresed on 1% agarose gel, and the gels were stained in ethidium bromide to visualize bands as mentioned earlier.

The ovary-specific promoter region of R. rugosa Cyp19 (AB379847) was isolated from a genomic library by using the sequence information on R. rugosa Cyp19 cDNA. Based on the sequence of its promoter region, primers were designed to amplify seven different DNA fragments of the promoter [− 5242/+48, 5.2 K; − 2734/+48, 2.7 K; − 2033/+48, 2.0 K; − 1529/+48, 1.5 K; − 1015/+48, 1.0 K; −501/+ 48, 0.50 K; and − 191/+48, 0.19 K (Table 1)]. All of the DNA fragments were ligated into KpnI/XhoI (TaKaRa) sites of a pGL3-Luciferase (pGL3-Luc) Vector (Promega) and used to transform E. coli DH5α (TaKaRa). These samples were grown at 37 °C overnight in the LB medium supplemented with ampicillin (100 μg/ml). The plasmids then were purified using the Wizard Plus Minipreps DNA Purification System (Promega). To obtain the 2.4 kbp promoter region of tilapia Cyp19a (AF472620), we prepared genomic DNA from the liver of tilapia fish by the conventional method. The Cyp19a promoter then was amplified by PCR using a set of primers (Table 1). A tilapia Cyp19a-luciferase reporter was prepared by the same method as described above. The reporter was used to transfect E. coli DH5α, and the plasmids were purified as described earlier.

2.6. Construction of genomic library and isolation of Cyp19 promoter

2.9. Site-directed mutagenesis

A fosmid genomic library was constructed from genomic DNA of an adult male of the ZZ/ZW type R. rugosa using a CopyControl Fosmid Library Production kit (Epicentre), according to the manufacturer's protocol. To prepare genomic DNA, we excised all tissues (except the stomach and guts) from the abdomen of an adult male R. rugosa and immediately froze them in liquid nitrogen (Hårdeman and Sjöling, 2007). All of the fosmid clones were used to transfect Escherichia coli EPI300 (Epicentre). The bacteria were grown at 37 °C for 24 h in Luria–Bertani (LB) medium supplemented with chloramphenicol (12.5 μg/ml), and then divided into 192 tubes, each containing 2000 colonies in 2 ml of the LB medium/glycerol (1:1) with chloramphenicol (12.5 μg/ml). The tubes were stored at −80 °C until used.

The pGL3-Luc-2.7 K plasmid contains the 2.7 kb upstream region from the XhoI site in exon 2 of R. rugosa Cyp19/luciferase reporter genes. Mutated pGL3-Luc 2.7K carrying a 7-nucleotide substitution from 5′-AACAAAG-3′ to 5′-GGTGGGA-3′ within a putative Sox3 binding site situated at − 2527 or −57 was constructed from pGL3Luc-2.7 K. Site-directed mutagenesis within the Sox3 binding site was performed as follows: a 2.7 kb DNA fragment cut with KpnI and XhoI

2.7. Colony-direct PCR A fosmid clone containing the 5′-flanking region of R. rugosa Cyp19 was screened by colony-direct PCR (Singh and Ramesh, 2008). For this PCR, 1 μl of the LB/glycerol solution in each tube containing 2000 transfected bacteria was first diluted 100-fold with water. All of the 192 diluted samples were used as the template for PCR analysis. The PCR solution contained 1 μl of 10-strength ExTaq buffer (TaKaRa), 0.8 μl of 2.5 mM dNTP, 0.05 μl of ExTaq Hot Start Version (5 U/μl, TaKaRa), 1 μl of a 2.5 mM concentration of each forward and backward primer (forward 5′-TCCCTTCTGCACGGATACCTAGAA-3′ and backward 5′-TCCAGATGATGATCAGCAGAAATACGA-3′), and 5.15 μl water. The primers were designed based on the nucleotide sequence of the Cyp19 promoter (AB440158). The reaction consisted of 9 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 65 °C, and 1 min at 72 °C, ending with a step at 4 °C. PCR products were subjected to 1% agarose gel electrophoresis, and the gels were then stained with ethidium bromide. When a DNA band of a desired size was obtained by PCR, the transformed bacteria (2000 colonies per tube, developed as described above), one of which was carrying the 5′-flanking region of Cyp19 in the pCC1FOS vector, were spread on 48 LB-plates, and supplemented with chloramphenicol (12.5 μg/ml) in order to inoculate 200 clones per plate. By repeating the colony-direct PCR analysis, a positive clone carrying the Cyp19 promoter was obtained. The cloned fosmid was digested with NotI and other restriction enzymes, and analyzed by electrophoresis on 1% agarose.

Table 1 Primer sequences for preparation of Cyp19 promoter constructs. Ovary-type R. rugosa Cyp19 (− 5242 to + 48) 5′-TGCTAGCACGCGTACAAATAGAGCTTTCTTTTGGTGGT-3′ 5′-AAGCTTCTCGAGCTCTCCTGAGAGGTCTCCTCTTCT-3′ Cyp19 (− 2734 to + 48) 5′-TTCTAGAGGTACCTGTGAACCCAGGCTCAGTAAGAAA-3′ 5′-AAGCTTCTCGAGCTCTCCTGAGAGGTCTCCTCTTCT-3′ Cyp19 (− 2033 to + 48) 5′-TGGTACCTTTCGGATAAAAGTGGTCTCTGTGG-3′ 5′-AAGCTTCTCGAGCTCTCCTGAGAGGTCTCCTCTTCT-3′ Cyp19 (− 1529 to + 48) 5′-AAGCTTGGTACCGAGCTCACGTGCAGAAGTGATTG-3′ 5′-AAGCTTCTCGAGCTCTCCTGAGAGGTCTCCTCTTCT-3′ Cyp19 (− 1015 to + 48) 5′-TGGTACCTCACTTCCGGCTCTCCTATCTCCT-3′ 5′-AAGCTTCTCGAGCTCTCCTGAGAGGTCTCCTCTTCT-3′ Cyp19 (− 501 to + 48) 5′-TGGTACCAGCTAAAGTGATCTGGTGATTCAAGG-3′ 5′-AAGCTTCTCGAGCTCTCCTGAGAGGTCTCCTCTTCT-3′ Cyp19 (− 191 to + 48) 5′-TGAGCTCGGTACCTCCTAAACAGCTGGCACTTGCTC-3′ 5′-AAGCTTCTCGAGCTCTCCTGAGAGGTCTCCTCTTCT-3′ Cyp19 (− 2734 to + 48) (Δ − 2527, mutated) 5′-GGGAGTATGCTGTAAACTATGCCCAG-3′ 5′-ACCACATTGCATCAAGCATGAGTAT-3′ Cyp19 (− 2734 to + 48) (Δ − 57, mutated) 5′-GGGACAGCGCTTGATGTCGCATA-3′ 5′-ACCGGCATTTACCCTTTTTGTGTTTTG-3′ tilapia Cyp19a (− 2414 to + 36) 5′-TGGTACCCTGCTGGGAAAGCAGCTTTATCTCT-3′ 5′- TCTCGAGGAGAAGGGTGATGATGTAGAACAGCC-3′ Brain-type R. rugosa Cyp19 (− 1501 to + 43) 5′-TGGTACCACATGAAACCGGGGAGGGAAA-3′ 5′-TCCCGGGCTCGAGGAACCCTTGAAGTTCCTACCCCTTT-3′

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enzymes was subcloned into KpnI/XhoI sites of a pGL3 basic vector and was used as the template for mutagenesis. For mutagenesis of the Sox3 binding site at −2527, forward (5′-GGGAGTATGCTGTAAACTATG CCCAG-3′) and backward (5′-ACCACATTGCATCAAGCATGAGTAT-3′) primers, and for the binding site at −57, forward (5′-GGGACAGCGCTTGATGTCGCATA-3′) and backward (5′-ACCGGCATTTACCCTTTTTGTGTTTTG-3′) primers were used for inverse PCR (mutated sites are shown as underlined letters) (Ochman et al., 1990). KOD-Plus-DNA polymerase (Toyobo) was used to synthesize a mutated promoter according to the manufacturer's instructions. Mutated DNA fragments were self-ligated and used to transfect INVαF′ competent cells

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(Invitrogen). Multicopied plasmids were isolated routinely and sequenced to confirm the mutations. 2.10. Construction of truncated form of Sox3 To prepare wild-type Sox3 and Sf1, and truncated Sox3 with no HMG box, we amplified Sox3 (AB295441) and Sf1 (AB017352) cDNAs derived from the R. rugosa ovary by PCR. For this amplification, the following three sets of gene-specific primers carrying KpnI or XhoI site (mutated sites, underlined) were used: Sox3 (forward 5′-TGGTACCACAATGGATAGVATGGTGGACACTGATA-3′ and backward 5′-TCTCGAGT-

Fig. 1. Analysis of Sox3 and Cyp19 mRNA levels. Total RNA was prepared from different tissues of adult frogs (A) and from gonad/mesonephros complexes of tadpoles (C) at 3 weeks after they had reached stage 25 (St.25 3W), and at stages I, III, and V (St. I, III, and V). (A)Sox3 and Cyp19 mRNA levels in different adult tissues. RT-PCR was carried out for 30 cycles for Sox3 and Cyp19 and for 15 cycles for 18S rRNA. NTC, no template DNA added. (B) Histology of the gonad of tadpoles at stages 25 to V (a–d), Male (M), and (e–h) female (F). Black arrows indicate the ovarian cavity. Scale bars = 10 μm. (C) Sox3 and Cyp19 mRNA levels in the gonad during sex determination. Open and blackened columns indicate male and female, respectively. Each bar represents the mean ± SEM from three independent determinations by real-time PCR analysis. Asterisks (⁎,⁎⁎) indicate that the mean (± SEM) differs from its respective control (⁎P b 0.05, ⁎⁎P b 0.01).

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AAATGTGCGTTAGTGGTACTGTTCC-3′), truncated Sox3 (forward 5′TGGTACCAACATGGAGGAATACCCGGATTAC-3′ and backward 5′-TCTCGAGTAAATGTGCGTTAGTGGTACTGTTCC-3′), and Sf1 (forward 5′-TAAGCTTGGTACCACCATGGATTATAGCTACGAC-3′ and backward 5′-TCTCGAGGATCGCTTAGCATGGAGCATTT-3′). After amplification of each cDNA by PCR, DNA fragments were digested with KpnI and XhoI to obtain a DNA fragment with a KpnI site (-GGTACC-) at one end and an XhoI site (-CTCGAG-) at the other end. The DNA fragment then was ligated into KpnI/XhoI sites of a pcDNA3.1/V5-His expression vector (Invitrogen) and used to transfect E. coli DH5α. The transfected bacteria were grown at 37 °C overnight in the LB medium supplemented with ampicillin (100 μg/ml). Plasmids were purified by using a Wizard Plus Minipreps DNA Purification System (Promega). By following this procedure, we obtained plasmids into which had been inserted wild-type Sox3 encoding 1–306 amino acids, truncated Sox3 encoding 101–306 amino acids, and wild-type Sf1 encoding 1–468 amino acids.

measuring luciferase activity with a GloMax 20/20n Luminometer (Turner BioSystems) and the Dual-luciferase Reporter Assay System (Promega). Firefly luciferase activity reflects Cyp19 promoter activity, and sea pansy luciferase activity was used to normalize the data.

2.11. Cell culture, transient transfection, and luciferase assay

2.13. Analysis of a direct interaction of Sox3 with its binding site by using a chromatin immunoprecipitation (ChIP) assay

Luciferase assays were carried out as described elsewhere (Oshima et al., 2006). For this assay, we used A6 cells (gift from Drs. M. Ikusawa and M. Asashima). established from X. laevis kidney cells. A6 cells were allowed to grow to 80% confluence after transfection with the following plasmids: (1) sequentially deleted DNA constructs of R. rugosa Cyp19 or tilapia Cyp19a promoter inserted in the pGL3-basic luciferase reporter plasmid; (2) pcDNA3.1 plasmid (Invitrogen) expressing R. rugosa wild-type and truncated Sox3, or wildtype Sf1; and (3) pRL-TK vector as an internal control (50 ng/well, Promega). Transfections were conducted as reported elsewhere (Oshima et al., 2006). The level of reporter gene expression was quantified by

2.12. Immunohistology Immunostaining was performed to detect Sox3 and Sf1 by using anti-His (C-term) antibody against His-tagged Sox3 or Sf1 after A6 cells were transfected with Sox3 or Sf1 expression vector. Transfected A6 cells on a glass slide were washed with PBS for 5 min twice, treated with 100% methanol for 5 min at room temperature, and then washed with PBS for 5 min once. Then, the cells were reacted with the anti-His antibody at a dilution of 1:300, and then with anti-mouse IgG antibody conjugated with FITC (Sigma-Aldrich). Fluorescence signals were detected under a fluorescence microscope (model ECLIPSE E600, Nikon).

To examine whether Sox3 bound directly to its binding site in the R. rugosa Cyp19 promoter, we performed a ChIP assay in A6 cells by using a LowCell# ChIP Kit (Diagenode), according to the manufacturer's protocol. The assay consisted of the following six steps. 2.13.1. Step 1: binding antibodies to magnetic beads This step consisted of binding the anti-His (C-term) antibody (Invitrogen) and normal rabbit IgG (Diagenode) as a negative control to the Protein A-coated paramagnetic beads in the kit. Antibodies were bound to the beads according to the manufacturer's protocol and used for immunoprecipitation.

Fig. 2. In situ hybridization analysis of Sox3 and Cyp19 expression. In situ hybridization was carried out on the undifferentiated gonad of a female tadpole at stage I. Positive signals for Cyp19 (A) and Sox3 (C) were observed in somatic cells with the anti-sense probe (AS), but no signals appeared with the sense probe (S, B and D). The area delimited by the small rectangle in each panel is magnified in the corresponding inset. Arrows indicate positive signals in somatic cells. Scale bars = 10 μm.

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2.13.2. Step 2: cell collection and DNA–Sox3 cross-linking A6 cells in 25-cm2 flasks (Corning) were transfected with (1) the pGL3 vector to which had been added the R. rugosa Cyp19 promoter comprising nucleotide positions − 1015 to +48 (7.5 μg), and (2) the pcDNA3.1-V5-His vector (Invitrogen) expressing His-tagged Sox3 of R. rugosa (15 μg). Forty-eight hours after transfection, the cells were harvested in PBS at pH 7.5 and counted with a hematocytometer to adjust to 2 × 106/ml of PBS. According to the manufacturer's protocol, the cells were fixed with formaldehyde for 8 min at room temperature and centrifuged at 470 ×g for 10 min at 4 °C. 2.13.3. Step 3: cell lysis and chromatin shearing Cross-linked cells obtained in step 2 were washed twice with icecold PBS. PBS-20 mM sodium butyrate (NaBu) was then added to the cells. After centrifugation at 470 ×g for 10 min at 4 °C, the pellet was re-suspended in 130 μl of the solution containing protease inhibitor (Diagenode) and NaBu for cell lysis. The sample was sonicated to shear

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the chromatin by using an ULTRA S. HOMOGENIZER (model VP30S, TAITEC) for 12 cycles of 30 s “on,” and 30 s “off.” The sheared chromatin was used directly in the ChIP assay. 2.13.4. Step 4: magnetic immunoprecipitation of Sox3 and DNA complex Beads coated with antibodies from step 1 and sheared chromatins from step 3 were used in this step. Sox3–DNA complex was prepared according to the manufacturer's protocol by using the anti-His (C-term) antibody against His-tagged Sox3 and used for purification of the DNA. 2.13.5. Step 5: DNA purification DNA-purifying slurry (Diagenode) was added to the sample containing the beads bound to Sox3–DNA complexes, which was then boiled for 10 min. Proteinase K was added to the sample to remove Sox3 from the complex. Then, the sample was boiled for 10 min and centrifuged for 1 min at 14,000 ×g at 4 °C. The resultant supernatant was used for real-time PCR.

Fig. 3. Schematic map of the isolated DNA fragment. (A) Amplification of ovary-derived Cyp19 cDNA fragments. Upper panel: Numbers in parentheses and arrows indicate the position of each primer used to amplify ovary-derived Cyp19 cDNA. Lower panel: Each letter (a–e) indicates DNA fragments amplified by PCR using one of five sets of primers shown in the upper panel. The box and solid bar represent exon 2 and untranscribed regions of Cyp19, respectively. (B) Diagram of a partial Cyp19. Exons 1 to 3 are shown in this figure. By using the brain- and ovary-specific CYP19 promoter, exons 1 and 2 are transcribed from R. rugosa Cyp19 in the brain and the ovary, respectively. Regions indicated by the solid lines were completely sequenced, but regions shown by the dotted line were only partially sequenced. The length of intron 2 (dotted line) was deduced from the restriction enzyme map. Open and gray areas in the box indicate UTR and translated region, respectively. (C) Sox3 and Sf1-binding sites in the ovary-specific Cyp19 promoter. A schematic map of ovaryspecific promoter is shown with Sox3 (blackened ellipse) and Sf1 (dot-shaded ellipse) binding sites, and exons 1 and 2 (open box). Three putative Sox3 binding sites were found at − 11005, − 2527 and − 57 in the promoter region (11.8 kbp). Three Sf1 binding sites were also found at − 3813, − 481 and − 204 in its promoter region.

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2.13.6. Step 6: amplification of DNA by real-time PCR Immunoprecipitated DNA was amplified and analyzed by real-time PCR according to the manufacturer's instruction (Diagenode). DNA fragments (245 bp) of the Cyp19 promoter comprising nucleotide positions −225 to +19 were amplified by real-time PCR as described in Section 2.3. The concentration of a target DNA was calculated as mentioned earlier. For the analysis, a pair of primers specific for the Cyp19 promoter (forward, 5′-CAAGCTACTTAAGCTGCCCTGTCAA-3′ and backward, 5′-TCCGTGCAGAAGGGAAGACC-3′) were used. 2.14. Photography Images were scanned and adjusted by Adobe Photoshop (Adobe Systems Incorporated) for brightness and contrast. 3. Results 3.1. Sox3 expression during sex determination The Sox3 mRNA was detected by PCR exclusively in the ovary of an adult frog, as opposed to other tissues such as brain, heart, lung, liver, pancreas, kidney, muscle, and testis (Fig. 1A). Levels of Cyp19 transcripts were very high in the ovary and brain, but very low in the testis and other tissues (Fig. 1A). The phenotypic sex of R. rugosa tadpoles is determined at around stage II (Iwade et al., 2008). Histological observations in this study agreed with their results. As shown in Fig. 1B, no structural difference between male and female gonads was observed at 3 weeks after

tadpoles had reached stage 25 (St.25 3W; a and e) and at stage I (b and f). In the gonad at stage III, however, a morphological difference appeared (c and g): the ovarian cavity had been formed in the female gonad. At stage V, many oogonia were observed in the ovary (d and h). Next, we examined Sox3 expression in the undifferentiated gonad during sex determination by real-time PCR. The Sox3 mRNA was at higher levels in the gonad of a female tadpole before gonadal sex determination (Fig. 1C). Cyp19 was expressed at high levels during and after sex determination (Fig. 1C). When a set of primers specific for exon 1 was used, no Cyp19 mRNA was detectable in the ovary even after 35 cycles of PCR (data not shown). To identify the cells expressing Sox3 and Cyp19 in the undifferentiated ovary, we performed in situ hybridization analysis using the ovary of a tadpole at stage I. The anti-sense probe (AS) detected positive signals for Cyp19 (Fig. 2A) and Sox3 (Fig. 2C) in somatic cells of the ovary, but the sense probe (S) did not (Figs. 2B and D). To demonstrate whether both Sox3 and Cyp19 RNAs were present in the same cells, we carried out fluorescent in situ analysis by using antiDNP-Alexa488 (Molecular Probe) for Cyp19, and anti-DIG-AP and HNPP fluorescent detection kit (Roche) for Sox3, but were unsuccessful for both. Further study is needed to demonstrate that both genes are expressed in the same somatic cells. 3.2. Molecular cloning of exon 1 of Cyp19 By employing the 5′-RACE technique, we obtained a 243 bp cDNA fragment of R. rugosa Cyp19. Sequence analysis showed that the first 77 bp cDNA fragment was untranslated exon 1, which is expressed in

Fig. 4. Promotion of Cyp19 transcription by Sox3. (A) Activity of the Cyp19 promoter. A6 cells were transiently transfected with the Sox3 or Sf1 expression vector (each at 1 μg/well) and the ovary-specific R. rugosa Cyp19 promoter linked to luciferase in a pGL3 vector (500 ng/well) or with the Sf1 expression vector (100 ng/well) and tilapia cyp19a promoter linked to luciferase (500 ng/well). Luciferase activity was normalized to the internal transfection efficiency control (pRL-TK vector used for co-transfection) and it refers to the activity of the constructs in the absence of the Sox3 or Sf1 expression vector. Values (mean ± SE) were calculated from triplicate determinations. (B) Immunohistology of Sox3 and Sf1. After transfection of A6 cells with the Sox3 or Sf1 expression vector, both proteins were detected by immunostainings. Scale bars = 200 μm.

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the brain (AB383151; data not shown). The rest of the 166-bp cDNA coincided with the 5′ region of exon 2, indicating that it is expressed commonly in the brain and ovary. As mentioned above, R. rugosa probably carries a single Cyp19, as do humans and mice (Honda et al., 1996; Bulun et al., 2003). Based on the information obtained for the nucleotide sequence of R. rugosa Cyp19 cDNA, it may be inferred that exon 1 is untranslated in this species as it is in humans. The 5′-flanking region of Cyp19 contiguous with the 5′-end of exon 2 must be responsible for Cyp19 expression in the ovary of R. rugosa. This led us to use PCR with five sets of specific primers (a through e, upper panel in Fig. 3A) from which we confirmed that the ovary-derived Cyp19 cDNA (AB178482) obtained by 5′-RACE was the longest one extended in the 5′ direction. The PCR analysis revealed that two sets of primers (a and b, Fig. 3A) did not serve to amplify any cDNA fragments (lower panel, Fig. 3A). A forward primer at positions − 32 to − 5 did not serve to amplify any cDNA fragments either (data not shown). Thus, the 5′ end of the ovaryderived Cyp19 cDNA was probably the nucleotide at position + 1. 3.3. Isolation of brain-specific and ovary-specific Cyp19 promoters

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tilapia Sox3 (DQ632569), the HMG box of tilapia Sox3 had 96% identity with that of R. rugosa Sox3 at an amino acid level. Thus, the absence of activation of tilapia Cyp19a promoter by R. rugosa Sox3 might be due to the promoter having no response to R. rugosa Sox3. 3.5. Deletion analysis of the ovary-specific Cyp19 promoter We determined the functional significance of Sox3 by co-transfecting A6 cells with the 5.2 kbp Cyp19 promoter and Sox3 expression vector. The Cyp19 promoter was linked to the luciferase reporter in a pGL3 vector. Sox3 expression was directed by the CMV promoter. As shown in Fig. 5A, the reporter activity was increased in a Sox3dose-dependent manner. To clarify the location of the functional region of the R. rugosa Cyp19 promoter, we generated a series of luciferase reporter constructs containing deletions of the promoter region with 5′-termini at nucleotides − 5242, − 2734, −2033, −1529, −1015, −501, and − 191 and a common 3′-terminus at +48. The 5′-deleted mutants were verified by the luciferase reporter assay after A6 cells were transiently transfected with them. A promoter DNA fragment spanning nucleotides −191

We successfully isolated a 40 kbp genomic DNA fragment carrying the brain- and ovary-specific promoter regions of Cyp19 from the R. rugosa genomic library. Fig. 3B presents a schematic diagram of the DNA fragment that contains exons 1 to 3. Exon 2 was located at 11.8 kbp downstream from exon 1 and had the translation start-site of Cyp19. In humans, Cyp19 consists of 10 exons in common with other Cyp19 isoforms (Bulun et al., 2003). Thus, a 40 kbp R. rugosa DNA fragment lacks exons 4 to 10. Next, we determined the nucleotide sequence of the brain-specific (5.8 kbp) and ovary-specific (11.8 kbp) promoter regions of R. rugosa Cyp19 (Fig. 3C). Previously, we had established the promoter region (2.75 kbp) of R. rugosa Cyp19 (Oshima et al., 2006). In this study, we extended its promoter region considerably to the 5′ direction. However, neither the canonical TATA box nor the CCAAT box in the region was observed. When a computer search for regulatory elements was conducted, three Sox3 binding sites were found in the ovary-specific Cyp19 promoter region (Fig. 3C). In addition to Sox3 binding sites, three Sf1 binding sites were found in the ovary-specific promoter region (Fig. 3C). 3.4. Activation of Cyp19 transcription by Sox3 Cyp19 is expressed at high levels in the undifferentiated gonad of female tadpoles during and after sex determination (Fig. 1C). Thus, it was important to identify a factor that up-regulates Cyp19 transcription in R. rugosa. To estimate the transcription activity, we fused the brain- (1.5 kbp DNA fragment) or the ovary-specific promoter region of Cyp19 (5.2 to 0.19 kbp DNA fragments) to the luciferase reporter gene. As shown in Fig. 4A, Sox3, but not Sf1, increased the Cyp19 promoter activity by 6-fold. Our previous study showed that overexpression of R. rugosa Sf1 had no effect on the activation of Cyp19 transcription in either mammalian HEK293 or TM3 cells (Oshima et al., 2006). The present results were compatible with the previous results. The lack of increased Cyp19 transcription by Sf1 was not due to the absence of this protein, since Sf1 was detected in A6 cells immunohistochemically (Fig. 4B). In addition, the luciferase activity was enhanced by approximately 10-fold when the A6 cells were cotransfected with the tilapia 2.4 kbp Cyp19a promoter (500 ng/well) and R. rugosa Sf1 expression vector (100 ng/well, Fig. 4A). Unlike in the case of mammals and fish, Sf1 did not activate Cyp19 transcription in R. rugosa. The tilapia Cyp19a promoter contained two Sox3 binding sites at nucleotide positions at − 1246 to −1240 and −1068 to −1062. However, R. rugosa Sox3 did not activate the tilapia Cyp19a promoter in the luciferase assay conducted with A6 cells (data not shown). Based on a partial amino acid sequence of the HMG box of

Fig. 5. Functional region of the ovary-specific Cyp19 promoter. (A) Sox3-mediated activation of the Cyp19 promoter. The control reporter (pGL3-Luc-2.7 K, 500 ng/well) and Sox3 expression vector in increasing amounts (100–1000 ng/well) were used to cotransfect A6 cells. Values shown are the mean± SEM calculated from triplicate determinations. (B) Deletion analysis of the Cyp19 promoter. A6 cells were transiently transfected with a series of 5′-deleted promoter constructs (500 ng/well) and either mock vector (open columns) or Sox3 expression vector (blackened columns; 1000 ng/well). Fold induction by Sox3 is indicated. A6 cell were also transfected with the brain-specific R. rugosa Cyp19 promoter-Luc-1.5 k (500 ng/well) was also transfected into A6 cells either with mock vector (open column) or with Sox3 expression vector (blackened column; 1000 ng/well). All values are the mean ± SEM from triplicate determinations.

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Fig. 6. Localization of a functional binding site in the Cyp19 promoter. A6 cells were transiently co-transfected with a luciferase reporter gene containing the 2.7 kbp promoter region of R. rugosa Cyp19. A mutation was introduced in either of two Sox3 binding sites at −2529 or −57. Values are presented as the mean ± SEM from triplicate determinations. Blackened columns, Sox3 expression vector and open columns, mock vector.

to +48 was sufficient to drive Cyp19 transcription in the cells at levels of 4- to 6-fold over the promoter-less luciferase reporter (Fig. 5B). By contrast, no activity was observed when the brain-specific 1.5 kbp promoter was used (Fig. 5B). In addition, deletion analysis revealed that the reporter activity became lower when the promoter DNA was deleted from −501 to −191. There might be a repressor sequence between − 501 and −191. We have presently no data on what repressor it is if so. 3.6. Localization of a functional binding site in the ovary-specific Cyp19 promoter To identify the binding site interacting with Sox3, we introduced mutations into either of two Sox3 binding sites within the 2.7 kbp Cyp19 promoter. For detecting the Sox3 response to the binding site safely, we transfected A6 cells with a relatively large amount of Sox3 expression vector (1 μg/well) and mutated promoter (500 ng/well). The Cyp19 promoter appeared to be activated by approximately 6-fold when A6 cells were transfected with the wild-type Sox3 expression vector (Fig. 6). However, a complete loss of Sox3 responsiveness was observed with mutation in the binding site at −57, but no effect from the mutation at −2529 was observed (Fig. 6). Thus, it is concluded that the Sox3 binding site at −57 was critical for Sox3 interaction with the binding site. 3.7. Functional region of Sox3 for binding to its binding site in the Cyp19 promoter We constructed a truncated Sox3 with no HMG box in order to determine the functional region of Sox3 for transcriptional activity. The HMG box is located at amino acid positions 39 to 103 of R. rugosa Sox3. A

Fig. 8. Sox3 ChIP assay. Quantification of DNA fragments enriched by Sox3 ChIP assay is shown in this figure. The anti-His antibody was used in this assay, whereas normal rabbit IgG was used as a control. Fold enrichment is expressed relative to negative control cells, meaning that values greater than 1 represent specific enrichment. The location of Sox3 binding site in the enriched DNA fragment is shown in the lower panel. Arrows indicate nucleotide positions of specific primers to amplify DNA fragments. Results are presented as the mean ± SEM from triplicate determinations (⁎P b 0.05).

truncated Sox3 with an amino acid sequence at 101 to 306 was used for the luciferase assay. When the truncated Sox3 was expressed in A6 cells, the promoter activity fell to the null level (Fig. 7), indicating that the HMG box was essential for the transcriptional activity of Sox3. 3.8. Direct interaction of Sox3 with its binding site The question remained as to whether Sox3 physically interacted with its binding site in the promoter region to regulate Cyp19 expression directly. To address this question, we performed a ChIP assay in A6 cells according to the manufacturer's directions (Diagenode). Based on the nucleotide sequence of the ovary-specific Cyp19 promoter at nucleotide positions −1015 to +48, a pair of specific primers was designed to amplify immunoprecipitated DNA fragments (Fig. 8). In the ChIP assay, we used the anti-His antibody against His-tagged Sox3 (Invitrogen) that had been used for the immunohistological study (see Fig. 4B). As shown in Fig. 8, 245 bp DNA fragments were enriched by 8-fold by real-time PCR, when the sheared chromatin was immunoprecipitated with the antibody. By contrast, little enrichment of DNA fragments was observed with IgG. Taken together, Sox3 can bind directly to its binding site at nucleotide positions − 57 to −51 in the ovary-specific promoter of R. rugosa Cyp19. 4. Discussion 4.1. Sox3 expression in the gonad of R. rugosa

Fig. 7. Functional region of Sox3 to the Cyp19 responsiveness. The R. rugosa Cyp19Luc-2.7 K reporter vector and a wild-type Sox3 (amino acid sequence of 1–306) or a truncated Sox3 (101–306, ΔSox3) expression vector were used to transfect A6 cells. Results are the means ± SE from triplicate determinations in each treatment group. Blackened column, wild-type Sox3 expression vector: gray column, truncated Sox3 expression vector: and open columns, mock vector.

Estrogens are required for ovarian differentiation in vertebrates such as fish (Adkins-Regan,1987; Ijiri et al., 2008), amphibians (Mackenzie et al., 2003; Kuntz et al., 2003), reptiles (Crews et al., 1991; Ramsey et al., 2007), birds (Elbrecht and Smith, 1992), and mammals (Coveney et al., 2001). In this study, we showed by real-time PCR analysis that Cyp19 expression starts in the gonad of a R. rugosa tadpole before sex determination, and that its expression remains at high levels in female gonads during and after sex determination. Previous studies have shown that Cyp19 is located on an autosome in R. rugosa (Uno et al., 2008a), as it is in tilapia fish (Harvey et al., 2003) and in mice (Youngblood et al., 1989), implying that there must be a transcription factor that up-

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regulates Cyp19 expression in the gonad of R. rugosa. Sox3 may be a factor that up-regulates Cyp19 expression in R. rugosa. 4.2. No Cyp19 cDNA extended further to the 5′ direction in the ovary The ovary-derived Cyp19 cDNA (AB178482) obtained by 5′-RACE is probably the longest one extended to the 5′ direction, judged from PCR analysis using five sets of specific primers. The 5′ cap, referred to as a 7-methylguanosine cap, perhaps attaches to the first nucleotide (+ 1) at the 5′ end of ovary-derived Cyp19 cDNA (AB178482). However, we cannot totally exclude the possibility that the 5′ cap attaches to a nucleotide at a position between −5 to −1, since the forward primer at position −17 to +10 served to amplify cDNA fragments, but primers at −22 to +5 and at −27 to −1 did not. A Sox3 binding site is located at − 57 to −51 in the ovary-specific promoter region of R. rugosa Cyp19. Thus, by a direct binding to the binding site in the ovary-specific Cyp19 promoter region, Sox3 activates its transcription in the gonad of a tadpole in this species. 4.3. Promotion of Cyp19 transcription by Sox3 In Sox3-deficient mice, females develop ovaries but exhibit severely reduced fertility. In Sox3-hemizygous mice, on the other hand, males develop testes but are hypogonadal. Testis weight is severely reduced. Thus, Sox3 is not required for sex determination, but is important for normal oocyte development and male testis differentiation (Weiss et al., 2003). Recently, Yao et al. (2007) showed that Sox3 has more important roles in oogenesis of the fish Epinephelus coioides than in spermatogenesis. Thus, there is no doubt that Sox3 participates in gametogenesis in vertebrates. In this study, we sought a regulator for Cyp19 transcription in the gonad of R. rugosa. As differing from mammals, there is no dosage compensation for X-linked or Z-linked genes by the sex chromosome inactivation mechanism in the sex chromosomes of amphibians (Schempp and Schmid 1981). Thus, a target gene will be expressed differentially between males and females in amphibians if the regulatory gene on the sex chromosome is expressed. Interestingly, Sox3 is located on the sex chromosome in R. rugosa (Uno et al., 2008a,b). The Sox3 expression occurs at high levels in the female gonad of a R. rugosa tadpole. Therefore, it was a matter of course to examine whether Sox3 had the ability to promote Cyp19 transcription in the reporter assay with Xenopus A6 cells. As expected, Sox3 activated Cyp19 transcription in the cells. Since no endogenous Sox3 and Cyp19 mRNA are detectable even after 45 cycles of PCR in A6 cells (data not shown), Sox3 in A6 cells must have been derived from the Sox3 expression vector. In the 0.2 kb human ovary-specific promoter region (Sebastian and Bulun, 2001), no Sox3 binding site was found in the promoter region when a search had been made. This indicates that the Sox3 binding site is not conserved in the ovary-specific Cyp19 promoter regions among vertebrates. To the best of our knowledge, this is the first report demonstrating that Sox3 promotes Cyp19 transcription in the gonad of vertebrates. Sox3 probably plays an important role, as a transcription factor, in feminization of the gonad of R. rugosa. The results also suggest that the factor regulating Cyp19 expression in the ovary of R. rugosa differs from that in the ovary of humans. 4.4. Direct interaction between Sox3 and its binding site When sequential deletion was undertaken in the ovary-specific Cyp19 promoter, the promoter continued to respond to Sox3 unless the Sox3 binding site at −57 to −51 had been deleted. Interestingly, a truncated Sox3 lacking the HMG box had no ability to activate Cyp19 promoter, indicating that the HMG box is responsible for the interaction with its binding site in the promoter. To clarify whether Sox3 interacts directly with its binding site in the ovary-specific Cyp19 promoter of R. rugosa, a ChIP assay was performed. The assay revealed

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that the 245 bp DNA fragment was enriched by 8-fold. Thus, Sox3 can interact directly with the binding site at −57 to −51 in the Cyp19 promoter. This is the first report showing a direct binding of Sox3 to its binding site in the amphibian Cyp19 promoter. A direct interaction between Sox3 and its binding site may be necessary for promotion of Cyp19 transcription by Sox3 in the gonad of R. rugosa. 4.5. No promotion of Cyp19 transcription by Sf1 Sf1 is a gene encoding a transcription factor of cytochrome P450s in mammals (Morohashi et al.,1992) and tilapia fish (Chang et al., 2005). In the tilapia Oreochromis niloticus, a few Sf1 binding sites were found in the ovary-type Cyp19 promoter region (Chang et al., 2005). Tilapia Sf1 promoted the ovary-type cyp19 transcription in the luciferase reporter assay in HEK293 cells (Wang et al., 2007). In contrast, R. rugosa Sf1 by itself activated transcription of tilapia, but not R. rugosa CYP19 in the HEK293 cells (Oshima et al., 2006). Sf1 did not activate Cyp19 transcription in A6 cells either. Thus, it is unlikely that Sf1 directly promotes Cyp19 transcription in the gonad of R. rugosa. The function of Sf1 may not be always conserved in all phyla of vertebrates. Factors regulating Cyp19 transcription in vertebrates may be species-dependent. 5. Conclusion This study provides the following new findings: (1) Sox3 and Cyp19 are expressed at high levels in the gonad of a female tadpole before and, during and after gonadal sex determination of R. rugosa, respectively. (2) Sox3 is capable of promoting R. rugosa Cyp19 transcription. (3) Sox3 binds directly to its binding site in the ovary-specific Cyp19 promoter region through the HMG box. (4) Sf1 is not an activator of Cyp19 transcription in R. rugosa. Taken together, these findings indicate that Sox3 probably directs the undifferentiated gonad to develop into an ovary in R. rugosa. In the near future, we will turn our attention to identifying the factor that up-regulates Sox3 expression in the gonad of R. rugosa. Acknowledgments This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to M.N. (No. 19370026). References Adkins-Regan, E., 1987. In: Norris, D.O., Jones, R.E. (Eds.), Hormones and Sexual Differentiation. Plenum Press, New York, pp. 1–29. Biason-Lauber, A., Schoenle, E.J., 2000. Apparently normal ovarian differentiation in a prepubertal girl with transcriptionally inactive steroidogenic factor 1 (NR5A1/SF-1) and adrenocortical insufficiency. Am. J. Hum. Genet. 67, 1563–1568. Bowles, J., Schepers, G., Koopman, P., 2000. Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev. Biol. 227, 239–255. Bull, J.J., Gutzke, W.H., Crews, D., 1998. Sex reversal by estradiol in three reptilian orders. Gen. Comp. Endocrinol. 70, 425–428. Bulun, S.E., Sebastian, S., Takayama, K., Suzuki, T., Sasano, H., Shozu, M., 2003. The human CYP19 (aromatase P450) gene: update on physiologic roles and genomic organization of promoters. J. Steroid Biochem. Mol. Biol. 86, 219–224. Burns, R.K., 1961. In: Young, W.C. (Ed.), Role of Hormones in the Differentiation of Sex. Sex and Internal Secretions 1. Waverly Press, MD, pp. 86–95. Chang, X., Kobayashi, T., Senthilkumaran, B., Kobayashi-Kajura, H., Sudhakumari, C.C., Nagahama, Y., 2005. Two types of aromatase with different encoding genes, tissue distribution and developmental expression in Nile tilapia (Oreochromis niloticus). Gen. Comp. Endocrinol. 141, 101–115. Chiang, E.F., Yan, Y.L., Guiguen, Y., Postlethwait, J., Chung, B., 2001. Two Cyp19 (P450 aromatase) genes on duplicated zebrafish chromosomes are expressed in ovary or brain. Mol. Biol. Evol. 18, 542–550. Corbin, C.J., Khalil, M.W., Conley, A.J., 1995. Functional ovarian and placental isoforms of porcine aromatase. Mol. Cell. Endocrinol. 113, 29–37. Coveney, D., Shaw, G., Renfree, M.B., 2001. Estrogen-induced gonadal sex reversal in the tammar wallaby. Biol. Reprod. 65, 613–621. Crews, D., Bull, J.J., Wibbels, T., 1991. Estrogen and sex reversal in turtles: a dosedependent phenomenon. Gen. Comp. Endocrinol. 81, 357–364. Elbrecht, A., Smith, R.G., 1992. Aromatase enzyme activity and sex determination in chickens. Science 255, 467–470.

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Y. Oshima et al. / Gene 445 (2009) 38–48

Endo, D., Park, M.K., 2005. Molecular cloning of P450 aromatase from the leopard gecko and its expression in the ovary. J. Steroid Biochem. Mol. Biol. 96, 131–140. Foster, J.W., Graves, J.A., 1994. An SRY-related sequence on the marsupial X chromosome: implications for the evolution of the mammalian testis-determining gene. Proc. Natl. Acad. Sci. U. S. A. 91, 1927–1931. Gimelli, G., et al., 2007. Identification and molecular modelling of a novel familial mutation in the SRY gene implicated in the pure gonadal dysgenesis. Eur. J. Hum. Benet. 15, 76–80. Harada, N., Utsumi, T., Takagi, Y., 1993. Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis. Proc. Natl. Acad. Sci. U. S. A. 90, 11312–11316. Hårdeman, F., Sjöling, S., 2007. Metagenomic approach for the isolation of a novel lowtemperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol. Ecol. 59, 524–534. Hargrave, M., Wright, E., Kun, J., Emery, J., Cooper, L., Koopman, P., 1997. Expression of the Sox11 gene in mouse embryos suggests roles in neuronal maturation and epithelio-mesenchymal induction. Dev. Dyn. 210, 79–86. Harley, V.R., et al., 1992. DNA binding activity of recombinant SRY from normal males and XY females. Science 255, 453–456. Harvey, S.C., Kwon, J.Y., Penman, D.J., 2003. Physical mapping of the brain and ovarian aromatase genes in the Nile tilapia, Oreochromis niloticus, by fluorescence in situ hybridization. Anim. Genet. 34, 62–64. Hayes, T.B., 1998. Sex determination and primary sex differentiation in amphibians: genetic and developmental mechanisms. J. Exp. Zool. 281, 373–399. Honda, S., Harada, N., Takagi, Y., 1996. The alternative exons 1 of the mouse aromatase cytochrome P-450 gene. Biochim. Biophys. Acta 1305, 145–150. Ijiri, S., et al., 2008. Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus. Biol. Reprod. 78, 333–341. Iwade, R., Maruo, K., Okada, G., Nakamura, M., 2008. Elevated expression of P450c17 (CYP17) during testicular formation in the frog. Gen. Comp. Endocrinol. 155, 79–87. Jantzen, H.M., Admon, A., Bell, S.P., Tjian, R., 1990. Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins. Nature 26, 830–836. Kamachi, Y., Cheah, K.S., Kondoh, H., 1999. Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealed by comparison of SOX1/ 2/3 and SOX9. Mol. Cell Biol. 19, 107–120. Kato, T., Matsui, K., Takase, M., Kobayashi, M., Nakamura, M., 2004. Expression of P450 aromatase protein in developing and in sex-reversed gonads of the XX/XY type of the frog Rana rugosa. Gen. Comp. Endocrinol. 137, 227–236. Koopman, P., Gubbay, J., Vivian, N., Goodfellow, P., Lövell-Badge, R., 1991. Male development of chromosomally female mice transgenic for Sry. Nature 351, 117–121. Kuntz, S., et al., 2003. Differential expression of P450 aromatase during gonadal sex differentiation and sex reversal of the newt Pleurodeles waltl. J. Steroid Biochem. Mol. Biol. 84, 89–100. Lee, Y.H., et al., 2001. Sex change in the protandrous black porgy, Acanthopagrus schlegeli: a review in gonadal development, estradiol, estrogen receptor, aromatase activity and gonadotropin. J. Exp. Zool. 290, 715–726. Lynch, J.P., Lala, D.S., Peluso, J.J., Luo, W., Parker, K.L., White, B.A., 1993. Steroidogenic factor-1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues. Mol. Endocrinol. 7, 776–786. Mackenzie, C.A., Berrill, M., Metcalfe, C., Pauli, B.D., 2003. Gonadal differentiation in frogs exposed to estrogenic and antiestrogenic compounds. Environ. Toxicol. Chem. 22, 2466–2475. Mahendroo, M.S., Means, G.D., Mendelson, C.R., Simpson, E.R., 1991. Tissue-specific expression of human P-450AROM. The promoter responsible for expression in adipose tissue is different from that utilized in placenta. J. Biol. Chem. 266, 11276–11281. Maruo, K., Suda, M., Yokoyama, S., Oshima, Y., Nakamura, M., 2008. Steroidogenic gene expression during sex determination in the frog Rana rugosa. Gen. Comp. Endocrinol. 158, 87–94. Matthiessen, P., Sumpter, J.P., 1998. Effects of estrogenic substances in the aquatic environment. EXS 86, 319–335. Morohashi, K., Honda, S., Inomata, Y., Handa, H., Omura, T., 1992. A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s. J. Biol. Chem. 267, 17913–17919.

Ochman, H., Medhora, M.M., Garza, D., Hartl, D.L.,1990. Amplification of flanking sequences by inverse PCR. PCR Protocols. Academic Press, San Diego, CA, pp. 219–227. Oshima, Y., et al., 2006. Promoter activity and chromosomal location of the Rana rugosa P450 aromatase (P450arom) gene. Zool. Sci. 23, 79–85. Ramsey, M., Shoemaker, C., Crews, D., 2007. Gonadal expression of Sf1 and aromatase during sex determination in the red-eared slider turtle (Trachemys scripta), a reptile with temperature-dependent sex determination. Differentiation 75, 978–991. Schempp, W., Schmid, M., 1981. Chromosome banding in Amphibia. VI. BrdU-replication patterns in anura and demonstration of XX/XY sex chromosomes in Rana esculenta. Chromosoma 83, 697–710. Schmid, M., Nada, I., Steinlein, C., Kausch, K., Haaf, T., Epplen, J.T., 1991. In: Green, D.M., Sessions, S.K. (Eds.), Sex-Determining Mechanisms and Sex Chromosomes in Amphibia. Amphibian Cytogenetics and Evolution. Academic Press, New York, pp. 393–428. Sebastian, S., Bulun, S.E., 2001. A highly complex organization of the regulatory region of the human CYP19 (aromatase) gene revealed by the Human Genome Project. J. Clin. Endocrinol. Metab. 86, 4600–4602. Shimada, K., 1998. Gene expression of steroidogenic enzymes in chicken embryonic gonads. J. Exp. Zool. 281, 450–456. Shumway, W., 1940. Stages in the normal development of Rana pipiens. I. External form. Anat. Rec. 78, 139–147. Sinclair, A.H., et al., 1990. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346, 240–244. Singh, A.K., Ramesh, A., 2008. Succession of dominant and antagonistic lactic acid bacteria in fermented cucumber: insights from a PCR-based approach. Food Microbiol. 25, 278–287. Stevanović, M., Lövell-Badge, R., Collignon, J., Goodfellow, P.N., 1993. SOX3 is an X-linked gene related to SRY. Hum. Mol. Genet. 2, 2013–2018. Taylor, A.C., Kollros, J.J., 1946. Stages in the normal development of Rana pipiens larvae. Anat. Rec. 94, 7–23. Tchoudakova, A., Kishida, M., Wood, E., Callard, G.V., 2001. Promoter characteristics of two cyp19 genes differentially expressed in the brain and ovary of teleost fish. J. Steroid Biochem. Mol. Biol. 78, 427–439. Tong, S.K., Chung, B.C., 2003. Analysis of zebrafish cyp19 promoters. J. Steroid Biochem. Mol. Biol. 86, 381–386. Uno, Y., et al., 2008a. Comparative chromosome mapping of sex-linked genes and identification of sex chromosomal rearrangements in the Japanese wrinkled frog (Rana rugosa, Ranidae) with ZW and XY sex chromosome systems. Chromosom. Res. 16, 637–647. Uno, Y., et al., 2008b. Diversity in the origins of sex chromosomes in anurans inferred from comparative mapping of sexual differentiation genes for three species of the Raninae and Xenopodinae. Chromosom. Res. 16, 999–1011. Villalpando, I., Sánchez-Bringas, G., Sánchez-Vargas, I., Pedernera, E., Villafán-Monroy, H., 2000. The P450 aromatase (P450 arom) gene is asymmetrically expressed in a critical period for gonadal sexual differentiation in the chick. Gen. Comp. Endocrinol. 117, 325–334. Wang, D.S., et al., 2007. Foxl2 up-regulates aromatase gene transcription in a femalespecific manner by binding to the promoter as well as interacting with Ad4 binding protein/steroidogenic factor 1. Mol. Endocrinol. 21, 712–725. Weiss, J., Meeks, J.J., Hurley, L., Raverot, G., Frassetto, A., Jameson, J.L., 2003. Sox3 is required for gonadal function, but not sex determination, in males and females. Mol. Cell. Biol. 23, 8084–8091. Wright, E.M., Snopek, B., Koopman, P., 1993. Seven new members of the Sox gene family expressed during mouse development. Nucleic Acids Res. 21 (744). Yamamoto, T., Kajishima, T., 1968. Sex hormone induction of sex reversal in the goldfish and evidence for male heterogamity. J. Exp. Zool. 168, 215–221. Yao, B., Zhou, L., Wang, Y., Xia, W., Gui, J.F., 2007. Differential expression and dynamic changes of SOX3 during gametogenesis and sex reversal in protogynous hermaphroditic fish. J. Exp. Zool. Part A Ecol. Genet. Physiol. 307, 207–219. Yoshimoto, S., et al., 2008. A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis. Proc. Natl. Acad. Sci. U. S. A. 105, 2469–2474. Youngblood, G.L., Nesbitt, M.N., Payne, A.H., 1989. The structural genes encoding P450scc and P450arom are closely linked on mouse chromosome 9. Endocrinology 125, 2784–2786.