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Cloning and characterization of mouse mSox13 cDN
Gene 208 (1998) 201–206
Cloning and characterization of mouse mSox13 cDNA Susumu Kido a,1, Yoshiki Hiraoka b,*, Motoyuki Ogawa b, Yukinao Sakai b, Yasunori Yoshimura c, Sadakazu Aiso b a Tokyo Electric Power Hospital, Shinjuku-ku, Tokyo, 160, Japan b Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160, Japan c Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160, Japan Received 25 August 1997; accepted 13 November 1997; Received by A. Fujiyama
Abstract A novel SRY-related cDNA, mSox13, was isolated from a l phage library derived from mouse embryo. The cDNA encodes a protein of 595 amino acids containing the SRY-type high mobility group (HMG) box and a putative leucine zipper motif. A sequence comparison of mSox13 and other type-D SOX proteins shows that the leucine zipper and a neighboring glutamine-rich sequence stretch, which was named Q box, are well conserved among known type-D SOX proteins. The expression of mSox13 is restricted to the kidney and ovary. The electrophoretic mobility shift assay indicates that the recombinant mSox13 protein is capable of binding to the AACAAT sequence. © 1998 Elsevier Science B.V. Keywords: DNA binding; Embryo; HMG box; Leucine zipper; SRY
1. Introduction SOX is a family of genes related to the testis determining gene, SRY, and its members have been isolated from a wide variety of organisms (Sinclair et al., 1990; Denny et al., 1992a; Laudet et al., 1993; Stevanovic et al., 1993). The SOX genes encode transcriptional factors with a characteristic DNA-binding motif known as the HMG box. The SRY-type HMG domains interact with DNA in a sequence-specific manner (Nasrin et al., 1991; Denny et al., 1992b; Ferrari et al., 1992; Giese et al., 1992; Harley et al., 1992; King and Weiss, 1993; Wetering et al., 1993; Kamachi et al., 1995; Wotton et al., 1995; Shiozawa et al., 1996; Hiraoka et al., 1997; Sakai et al., 1997). SOX proteins have been reported to participate * Corresponding author. Tel: +81 3 33531211; Fax: +81 3 53791977; e-mail: [email protected] 1 Present address: Kawasaki Municipal Hospital, 12-1, Shinkawadori, Kawasaki-ku, Kawasaki City, Kanagawa 210, Japan. Abbreviations: aa, amino acid(s); b, base(s); cDNA, complementary DNA; EMSA, electrophoretic mobility shift assay; GST, glutathioneS-transferase; kb, kilobase(s) or 1000 b; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; RT/PCR, reverse transcription/PCR; SOX, SRY-related protein(s); SOX, gene (DNA, RNA) encoding SOX; SRY, sex-determining region Y gene; SRY, protein encoded by SRY; UTR, untranslated region. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 6 67 - 7
in controls of various developmental processes. The SOX9 gene was identified as a causal gene for autosomal sex reversal and campomelic dysplasia. This suggests that SOX9 plays important parts in both gonadal development and chondrogenesis (Foster et al., 1994; Wagner et al., 1994; Wright et al., 1995). Very recent studies have demonstrated that mouse Sox9 functions as a transcriptional activator in the expression of the type II collagen gene which is involved in cartilage development (Lefebvre et al., 1997; Ng et al., 1997). Sox4 is expressed in T and pre-B cells and functions as a transcriptional activator ( Wetering et al., 1993; Wotton et al., 1995). A study using gene targeting has revealed that Sox4deficient mice die prematurely due to malformation of the valves in the outflow tract of the heart, and therefore, the gene plays a crucial role in cardiac development as well as lymphocyte differentiation (Schilham et al., 1996). In adult mouse testis, Sox5 is expressed in a stage-specific manner during spermatogenesis (Denny et al., 1992b). Mouse testis also synthesizes Sox17 protein that functions as a transcriptional factor activating expression of other genes associated with premeiotic phase of spermatogenesis ( Kanai et al., 1996). Sox2, Sox3 and Sox11 are expressed in the developing nervous system ( Uwanogho et al., 1995). In recent studies, we have cloned and characterized Sox cDNAs derived from
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Xenopus laevis ovary ( Komatsu et al., 1996; Shiozawa et al., 1996; Hiraoka et al., 1997; Sakai et al., 1997). The Xenopus Sox genes are involved in oogenesis and/or early embryogenesis. In this study, we isolated and analyzed a cDNA encoding a novel mouse Sox protein, mSox13, containing an SRY-type HMG box and a leucine zipper motif.
2. Materials and methods 2.1. Isolation of mouse Sox cDNAs A cDNA library of mouse 8.5-day embryos was provided by Dr H. Ikeshima (Ikeshima et al., 1995) and screened with a mixture of partial cDNAs encoding mouse Sox5, Sox6 and Sox13 HMG boxes as probes. The partial cDNAs were obtained by RT/PCR, and the method was essentially the same as described previously (Denny et al., 1992a). 2.2. Northern analysis A Northern analysis was carried out using the same method as described elsewhere ( Komatsu et al., 1996). A fragment of 1593 bp (Fig. 1; nt 1597–3189) derived from the mSox13 cDNA was used as a probe. 2.3. EMSA Nucleotides 1225–1470 of the mSox13 cDNA, encoding aa 393–474, were amplified by PCR using primers: msox13BmU (5∞-AAGGATCCGGAACAGCAGCCACATCAAGAGA-3∞) and msox13R1D (5∞-TTGAATTCAGGTACGCTTAGGCCTTGGCTTGTAC-3∞). The PCR fragment was digested with BamHI and EcoRI, and subcloned into the pGEX-3X expression vector (Pharmacia). Recombinant polypeptide of mSox13 HMG domain was expressed in E.coli as a GST fusion protein. GST-mSox13 fusion and non-fused GST were purified by affinity chromatography on glutathioneSepharose 4B (Pharmacia). The purified proteins were used in EMSA. EMSA procedures have been described elsewhere (Shiozawa et al., 1996).
3. Results and discussion 3.1. Cloning and sequencing of mSox13 A mouse (8.5-day embryo) cDNA library was screened using a mixture of partial cDNAs encoding mouse Sox5, Sox6 and Sox13 HMG boxes as probes. The screening yielded 15 positive clones. To categorize the yielded cDNA clones, all of them were subjected to sequence analyses. Based on their partial sequences, the cDNAs were categorized into two groups. A cDNA clone was assigned to mSox5 (Denny et al., 1992b), and
the rest were all mSox13. The longest cDNA clone (3.2 knt) contained the same sequence as a partial mSox13 cDNA. The partial mSox13 cDNA reported by Wright et al. (1993) contains only 170 nt encoding 56 aa of an SRY-type HMG box, and therefore, the sequences flanking the HMG box of mSox13 are not known. Thus, we further analyzed our cDNA clone to determine the entire primary structure of mSox13 protein. Fig. 1 shows the nt sequence of the mSox13 cDNA and its deduced aa sequence. A putative poly(A) signal, AATAAA, was found upstream of the poly(A)-attached 3∞ end of the cDNA in its 3∞ UTR. An ORF encodes a protein of 595 aa containing the HMG box. Another characteristic sequence found in the primary structure of mSox13 is a leucine zipper motif located in the N-terminal region. 3.2. Comparison of sequences of type-D Sox proteins The family members of type-D SOX appear to have very similar sequence features. We compare the sequence of mSox13 with those of mouse Sox6/SOX-LZ (Connor et al., 1995; Takamatsu et al., 1995) and frog xSox12 ( Komatsu et al., 1996), which belong to the type-D family. As seen in Fig. 2a, the SRY-type HMG box is located at the center of the C-terminal half of each SOX protein. The percentage score of aa conservation is over 90% in any pair of HMG box (Fig. 2b, d). The SOX proteins are not uniform in spacing between the HMG box and the leucine zipper. The most N-terminal conserved motif is the leucine zipper ( Fig. 2a, c). Out of the four leucine residues, each of the last three is accompanied with a glutamine residue, and the consensus is LX QLX QLX QL (L=leucine, Q=glutamine 5 5 5 and X=any residue), which is completely conserved in all the type-D SOX proteins. There are several other residues conserved completely within the leucine zipper. The homology scores of the motif are more than 68% in any pair (Fig. 2c, d). A sequence stretch to the C-terminal side of the leucine zipper is also well conserved among the proteins and is remarkably rich in the content of glutamine residue (Fig. 2a, c, d). Thus, the glutamine-rich sequence motif was designated as Q box here in this study. Judging from the degree of aa conservation, the Q box may play a functionally important role. We therefore surveyed DNA and protein sequence data banks (GenBank, NBRF-PIR and SWISS-PROT ) to find proteins with similar sequence motifs to the Q box, but failed. Although we found many proteins with glutamine-rich sequences exhibiting 40–50% homologies to the Q box sequence, none of the alignments between the Q box and such glutamine-rich sequences suggested any significant structural similarity. The leucine zipper is known to serve as a protein–protein interaction. In a previous study, it has been reported that Sox6/SOX-LZ can dimerize homophilically in vitro, and the leucine zipper is responsible for the dimerization ( Takamatsu et al., 1995). There is, so far, no report on
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Fig. 1. Nucleotide sequence of mSox13 cDNA and its deduced amino-acid sequence. Nucleotides are numbered on the right. The deduced aa sequence is shown under the nt sequence. The numbers of aa are indicated in parentheses on the right. An in-frame stop codon is indicated with an asterisk. The SRY-related HMG box is underlined. A putative leucine zipper motif is marked with arrow heads and dots. The polyadenylation signal AATAAA is double-underlined. The nucleotide sequence has been submitted to the DDBJ, EMBL and GenBank Databases under the Accession Number AB006329.
target proteins of Sox6/SOX-LZ in its heterophilic interactions, but it might be possible. The highly conserved Q box is located immediately adjacent to the leucine zipper motif of type-D SOX proteins and may reinforce the leucine zipper-mediated protein–protein interaction or function to specify target proteins in its interaction.
( Fig. 3). As described above, the mSox13 cDNA is 3.2 knt in size, and therefore, the cDNA covers nearly the entire sequence of mSox13 mRNA. As shown in Fig. 3, the expression of mSox13 mRNA is restricted to the ovary and kidney. Further work is necessary to elucidate whether the mSox13 gene participates in development of kidney and ovary.
3.3. Tissue-specific expression of mSox13 3.4. DNA binding of mSox13 The tissue distribution of mSox13 mRNA in adult mouse was investigated by Northern blot analysis. As a Northern probe, we used a 3∞-half fragment of the mSox13 cDNA that was devoid of the highly conserved sequence motifs. The size of mSox13 mRNA was 3.3 knt
It has been believed that all SOX proteins including SRY bind to the same consensus sequence, AACAAT (Denny et al., 1992b; Lovell-Badge, 1993; Connor et al., 1995; Kamachi et al., 1995; Takamatsu et al., 1995;
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Fig. 2. Comparison of sequence features between mSox13 and other SOX proteins. (a) Schematic illustration of type-D SOX proteins. The leucine zipper, Q box and HMG box are indicated by open, dotted and closed boxes, respectively. Amino acids of N- and C-termini and at the boundaries of each boxes are numbered. (b) Sequence comparison of HMG box amino acids of type-D SOX proteins. Amino-acid variations among the three proteins are indicated by highlighted letters. (c) Leucine zipper motif and its flanking sequences of type-D SOX proteins. Hyphens represent sequence gaps. Amino acids shared by all the three SOX proteins are indicated by highlighted letters. The leucine zipper and a highly conserved sequence stretch, named Q box, are boxed. (d ) Homology scores of mSox13 to the other members of the type-D SOX family. Regional homologies (defined as the percentage amino-acid identity) of a protein shown in each column to a protein in a row are calculated based on the alignments shown in (b) and (c).
Fig. 3. Tissue-specific expression of mSox13. Poly(A) RNAs (3 mg) from adult mouse tissues were subjected to Northern analysis. As size markers, human 18S and 28S rRNAs derived from HL-60 cells were electrophoresed on the same gel. The last 1593 nt of the mSox13 cDNA were used as a probe. As a control for mRNA integrity, the RNA blot was rehybridized with a mouse b-actin probe (ACT ).
Collignon et al., 1996; Kanai et al., 1996; Shiozawa et al., 1996; Hiraoka et al., 1997; Sakai et al., 1997). Thus, we tested whether or not the potential DNAbinding domain of mSox13 also recognizes the sequence.
A fusion polypeptide of mSox13 HMG box and GST was expressed in E. coli, and its DNA binding was assessed with EMSA. Oligonucleotide probes used in EMSA are listed in Fig. 4a. As seen in Fig. 4b, the fusion protein recognized a probe WT containing the canonical target sequence, AACAAT, whereas nonfused GST did not bind to the sequence. The specificity of the target sequence was assessed by EMSA with a series of oligonucleotide probes including WT and its variants (Fig. 4c). The fusion protein binds most strongly to probe WT. Although probe MTALL does not bind, mutant probes MT1 and MT3 can be recognized by mSox13. The rest of the mutant probes were not recognizable by the protein. Our data demonstrate that mSox13 interacts specifically with the consensus sequence AACAAT. 3.5. Conclusions (1) We isolated mouse SRY-related cDNA, mSox13, and determined its nucleotide sequence. Its deduced
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Acknowledgement We thank Dr H. Ikeshima for her generous gift of the mouse cDNA library. This work was supported in part by a National Grant-in-Aid for the Establishment of High-Tech Research Center in a Private University from Ministry of Education, Science and Culture to S.A., Grants-in-Aids from Ministry of Education, Science and Culture to S.A. and Y.H., and by Keio Gakuji Fukuzawa Memorial Funds for the Advancement of Education and Research and Keio Gakuji Academic Developmental Funds from Keio University to S.A. and Y.H.
References
Fig. 4. EMSAs of oligonucleotide binding by GST-mSox13. (a) DNA sequences of EMSA probes are aligned. Mutated nucleotide in each probe is shown in upper-case letters. Nucleotides flanking the mutated position in each probe are identical to those of WT probe and indicated by a hyphen. Names of probes are indicated on the right. (b) Purified GST (1 mg; lane 2) or GST-mSox13 (1 mg; lanes 3 and 4) was preincubated in 10 ml of binding buffer (10 mM HEPES, pH 7.1/60 mM KCl/1 mM EDTA/1 mM DTT/12% glycerol ) with 10 fmol (approx. 20 000 cpm) of 32P-labeled oligonucleotide probe WT for 15 min at room temperature. The reaction for lane 1 was performed in the absence of protein. Unlabeled competitor oligonucleotide WT (1000-times excess over the labeled probe) was included in the control reaction ( lane 4). The reactions were electrophoresed on a 5% polyacrylamide gel in 0.5× TBE buffer at room temperature. The presence of three retarded molecular species in lane 3 is presumed to be due to proteolytic clipping of the amino-terminus of the GST moiety during the purification process. (c) The specificity of the sequence recognized by mSox13 was assessed by EMSA using a series of probes shown in (a). Concentrations of GST-mSox13 and 32P-labeled probes are the same as those in the experiment shown in (b). The probes used are indicated above the respective lanes.
amino-acid sequence contains the SRY-related type-D HMG box and a leucine zipper motif. (2) A novel sequence motif, named Q box, is well conserved among the three members of the type-D SOX subfamily. (3) The expression of mSox13 mRNA is restricted to the kidney and ovary. (4) The HMG domain of mSox13 binds to the canonical target sequence of SRY and SOX proteins, AACAAT.
Collignon, J., Sockanathan, S., Hacker, A., Cohen-Tannoudji, M., Norris, D., Rastan, S., Stevanovic, M., Goodfellow, P.N., LovellBadge, R., 1996. A comparison of the properties of Sox-3 with Sry and two related genes, Sox-1 and Sox-2. Development 122, 509–520. Connor, F., Wright, E., Denny, P., Koopman, P., Ashworth, A., 1995. The Sry-related HMG box-containing gene Sox6 is expressed in the adult testis and developing nervous system of the mouse. Nucleic Acids Res. 17, 3365–3372. Denny, P., Swift, S., Brand, N., Dabhade, N., Barton, P., Ashworth, A., 1992a. A conserved family of genes related to the testis determining gene, SRY. Nucleic Acids Res. 21, 2887 Denny, P., Swift, S., Connor, F., Ashworth, A., 1992b. An SRY-related gene expressed during spermatogenesis in the mouse encodes a sequence-specific DNA-binding protein. EMBO J. 11, 3705–3712. Ferrari, S., Harley, V.R., Pontiggia, A., Goodfellow, P.N., LovellBadge, R., Bianchi, M.E., 1992. SRY, like HMG1, recognizes sharp angles in DNA. EMBO J. 11, 4497–4506. Foster, J.W., Dominguez-Steglich, M.A., Guioli, S., Kwok, C., Weller, P.A., Stevanovic, M., Weissenbach, J., Mansour, S., Young, I.D., Goodfellow, P.N., Brook, J.D., Schafer, A.J., 1994. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature 372, 525–530. Giese, K., Cox, J., Grosschedl, R., 1992. The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell 69, 185–195. Harley, V.R., Jackson, D.I., Hextall, P.J., Hawkins, J.R., Berkovitz, G.D., Sockanathan, S., Lovell-Badge, R., Goodfellow, P.N., 1992. DNA binding activity of recombinant SRY from normal males and XY females. Science 255, 453–456. Hiraoka, Y., Komatsu, N., Sakai, Y., Ogawa, M., Shiozawa, M., Aiso, S., 1997. XLS13A and XLS13B: SRY-related genes of Xenopus laevis. Gene 197, 65–71. Ikeshima, H., Imai, S., Shimoda, K., Hata, J., Takano, T., 1955. Expression of a MADS box gene, MEF2, in neurons of the mouse central nervous system: implication of its binary function in myogenic and neurogenic cell lineages. Neurosci. Lett. 200, 117–120. Kamachi, Y., Sockanathan, S., Liu, Q., Breitman, M., Lovell-Badge, R., Kondoh, H., 1995. Involvement of Sox proteins in lens-specific activation of crystallin genes. EMBO J. 14, 3510–3519. Kanai, Y., Kanai-Azuma, M., Noce, T., Saido, T.C., Shiroishi, T., Hayashi, Y., Yazaki, K., 1996. Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis. J. Cell Biol. 133, 1–15. King, C.-Y., Weiss, M.A., 1993. The SRY high-mobility-group box
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recognizes DNA by partial intercalation in the minor groove: A topological mechanism of sequence specificity. Proc. Natl. Acad. Sci. USA 90, 11990–11994. Komatsu, N., Hiraoka, Y., Shiozawa, M., Ogawa, M., Aiso, S., 1996. Cloning and expression of Xenopus laevis xSox12 cDNA. Biochim. Biophys. Acta 1305, 117–119. Laudet, V., Stehelin, D., Clevers, H., 1993. Ancestry and diversity of the HMG box superfamily. Nucleic Acids Res. 21, 2493–2501. Lefebvre, V., Huang, W., Harley, V., Goodfellow, P.N., Crombrugghe, B., 1997. SOX9 is a potent activator of the chondrocyte-specific enhancer of the Proa1(II ) collagen gene. Mol. Cel. Biol. 17, 2336–2346. Lovell-Badge, R., 1993. Sex determining gene expression during embryogenesis. Phil. Trans. R. Soc. Lond. B 339, 159–164. Nasrin, N., Buggs, C., Kong, X.F., Carnazza, J., Goebl, M., Alexander-Bridges, M., 1991. DNA-binding properties of the product of the testis-determining gene and a related protein. Nature 354, 317–320. Ng, L.-J., Wheatley, S., Muscat, G.E.O., Conway-Campbell, J., Bowles, J., Wright, E., Bell, D.M., Tam, P.P.L., Cheah, K.S.E., Koopman, P., 1997. SOX9 binds DNA, activates transcription, and coexpresses with type II collagen during chondrogenesis in the mouse. Dev. Biol. 183, 108–121. Sakai, Y., Hiraoka, Y., Konishi, M., Ogawa, M., Aiso, S., 1997. Isolation and characterization of Xenopus laevis xSox-B1 cDNA. Arch. Biochem. Biophys. 346, 1–6. Schilham, M.W., Oosterwegel, M.A., Moerer, P., Ya, J., Boer, P.A.J.-d., Wetering, M.v.-d., Verbeek, S., Lamers, W.H., Keuisbbek, A.M., Cumano, A., Clevers, H., 1996. Defects in cardiac outflow tract formation and pre-B-lymphocyte expansion in mice lacking Sox-4. Nature 380, 711–714. Shiozawa, M., Hiraoka, Y., Komatsu, N., Ogawa, M., Sakai, Y., Aiso, S., 1996. Cloning and characterization of Xenopus laevis xSox7 cDNA. Biochim. Biophys. Acta 1309, 73–76.
Sinclair, A.H., Berta, P., Palmer, M.S., Hawkins, J.R., Griffiths, B.L., Smith, M.J., Foster, J.W., Frischauf, A.-M., Lovell-Badge, R., Goodfellow, P.N., 1990. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346, 240–244. Stevanovic, M., Lovell-Badge, R., Collignon, J., Goodfellow, P.N., 1993. SOX3 is an X-linked gene related to SRY. Hum. Mol. Gen. 2, 2013–2018. Takamatsu, N., Kanda, H., Tsuchiya, I., Yamada, S., Ito, M., Kabeno, S., Shiba, T., Yamashita, S., 1995. A gene that is related to SRY and is expressed in the testes encodes a leucine zipper-containing protein. Mol. Cell. Biol. 15, 3759–3766. Uwanogho, D., Rex, M., Cartwright, E.J., Pearl, G., Healy, C., Scotting, P.J., Sharpe, P.T., 1995. Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development. Mech. Dev. 49, 23–36. Wagner, T., Wirth, J., Meyer, J., Zabel, B., Held, M., Zimmer, J., Pasantes, J., Bricarelli, F.D., Keutel, J., Hustert, E., Wolf, U., Tommerup, N., Schempp, W., Scherer, G., 1994. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79, 1111–1120. Wetering, M.v.-d., Oosterwegel, M., Norren, K.v., Clevers, H., 1993. Sox-4, an Sry-like HMG box protein, is a transcriptional activator in lymphocytes. EMBO J. 12, 3847–3854. Wotton, D., Lake, R.A., Farr, C.J., Owen, M.J., 1995. The high mobility group transcription factor, SOX4, transactivates the human CD2 enhancer. J. Biol. Chem. 270, 7515–7522. 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 Wright, E.M., Hargrave, R., Christiansen, J., Cooper, L., Kun, J., Evans, T., Gangadharan, U., Greenfield, A., Koopman, P., 1995. The SRY-related gene SOX9 is expressed during chondrogenesis in mouse embryos. Nat. Genet. 9, 15–20.
Cloning and characterization of mouse mSox13 cDNA Susumu Kido a,1, Yoshiki Hiraoka b,*, Motoyuki Ogawa b, Yukinao Sakai b, Yasunori Yoshimura c, Sadakazu Aiso b a Tokyo Electric Power Hospital, Shinjuku-ku, Tokyo, 160, Japan b Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160, Japan c Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160, Japan Received 25 August 1997; accepted 13 November 1997; Received by A. Fujiyama
Abstract A novel SRY-related cDNA, mSox13, was isolated from a l phage library derived from mouse embryo. The cDNA encodes a protein of 595 amino acids containing the SRY-type high mobility group (HMG) box and a putative leucine zipper motif. A sequence comparison of mSox13 and other type-D SOX proteins shows that the leucine zipper and a neighboring glutamine-rich sequence stretch, which was named Q box, are well conserved among known type-D SOX proteins. The expression of mSox13 is restricted to the kidney and ovary. The electrophoretic mobility shift assay indicates that the recombinant mSox13 protein is capable of binding to the AACAAT sequence. © 1998 Elsevier Science B.V. Keywords: DNA binding; Embryo; HMG box; Leucine zipper; SRY
1. Introduction SOX is a family of genes related to the testis determining gene, SRY, and its members have been isolated from a wide variety of organisms (Sinclair et al., 1990; Denny et al., 1992a; Laudet et al., 1993; Stevanovic et al., 1993). The SOX genes encode transcriptional factors with a characteristic DNA-binding motif known as the HMG box. The SRY-type HMG domains interact with DNA in a sequence-specific manner (Nasrin et al., 1991; Denny et al., 1992b; Ferrari et al., 1992; Giese et al., 1992; Harley et al., 1992; King and Weiss, 1993; Wetering et al., 1993; Kamachi et al., 1995; Wotton et al., 1995; Shiozawa et al., 1996; Hiraoka et al., 1997; Sakai et al., 1997). SOX proteins have been reported to participate * Corresponding author. Tel: +81 3 33531211; Fax: +81 3 53791977; e-mail: [email protected] 1 Present address: Kawasaki Municipal Hospital, 12-1, Shinkawadori, Kawasaki-ku, Kawasaki City, Kanagawa 210, Japan. Abbreviations: aa, amino acid(s); b, base(s); cDNA, complementary DNA; EMSA, electrophoretic mobility shift assay; GST, glutathioneS-transferase; kb, kilobase(s) or 1000 b; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; RT/PCR, reverse transcription/PCR; SOX, SRY-related protein(s); SOX, gene (DNA, RNA) encoding SOX; SRY, sex-determining region Y gene; SRY, protein encoded by SRY; UTR, untranslated region. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 6 67 - 7
in controls of various developmental processes. The SOX9 gene was identified as a causal gene for autosomal sex reversal and campomelic dysplasia. This suggests that SOX9 plays important parts in both gonadal development and chondrogenesis (Foster et al., 1994; Wagner et al., 1994; Wright et al., 1995). Very recent studies have demonstrated that mouse Sox9 functions as a transcriptional activator in the expression of the type II collagen gene which is involved in cartilage development (Lefebvre et al., 1997; Ng et al., 1997). Sox4 is expressed in T and pre-B cells and functions as a transcriptional activator ( Wetering et al., 1993; Wotton et al., 1995). A study using gene targeting has revealed that Sox4deficient mice die prematurely due to malformation of the valves in the outflow tract of the heart, and therefore, the gene plays a crucial role in cardiac development as well as lymphocyte differentiation (Schilham et al., 1996). In adult mouse testis, Sox5 is expressed in a stage-specific manner during spermatogenesis (Denny et al., 1992b). Mouse testis also synthesizes Sox17 protein that functions as a transcriptional factor activating expression of other genes associated with premeiotic phase of spermatogenesis ( Kanai et al., 1996). Sox2, Sox3 and Sox11 are expressed in the developing nervous system ( Uwanogho et al., 1995). In recent studies, we have cloned and characterized Sox cDNAs derived from
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Xenopus laevis ovary ( Komatsu et al., 1996; Shiozawa et al., 1996; Hiraoka et al., 1997; Sakai et al., 1997). The Xenopus Sox genes are involved in oogenesis and/or early embryogenesis. In this study, we isolated and analyzed a cDNA encoding a novel mouse Sox protein, mSox13, containing an SRY-type HMG box and a leucine zipper motif.
2. Materials and methods 2.1. Isolation of mouse Sox cDNAs A cDNA library of mouse 8.5-day embryos was provided by Dr H. Ikeshima (Ikeshima et al., 1995) and screened with a mixture of partial cDNAs encoding mouse Sox5, Sox6 and Sox13 HMG boxes as probes. The partial cDNAs were obtained by RT/PCR, and the method was essentially the same as described previously (Denny et al., 1992a). 2.2. Northern analysis A Northern analysis was carried out using the same method as described elsewhere ( Komatsu et al., 1996). A fragment of 1593 bp (Fig. 1; nt 1597–3189) derived from the mSox13 cDNA was used as a probe. 2.3. EMSA Nucleotides 1225–1470 of the mSox13 cDNA, encoding aa 393–474, were amplified by PCR using primers: msox13BmU (5∞-AAGGATCCGGAACAGCAGCCACATCAAGAGA-3∞) and msox13R1D (5∞-TTGAATTCAGGTACGCTTAGGCCTTGGCTTGTAC-3∞). The PCR fragment was digested with BamHI and EcoRI, and subcloned into the pGEX-3X expression vector (Pharmacia). Recombinant polypeptide of mSox13 HMG domain was expressed in E.coli as a GST fusion protein. GST-mSox13 fusion and non-fused GST were purified by affinity chromatography on glutathioneSepharose 4B (Pharmacia). The purified proteins were used in EMSA. EMSA procedures have been described elsewhere (Shiozawa et al., 1996).
3. Results and discussion 3.1. Cloning and sequencing of mSox13 A mouse (8.5-day embryo) cDNA library was screened using a mixture of partial cDNAs encoding mouse Sox5, Sox6 and Sox13 HMG boxes as probes. The screening yielded 15 positive clones. To categorize the yielded cDNA clones, all of them were subjected to sequence analyses. Based on their partial sequences, the cDNAs were categorized into two groups. A cDNA clone was assigned to mSox5 (Denny et al., 1992b), and
the rest were all mSox13. The longest cDNA clone (3.2 knt) contained the same sequence as a partial mSox13 cDNA. The partial mSox13 cDNA reported by Wright et al. (1993) contains only 170 nt encoding 56 aa of an SRY-type HMG box, and therefore, the sequences flanking the HMG box of mSox13 are not known. Thus, we further analyzed our cDNA clone to determine the entire primary structure of mSox13 protein. Fig. 1 shows the nt sequence of the mSox13 cDNA and its deduced aa sequence. A putative poly(A) signal, AATAAA, was found upstream of the poly(A)-attached 3∞ end of the cDNA in its 3∞ UTR. An ORF encodes a protein of 595 aa containing the HMG box. Another characteristic sequence found in the primary structure of mSox13 is a leucine zipper motif located in the N-terminal region. 3.2. Comparison of sequences of type-D Sox proteins The family members of type-D SOX appear to have very similar sequence features. We compare the sequence of mSox13 with those of mouse Sox6/SOX-LZ (Connor et al., 1995; Takamatsu et al., 1995) and frog xSox12 ( Komatsu et al., 1996), which belong to the type-D family. As seen in Fig. 2a, the SRY-type HMG box is located at the center of the C-terminal half of each SOX protein. The percentage score of aa conservation is over 90% in any pair of HMG box (Fig. 2b, d). The SOX proteins are not uniform in spacing between the HMG box and the leucine zipper. The most N-terminal conserved motif is the leucine zipper ( Fig. 2a, c). Out of the four leucine residues, each of the last three is accompanied with a glutamine residue, and the consensus is LX QLX QLX QL (L=leucine, Q=glutamine 5 5 5 and X=any residue), which is completely conserved in all the type-D SOX proteins. There are several other residues conserved completely within the leucine zipper. The homology scores of the motif are more than 68% in any pair (Fig. 2c, d). A sequence stretch to the C-terminal side of the leucine zipper is also well conserved among the proteins and is remarkably rich in the content of glutamine residue (Fig. 2a, c, d). Thus, the glutamine-rich sequence motif was designated as Q box here in this study. Judging from the degree of aa conservation, the Q box may play a functionally important role. We therefore surveyed DNA and protein sequence data banks (GenBank, NBRF-PIR and SWISS-PROT ) to find proteins with similar sequence motifs to the Q box, but failed. Although we found many proteins with glutamine-rich sequences exhibiting 40–50% homologies to the Q box sequence, none of the alignments between the Q box and such glutamine-rich sequences suggested any significant structural similarity. The leucine zipper is known to serve as a protein–protein interaction. In a previous study, it has been reported that Sox6/SOX-LZ can dimerize homophilically in vitro, and the leucine zipper is responsible for the dimerization ( Takamatsu et al., 1995). There is, so far, no report on
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Fig. 1. Nucleotide sequence of mSox13 cDNA and its deduced amino-acid sequence. Nucleotides are numbered on the right. The deduced aa sequence is shown under the nt sequence. The numbers of aa are indicated in parentheses on the right. An in-frame stop codon is indicated with an asterisk. The SRY-related HMG box is underlined. A putative leucine zipper motif is marked with arrow heads and dots. The polyadenylation signal AATAAA is double-underlined. The nucleotide sequence has been submitted to the DDBJ, EMBL and GenBank Databases under the Accession Number AB006329.
target proteins of Sox6/SOX-LZ in its heterophilic interactions, but it might be possible. The highly conserved Q box is located immediately adjacent to the leucine zipper motif of type-D SOX proteins and may reinforce the leucine zipper-mediated protein–protein interaction or function to specify target proteins in its interaction.
( Fig. 3). As described above, the mSox13 cDNA is 3.2 knt in size, and therefore, the cDNA covers nearly the entire sequence of mSox13 mRNA. As shown in Fig. 3, the expression of mSox13 mRNA is restricted to the ovary and kidney. Further work is necessary to elucidate whether the mSox13 gene participates in development of kidney and ovary.
3.3. Tissue-specific expression of mSox13 3.4. DNA binding of mSox13 The tissue distribution of mSox13 mRNA in adult mouse was investigated by Northern blot analysis. As a Northern probe, we used a 3∞-half fragment of the mSox13 cDNA that was devoid of the highly conserved sequence motifs. The size of mSox13 mRNA was 3.3 knt
It has been believed that all SOX proteins including SRY bind to the same consensus sequence, AACAAT (Denny et al., 1992b; Lovell-Badge, 1993; Connor et al., 1995; Kamachi et al., 1995; Takamatsu et al., 1995;
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Fig. 2. Comparison of sequence features between mSox13 and other SOX proteins. (a) Schematic illustration of type-D SOX proteins. The leucine zipper, Q box and HMG box are indicated by open, dotted and closed boxes, respectively. Amino acids of N- and C-termini and at the boundaries of each boxes are numbered. (b) Sequence comparison of HMG box amino acids of type-D SOX proteins. Amino-acid variations among the three proteins are indicated by highlighted letters. (c) Leucine zipper motif and its flanking sequences of type-D SOX proteins. Hyphens represent sequence gaps. Amino acids shared by all the three SOX proteins are indicated by highlighted letters. The leucine zipper and a highly conserved sequence stretch, named Q box, are boxed. (d ) Homology scores of mSox13 to the other members of the type-D SOX family. Regional homologies (defined as the percentage amino-acid identity) of a protein shown in each column to a protein in a row are calculated based on the alignments shown in (b) and (c).
Fig. 3. Tissue-specific expression of mSox13. Poly(A) RNAs (3 mg) from adult mouse tissues were subjected to Northern analysis. As size markers, human 18S and 28S rRNAs derived from HL-60 cells were electrophoresed on the same gel. The last 1593 nt of the mSox13 cDNA were used as a probe. As a control for mRNA integrity, the RNA blot was rehybridized with a mouse b-actin probe (ACT ).
Collignon et al., 1996; Kanai et al., 1996; Shiozawa et al., 1996; Hiraoka et al., 1997; Sakai et al., 1997). Thus, we tested whether or not the potential DNAbinding domain of mSox13 also recognizes the sequence.
A fusion polypeptide of mSox13 HMG box and GST was expressed in E. coli, and its DNA binding was assessed with EMSA. Oligonucleotide probes used in EMSA are listed in Fig. 4a. As seen in Fig. 4b, the fusion protein recognized a probe WT containing the canonical target sequence, AACAAT, whereas nonfused GST did not bind to the sequence. The specificity of the target sequence was assessed by EMSA with a series of oligonucleotide probes including WT and its variants (Fig. 4c). The fusion protein binds most strongly to probe WT. Although probe MTALL does not bind, mutant probes MT1 and MT3 can be recognized by mSox13. The rest of the mutant probes were not recognizable by the protein. Our data demonstrate that mSox13 interacts specifically with the consensus sequence AACAAT. 3.5. Conclusions (1) We isolated mouse SRY-related cDNA, mSox13, and determined its nucleotide sequence. Its deduced
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Acknowledgement We thank Dr H. Ikeshima for her generous gift of the mouse cDNA library. This work was supported in part by a National Grant-in-Aid for the Establishment of High-Tech Research Center in a Private University from Ministry of Education, Science and Culture to S.A., Grants-in-Aids from Ministry of Education, Science and Culture to S.A. and Y.H., and by Keio Gakuji Fukuzawa Memorial Funds for the Advancement of Education and Research and Keio Gakuji Academic Developmental Funds from Keio University to S.A. and Y.H.
References
Fig. 4. EMSAs of oligonucleotide binding by GST-mSox13. (a) DNA sequences of EMSA probes are aligned. Mutated nucleotide in each probe is shown in upper-case letters. Nucleotides flanking the mutated position in each probe are identical to those of WT probe and indicated by a hyphen. Names of probes are indicated on the right. (b) Purified GST (1 mg; lane 2) or GST-mSox13 (1 mg; lanes 3 and 4) was preincubated in 10 ml of binding buffer (10 mM HEPES, pH 7.1/60 mM KCl/1 mM EDTA/1 mM DTT/12% glycerol ) with 10 fmol (approx. 20 000 cpm) of 32P-labeled oligonucleotide probe WT for 15 min at room temperature. The reaction for lane 1 was performed in the absence of protein. Unlabeled competitor oligonucleotide WT (1000-times excess over the labeled probe) was included in the control reaction ( lane 4). The reactions were electrophoresed on a 5% polyacrylamide gel in 0.5× TBE buffer at room temperature. The presence of three retarded molecular species in lane 3 is presumed to be due to proteolytic clipping of the amino-terminus of the GST moiety during the purification process. (c) The specificity of the sequence recognized by mSox13 was assessed by EMSA using a series of probes shown in (a). Concentrations of GST-mSox13 and 32P-labeled probes are the same as those in the experiment shown in (b). The probes used are indicated above the respective lanes.
amino-acid sequence contains the SRY-related type-D HMG box and a leucine zipper motif. (2) A novel sequence motif, named Q box, is well conserved among the three members of the type-D SOX subfamily. (3) The expression of mSox13 mRNA is restricted to the kidney and ovary. (4) The HMG domain of mSox13 binds to the canonical target sequence of SRY and SOX proteins, AACAAT.
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