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Analysis of Temporal Expression Pattern andcis-Regulatory Sequences of Chicken Pepsinogen A and C
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
250, 420 – 424 (1998)
RC989290
Analysis of Temporal Expression Pattern and cisRegulatory Sequences of Chicken Pepsinogen A and C Nobuyuki Sakamoto,1 Hidetoshi Saiga, and Sadao Yasugi Department of Biology, Faculty of Science, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachioji, Tokyo 192-0397, Japan
Received August 5, 1998
Three groups of pepsinogens exist in vertebrates, namely, pepsinogen A, pepsinogen C, and prochymosin, which are produced at different developmental stages. In the chicken, prochymosin is expressed only in the embryonic stage, while pepsinogens A and C are secreted from adult chicken proventricular (glandular stomach) mucosa. In order to understand the mechanism of transcriptional regulation of these genes, we have cloned the genes encoding chicken pepsinogens A and C and analyzed the sequences possibly involved in their regulation. 5*-Upstream sequences of both genes contain putative binding motifs for transcription factors such as GATA, Sox, and HNF-3b, which are expressed in the chicken gut epithelium. Moreover, we found seven putative binding motifs for human MZF-1 in intron 8 of pepsinogen A gene. These transcription factors may act as regulators of expression of chicken pepsinogen genes. © 1998 Academic Press
Pepsinogens, zymogens of pepsins, are classified into three major groups: pepsinogens A, pepsinogens C (progastricsins) and prochymosins. These pepsinogens are secreted from gastric mucosa at different developmental stages in the vertebrates. In mammals, prochymosin is a major component of pepsinogen in the neonatal stage, and pepsinogens A and C constitute major components of adult pepsinogen [1, 2]. Such stagedependent production of pepsinogens has been attributed to their enzymatic properties, such as a high milk clotting activity of prochymosin [3] and substrate specificities, in relation to the physiological demand of the animals. In the chicken, prochymosin is produced only in embryonic stages and has been named ECPg (embryonic chicken pepsinogen) [4]. Chicken pepsinogen A (cPgA) is a major component of pepsinogens secreted from adult proventriculus (glandular stomach) [5]. Chicken pepsinogen C (cPgC) is a minor component of 1 Corresponding author. Fax: 181 (426) 77-2559. E-mail: [email protected].
0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
adult pepsinogens [5], and its protein being detected only in hatched chicken proventriculus [6]. To understand the molecular nature of mechanisms for differential usage of pepsinogens during development, it is necessary first to study how the transcription of pepsinogen genes is regulated. To date, molecular cloning of pepsinogen cDNAs and genes has been performed in various vertebrate species, especially in mammals, and studies on these genes have revealed that methylation of genes plays an important role in the regulation of their expression [7-9]. It has also been shown that glucocorticoid hormone has an upregulatory effect on pepsinogen gene expression [10]. However, the molecular basis of temporal regulation of pepsinogen genes is largely unknown. To elucidate the mechanism for switching of pepsinogens during development, we have cloned cPgA and cPgC genes expressed in the adult proventricular mucosa. Unexpectedly, cPgA and cPgC genes are also expressed at late embryonic stages. Analysis of 59-flanking region of cPgA and cPgC genes demonstrated the existence of putative binding sites for transcription factors, GATA, Sox and HNF-3b. We also found seven putative binding sites for transcription factor MZF-1 in intron 8 of the cPgA gene. Our results revealed that the switching of chicken pepsinogens occurs during the late embryonic period unlike mammalian pepsinogens and suggest that transcription factors such as GATA, Sox, HNF-3b and MZF-1 may regulate expression of chicken pepsinogen genes during development. MATERIALS AND METHODS Cloning and sequencing of cDNA and genes of cPgA and cPgC. A cDNA library was constructed in the lgt11 vector from post-hatching 14-day chicken proventriculus. The library was screened for cPgA using 32P-labeled oligonucleotides [59-RGGMACCCASARGTTRGAGGAKCCVGTGTC-39], a highly conserved sequence among vertebrate pepsinogen genes. The cDNAs were subcloned into EcoRI site of pBluescript SK1 (Stratagene) and sequenced. The cPgA cDNA clone obtained was labeled with a-[32P]dCTP using a random primer DNA labeling kit (Takara) and used to screen for cPgC cDNA. A chicken liver genomic library constructed in the lfixII vector was
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FIG. 1. Amino acids sequence alignments of vertebrate pepsinogens. Amino acids sequences of cPgA and cPgC, deduced from cDNA sequences obtained in the present study are aligned and gapped by the computer program CLUSTAL W (ver. 1.5) with other known vertebrate pepsinogen proteins. All insertion sites of introns indicated by arrowheads are coincident. Asterisks show identical residues. Thr of residue 88 of cPgA, which differs from a previously reported sequence 13, is bold. The amino acid sequence data used are ECPg [4], human PgA [29], human PgC [30], and rat PgC [17].
screened with the cPgA cDNA or cPgC cDNA clones labeled with a[32P]dCTP. The genomic clones isolated were subcloned into pBluescript SK1 (Stratagene) and sequenced with an automatic DNA sequencer (Applied Biosystems Inc.). Sequence comparison and phylogenetic analysis. Amino acid sequences of vertebrate pepsinogens were aligned and phylogenetic analysis was performed by neighbor-joining method [11] using computer program CLUSTAL W (ver. 1.5) [12]. For bootstrap analysis, the bootstrap program of CLUSTAL W was used to generate 1000 data sets. Northern hybridization. Total RNA samples were denatured and subjected to electrophoresis in a 1% agarose gel and blotted on a
nylon membrane (BIO-RAD) in 10x SSC (1x SSC contains 0.15 M NaCl, 0.015 M sodium citrate). The membranes were exposed to 312nm UV light for 5 min. To avoid cross-hybridization, specific probes were made from DNA fragments which containing 39 non-coding region of cPgA and cPgC cDNAs. To detect ECPg mRNA, we used ECP200 or ECP1.1K as a probe [4]. To prepare the radioactive probe, a random primer DNA labeling kit (Takara) was used. The blots were hybridized to radioactive probe in 5x SSPE (1x SSPE contains 0.18 M NaCl, 1 mM EDTA, 10 mM sodium phosphate), 50% formamide, 5x Denhardt9s solution, 0.5% SDS and 20 mg/ml denatured salmon sperm DNA, at 43°C for overnight, and washed twice in 0.1x SSC, 0.1% SDS at 60°C for 30 min. The hybrids were detected by autoradiography or a bioimaging analyzer BAS2000 (Fuji Photo Film).
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FIG. 2. Phylogenetic relationship of vertebrate pepsinogens. A molecular phylogenetic tree was constructed by the neighbor-joining method [11] using the computer program CLUSTAL W [12]. cPgA and cPgC are boxed. The scale bar indicates an evolutionary distance of 0.05 amino acid substitutions per position. The amino acid sequence data used are human PgA [29], monkey PgA [31], pig PgA [32], ECPg [4], bovine prochymosin [33], human PgC [30], rat PgC [17], and frog PgC [34].
Primer extension analysis. Synthetic oligonucleotide primers were labeled with [32P]ATP and T4 polynucleotide kinase (Takara). 1mg of total RNA of post-hatching 7-day chicken proventriculus and labeled primer were mixed in annealing buffer (250 mM KCl, 2 mM Tris-HCl(pH8.0), 0.2 mM EDTA) and heated at 60°C for 90 min. After precipitation with ethanol, cDNA was synthesized with SuperScriptII (Gibco BRL) at 50°C for 50 min. The cDNA products were analyzed by electrophoresis in a 6% polyacrylamide/7 M urea gel.
RESULTS AND DISCUSSION Cloning of cDNAs of chicken pepsinogens A and C. The cPgA cDNA cloned was of 1277bp and contained an open reading frame composed of 382 amino acids, the sequence of which was completely identical to the previously reported sequence of chicken pepsinogen A [13], except for a Thr in place of Ser at residue 88, probably due to polymorphism (Fig. 1). The cPgC cDNA was 1348bp in length and the deduced amino acid sequence consisted of 389 residues (Fig. 1). To investigate the phylogenetic relationship of these pepsinogens to other vertebrate pepsinogens, we constructed a molecular phylogenetic tree of vertebrate pepsinogens (Fig. 2). The tree revealed that the cPgA gene is included in the pepsinogen A group and the cPgC gene belongs to the pepsinogen C group, while the ECPg gene is related to the bovine prochymosin gene, as reported previously [4]. ECPg is more closely related to cPgA than to cPgC. Putative signal peptide cleavage sites of cPgA and cPgC proteins as conjectured with the (-3, -1) rule [14] are between residue 15 and 16, and residue 16 and 17, respectively. The estimated molecular weights of secreted proteins of cPgA and cPgC are 40.4kD and 40.7kD respectively. Genomic Southern analysis revealed that cPgA and cPgC genes exist as single copies in the chicken genome (data not shown). Genomic structures of chicken pepsinogen A and C genes. To investigate the genomic structure of chicken pepsinogen genes, a chicken genomic library
was made and screened with cPgA or cPgC cDNA as a probe, and obtained clones were subcloned and sequenced. Both genes consist of nine exons just as mammalian pepsinogens [15-17] and ECPg [18] genes (Fig. 1 and 5). Furthermore, it was revealed that the positions of introns are highly conserved among vertebrate pepsinogen genes (Fig. 1). We found some polymorphism of amino acid residues in exon 4 of cPgA and in exon 1 of cPgC. In the latter case, the residue 8 is Val when deduced from cDNA whereas it is Met when deduced from genomic sequence. Temporal expression patterns of cPgA and cPgC during development. In mammals and birds, it is known that members of the different pepsinogen groups are expressed at different developmental stages [1, 2]. To investigate the temporal expression patterns of cPgA and cPgC genes, total RNA of proventriculus of various stages from embryonic day 12 (E12) to post-hatching day 7 (A7) was subjected to Northern analysis. Transcripts of cPgA and cPgC were seen in both embryonic and adult proventriculus. cPgA mRNA was detected from E16 to E19, and disappeared temporarily just before hatching. Within one day after hatching, transcription of the cPgA gene started again and then increased rapidly (Fig. 3). Expression pattern of the cPgC gene was similar to but weaker than that of cPgA. It is noteworthy that the transcripts of cPgA and cPgC were detected after transcription of ECPg gene was downregulated. This suggest that there are mechanisms which switch major components of pepsinogens from ECPg to cPgA and cPgC during the late embryonic period although the functions of embryonic cPgA and cPgC proteins are unknown at present. Transient downregulation of cPgA and cPgC genes during the hatching period may be related to the reconstruction of the stomach mucosal epithelium [19]. According to phylogenetic relationship among chicken pepsinogen genes (Fig. 2), cPgA is closer to ECPg than to cPgC, as mentioned above. However, Northern analysis of chicken pepsinogen genes showed that transcriptional regulation of cPgA gene was similar to that of cPgC gene rather than that of ECPg gene, suggesting that only regulatory sequence(s) which determine the tem-
FIG. 3. Northern analysis of chicken pepsinogen expression during development. Total RNAs of provenrticuli from embryonic day (E) 14 to post-hatching day (A) 7 were analyzed by Northern hybridization. 28S rRNA was detected with ethidium bromide and used as a loading control.
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FIG. 4. 59-Upstream sequences of cPgA (A) and cPgC (B). Transcription start sites determined by primer extension analysis are shown by arrowheads. TATA-like boxes are underlined. The presumptive GATA-binding site is double underlined.
poral expression pattern of ECPg gene changed considerably after the ECPg gene had diverged from cPgA gene. cis-regulatory elements of cPgA and cPgC genes. To determine the transcription start sites of cPgA and cPgC genes, we performed primer extension analysis. The transcription start site of cPgA was located to 20bp upstream of the 59 cDNA end, while in cPgC gene, there were two transcription start sites at 12 and 14bp upstream of the 59 cDNA end (Fig. 4). TATA-like boxes were found 28bp and 30bp upstream of the transcription start site of cPgA and cPgC, respectively. Promoter regions of mammalian pepsinogen genes share a hexamer sequence, “AATCTT” [16], but it does not exist in chicken pepsinogen genes. Moreover, three oligonucleotide sequences commonly found in promoter regions of human and rat pepsinogen C genes [16] do not exist in cPgC gene. Together, this demonstrates that the promoter regions of avian and mammalian pepsinogens are not conserved, suggesting that promoter regions have changed after avian and mammalian pepsinogens diverged and this may have caused different temporal expression patterns of vertebrate pepsinogens. Some putative transcription factor binding sites of GATA, Sox and HNF-3b were found in 59-flanking regions of cPgA and cPgC genes (Fig. 5). Chicken GATA-5 is strongly expressed in chicken proventricular epithelium (Sakamoto, unpublished data), suggesting that it may in effect regulate the expression of cPgA and/or cPgC genes. Chicken GATA-5 may act as general regulator of chicken pepsinogen, since its four putative binding site was also found within 1.1kbp upstream of transcription start site of ECPg gene (Fig. 5) which is known to be enough to regulate proper expression of ECPg in the proventriculus [20]. In the chicken, Sox-2, a member of the Sox transcription factor family, expressed in the proventricular epithelium, ceases to be expressed specifically in glandular epithelium after gland formation occurs [21]. This suggests
that Sox-2 may suppress formation of gland and/or expression of pepsinogen. HNF-3b is also a candidate for regulator of chicken pepsinogen genes since it is expressed in gut epithelium in mouse [22] and chicken (our unpublished data). So far, the introns of mammalian pepsinogen genes have not been analyzed thoroughly in relation to their importance in the regulation of expression. However, data showing that intronic enhancers act as important cis-regulatory elements in many genes have recently been reported [23-25]. In this study, we found seven binding motifs human MZF-1 (myeloid zinc finger-1) [26], which is expressed in human myeloid cells and is necessary to granulopoiesis [27, 28] (Fig. 6), in intron 8 of cPgA gene. However, we could not find putative binding motif for MZF-1 in cPgC gene. A MZF-1-like factor might regulate the expression of cPgA, although
FIG. 5. Genomic structures and cis-elements in 59-upstream sequences of the cPgA gene (A), the cPgC gene (B), and the ECPg gene (C). The exons are indicated by solid boxes. Putative transcription factor-binding sites in 59-upstream sequences are indicated as follows: GATA, open ellipse; Sox, striped ellipse; HNF-3b, solid ellipse. The restriction enzyme recognition sites are: B, BamHI; E, EcoRI and H, HindIII.
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FIG. 6. Sequence of intron 8 of the cPgA gene. Seven putative MZF-1 binding motifs, “(A/G)TGGGGA,” are underlined. Sequences of exons 8 and 9 are boxed.
we do not know whether a chicken homolog of MZF-1 is expressed in chicken gut epithelium. Further experiments in which genes encoding the putative transcription regulating factors mentioned above, GATA, Sox, HNF-3b and MZF-1, are introduced into pepsinogen-nonproducing or -producing epithelial cells, will clarify the mechanisms of spatial and temporal regulation of expression patterns of pepsinogen genes among vertebrates. Such experiments are currently being carried out in our laboratory. ACKNOWLEDGMENTS We thank Dr. Paul J. Scotting for his manuscript. This work was supported in Scientific Research Grant from The Japan and by Grants-in-Aid from the Ministry of Culture of Japan to S.Y.
critical reading of the part by the Sasakawa Science Society to N.S. Education, Science and
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13. Baudys, M., and Kostka, V. (1983) Eur. J. Biochem. 136, 89 –99. 14. von Heijne, G. (1984) J. Mol. Biol. 173, 243–251. 15. Hayano, T., Sogawa, K., Ichihara, Y., Fujii–Kuriyama, Y., and Takahashi, K. (1986) Biochem. Biophys. Res. Commun. 138, 289 –296. 16. Hayano, T., Sogawa, K., Ichihara, Y., Fijii–Kuriyama, Y., and Takahashi, K. (1988) J. Biol. Chem. 263, 1382–1385. 17. Ishihara, T., Ichihara, Y., Hayano, T., Katsura, I., Sogawa, K., Fujii–Kuriyama, Y., and Takahashi, K. (1989) J. Biol. Chem. 264, 10193–10199. 18. Hayashi, K., Yasugi, S., and Mizuno, T. (1988) Biochem. Biophys. Res. Commun. 152, 776 –782. 19. Thomson, D. S. (1969) Ohio J. Sci. 69, 74 – 84. 20. Fukuda, K., Ishii, Y., Saiga, H., Shiokawa, K., and Yasugi, S. (1994) Development 120, 3487–3495. 21. Ishii, Y., Rex, M., Scotting, P. J., and Yasugi, S. Dev. Dyn., in press. 22. Ang, S.–L., Wierda, A., Wong, D., Stevens, K. A., Cascio, S., Rossant, J., and Zaret, K. S. (1993) Development 119, 1301–1315. 23. Hennig, G., Lowrick, O., Birchmeier, W., and Behrens, J. (1996) J. Biol. Chem. 271, 595– 602. 24. Sabourin, J. C., Kern, A. S., Gregori, C., Porteu, A., Cywiner, C., Chatelet, F. P., Kahn, A., and Pichard, A. L. (1996) J. Biol. Chem. 271, 3469 –3473. 25. Schlaeger, T. M., Bartunkova, S., Lawitts, J. A., Teichmann, G., Risau, W., Deutsch, U., and Sato, T. N. (1997) Proc. Natl. Acad. Sci. USA 94, 3058 –3063. 26. Morris, J. F., Hromas, R., and Rauscher III, F. J. (1994) Mol. Cell. Biol. 14, 1786 –1795. 27. Bavisotto, L., Kaushansky, K., Lin, N., and Hromas, R. (1991) J. Exp. Med. 174, 1097–1101. 28. Hromas, R., Collins, S. J., Hickstein, D., Raskind, W., Deaven, L. L., O9Hara, P., Hagen, F. S., and Kaushansky, K. (1991) J. Biol. Chem. 266, 14183–14187. 29. Sogawa, K., Fujii–Kuriyama, Y., Mizukami, Y., Ichihara, Y., and Takahashi, K. (1983) J. Biol. Chem. 258, 5306 –5311. 30. Taggart, R. T., Cass, L. G., Mohandas, T. K., Derby, P., Barr, P. J., Pals, G., and Bell, G. I. (1989) J. Biol. Chem. 264, 375–379. 31. Evers, M. P. J., Zelle, B., Bebelman, J. P., Pronk, J. C., Mager, W. H., Planta, R. J., Eriksson, A. W., and Frants, R. R. (1988) Gene 65, 179 –185. 32. Lin, X.–L., Wong, R. N. S., and Tang, J. (1989) J. Biol. Chem. 264, 4482– 4489. 33. Tan, S. Y., Zhang, Y. Y., Liu, T. Y., Liu, N. J., and Yang, K. Y. (1989) Chin. J. Biotechnol. 5, 328 –332. 34. Yakabe, E., Tanji, M., Ichinose, M., Goto, S., Miki, K., Kurokawa, K., Ito, H., Kageyama, T., and Takahashi, K. (1991) J. Biol. Chem. 266, 22436 –22443.
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RC989290
Analysis of Temporal Expression Pattern and cisRegulatory Sequences of Chicken Pepsinogen A and C Nobuyuki Sakamoto,1 Hidetoshi Saiga, and Sadao Yasugi Department of Biology, Faculty of Science, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachioji, Tokyo 192-0397, Japan
Received August 5, 1998
Three groups of pepsinogens exist in vertebrates, namely, pepsinogen A, pepsinogen C, and prochymosin, which are produced at different developmental stages. In the chicken, prochymosin is expressed only in the embryonic stage, while pepsinogens A and C are secreted from adult chicken proventricular (glandular stomach) mucosa. In order to understand the mechanism of transcriptional regulation of these genes, we have cloned the genes encoding chicken pepsinogens A and C and analyzed the sequences possibly involved in their regulation. 5*-Upstream sequences of both genes contain putative binding motifs for transcription factors such as GATA, Sox, and HNF-3b, which are expressed in the chicken gut epithelium. Moreover, we found seven putative binding motifs for human MZF-1 in intron 8 of pepsinogen A gene. These transcription factors may act as regulators of expression of chicken pepsinogen genes. © 1998 Academic Press
Pepsinogens, zymogens of pepsins, are classified into three major groups: pepsinogens A, pepsinogens C (progastricsins) and prochymosins. These pepsinogens are secreted from gastric mucosa at different developmental stages in the vertebrates. In mammals, prochymosin is a major component of pepsinogen in the neonatal stage, and pepsinogens A and C constitute major components of adult pepsinogen [1, 2]. Such stagedependent production of pepsinogens has been attributed to their enzymatic properties, such as a high milk clotting activity of prochymosin [3] and substrate specificities, in relation to the physiological demand of the animals. In the chicken, prochymosin is produced only in embryonic stages and has been named ECPg (embryonic chicken pepsinogen) [4]. Chicken pepsinogen A (cPgA) is a major component of pepsinogens secreted from adult proventriculus (glandular stomach) [5]. Chicken pepsinogen C (cPgC) is a minor component of 1 Corresponding author. Fax: 181 (426) 77-2559. E-mail: [email protected].
0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
adult pepsinogens [5], and its protein being detected only in hatched chicken proventriculus [6]. To understand the molecular nature of mechanisms for differential usage of pepsinogens during development, it is necessary first to study how the transcription of pepsinogen genes is regulated. To date, molecular cloning of pepsinogen cDNAs and genes has been performed in various vertebrate species, especially in mammals, and studies on these genes have revealed that methylation of genes plays an important role in the regulation of their expression [7-9]. It has also been shown that glucocorticoid hormone has an upregulatory effect on pepsinogen gene expression [10]. However, the molecular basis of temporal regulation of pepsinogen genes is largely unknown. To elucidate the mechanism for switching of pepsinogens during development, we have cloned cPgA and cPgC genes expressed in the adult proventricular mucosa. Unexpectedly, cPgA and cPgC genes are also expressed at late embryonic stages. Analysis of 59-flanking region of cPgA and cPgC genes demonstrated the existence of putative binding sites for transcription factors, GATA, Sox and HNF-3b. We also found seven putative binding sites for transcription factor MZF-1 in intron 8 of the cPgA gene. Our results revealed that the switching of chicken pepsinogens occurs during the late embryonic period unlike mammalian pepsinogens and suggest that transcription factors such as GATA, Sox, HNF-3b and MZF-1 may regulate expression of chicken pepsinogen genes during development. MATERIALS AND METHODS Cloning and sequencing of cDNA and genes of cPgA and cPgC. A cDNA library was constructed in the lgt11 vector from post-hatching 14-day chicken proventriculus. The library was screened for cPgA using 32P-labeled oligonucleotides [59-RGGMACCCASARGTTRGAGGAKCCVGTGTC-39], a highly conserved sequence among vertebrate pepsinogen genes. The cDNAs were subcloned into EcoRI site of pBluescript SK1 (Stratagene) and sequenced. The cPgA cDNA clone obtained was labeled with a-[32P]dCTP using a random primer DNA labeling kit (Takara) and used to screen for cPgC cDNA. A chicken liver genomic library constructed in the lfixII vector was
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FIG. 1. Amino acids sequence alignments of vertebrate pepsinogens. Amino acids sequences of cPgA and cPgC, deduced from cDNA sequences obtained in the present study are aligned and gapped by the computer program CLUSTAL W (ver. 1.5) with other known vertebrate pepsinogen proteins. All insertion sites of introns indicated by arrowheads are coincident. Asterisks show identical residues. Thr of residue 88 of cPgA, which differs from a previously reported sequence 13, is bold. The amino acid sequence data used are ECPg [4], human PgA [29], human PgC [30], and rat PgC [17].
screened with the cPgA cDNA or cPgC cDNA clones labeled with a[32P]dCTP. The genomic clones isolated were subcloned into pBluescript SK1 (Stratagene) and sequenced with an automatic DNA sequencer (Applied Biosystems Inc.). Sequence comparison and phylogenetic analysis. Amino acid sequences of vertebrate pepsinogens were aligned and phylogenetic analysis was performed by neighbor-joining method [11] using computer program CLUSTAL W (ver. 1.5) [12]. For bootstrap analysis, the bootstrap program of CLUSTAL W was used to generate 1000 data sets. Northern hybridization. Total RNA samples were denatured and subjected to electrophoresis in a 1% agarose gel and blotted on a
nylon membrane (BIO-RAD) in 10x SSC (1x SSC contains 0.15 M NaCl, 0.015 M sodium citrate). The membranes were exposed to 312nm UV light for 5 min. To avoid cross-hybridization, specific probes were made from DNA fragments which containing 39 non-coding region of cPgA and cPgC cDNAs. To detect ECPg mRNA, we used ECP200 or ECP1.1K as a probe [4]. To prepare the radioactive probe, a random primer DNA labeling kit (Takara) was used. The blots were hybridized to radioactive probe in 5x SSPE (1x SSPE contains 0.18 M NaCl, 1 mM EDTA, 10 mM sodium phosphate), 50% formamide, 5x Denhardt9s solution, 0.5% SDS and 20 mg/ml denatured salmon sperm DNA, at 43°C for overnight, and washed twice in 0.1x SSC, 0.1% SDS at 60°C for 30 min. The hybrids were detected by autoradiography or a bioimaging analyzer BAS2000 (Fuji Photo Film).
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FIG. 2. Phylogenetic relationship of vertebrate pepsinogens. A molecular phylogenetic tree was constructed by the neighbor-joining method [11] using the computer program CLUSTAL W [12]. cPgA and cPgC are boxed. The scale bar indicates an evolutionary distance of 0.05 amino acid substitutions per position. The amino acid sequence data used are human PgA [29], monkey PgA [31], pig PgA [32], ECPg [4], bovine prochymosin [33], human PgC [30], rat PgC [17], and frog PgC [34].
Primer extension analysis. Synthetic oligonucleotide primers were labeled with [32P]ATP and T4 polynucleotide kinase (Takara). 1mg of total RNA of post-hatching 7-day chicken proventriculus and labeled primer were mixed in annealing buffer (250 mM KCl, 2 mM Tris-HCl(pH8.0), 0.2 mM EDTA) and heated at 60°C for 90 min. After precipitation with ethanol, cDNA was synthesized with SuperScriptII (Gibco BRL) at 50°C for 50 min. The cDNA products were analyzed by electrophoresis in a 6% polyacrylamide/7 M urea gel.
RESULTS AND DISCUSSION Cloning of cDNAs of chicken pepsinogens A and C. The cPgA cDNA cloned was of 1277bp and contained an open reading frame composed of 382 amino acids, the sequence of which was completely identical to the previously reported sequence of chicken pepsinogen A [13], except for a Thr in place of Ser at residue 88, probably due to polymorphism (Fig. 1). The cPgC cDNA was 1348bp in length and the deduced amino acid sequence consisted of 389 residues (Fig. 1). To investigate the phylogenetic relationship of these pepsinogens to other vertebrate pepsinogens, we constructed a molecular phylogenetic tree of vertebrate pepsinogens (Fig. 2). The tree revealed that the cPgA gene is included in the pepsinogen A group and the cPgC gene belongs to the pepsinogen C group, while the ECPg gene is related to the bovine prochymosin gene, as reported previously [4]. ECPg is more closely related to cPgA than to cPgC. Putative signal peptide cleavage sites of cPgA and cPgC proteins as conjectured with the (-3, -1) rule [14] are between residue 15 and 16, and residue 16 and 17, respectively. The estimated molecular weights of secreted proteins of cPgA and cPgC are 40.4kD and 40.7kD respectively. Genomic Southern analysis revealed that cPgA and cPgC genes exist as single copies in the chicken genome (data not shown). Genomic structures of chicken pepsinogen A and C genes. To investigate the genomic structure of chicken pepsinogen genes, a chicken genomic library
was made and screened with cPgA or cPgC cDNA as a probe, and obtained clones were subcloned and sequenced. Both genes consist of nine exons just as mammalian pepsinogens [15-17] and ECPg [18] genes (Fig. 1 and 5). Furthermore, it was revealed that the positions of introns are highly conserved among vertebrate pepsinogen genes (Fig. 1). We found some polymorphism of amino acid residues in exon 4 of cPgA and in exon 1 of cPgC. In the latter case, the residue 8 is Val when deduced from cDNA whereas it is Met when deduced from genomic sequence. Temporal expression patterns of cPgA and cPgC during development. In mammals and birds, it is known that members of the different pepsinogen groups are expressed at different developmental stages [1, 2]. To investigate the temporal expression patterns of cPgA and cPgC genes, total RNA of proventriculus of various stages from embryonic day 12 (E12) to post-hatching day 7 (A7) was subjected to Northern analysis. Transcripts of cPgA and cPgC were seen in both embryonic and adult proventriculus. cPgA mRNA was detected from E16 to E19, and disappeared temporarily just before hatching. Within one day after hatching, transcription of the cPgA gene started again and then increased rapidly (Fig. 3). Expression pattern of the cPgC gene was similar to but weaker than that of cPgA. It is noteworthy that the transcripts of cPgA and cPgC were detected after transcription of ECPg gene was downregulated. This suggest that there are mechanisms which switch major components of pepsinogens from ECPg to cPgA and cPgC during the late embryonic period although the functions of embryonic cPgA and cPgC proteins are unknown at present. Transient downregulation of cPgA and cPgC genes during the hatching period may be related to the reconstruction of the stomach mucosal epithelium [19]. According to phylogenetic relationship among chicken pepsinogen genes (Fig. 2), cPgA is closer to ECPg than to cPgC, as mentioned above. However, Northern analysis of chicken pepsinogen genes showed that transcriptional regulation of cPgA gene was similar to that of cPgC gene rather than that of ECPg gene, suggesting that only regulatory sequence(s) which determine the tem-
FIG. 3. Northern analysis of chicken pepsinogen expression during development. Total RNAs of provenrticuli from embryonic day (E) 14 to post-hatching day (A) 7 were analyzed by Northern hybridization. 28S rRNA was detected with ethidium bromide and used as a loading control.
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FIG. 4. 59-Upstream sequences of cPgA (A) and cPgC (B). Transcription start sites determined by primer extension analysis are shown by arrowheads. TATA-like boxes are underlined. The presumptive GATA-binding site is double underlined.
poral expression pattern of ECPg gene changed considerably after the ECPg gene had diverged from cPgA gene. cis-regulatory elements of cPgA and cPgC genes. To determine the transcription start sites of cPgA and cPgC genes, we performed primer extension analysis. The transcription start site of cPgA was located to 20bp upstream of the 59 cDNA end, while in cPgC gene, there were two transcription start sites at 12 and 14bp upstream of the 59 cDNA end (Fig. 4). TATA-like boxes were found 28bp and 30bp upstream of the transcription start site of cPgA and cPgC, respectively. Promoter regions of mammalian pepsinogen genes share a hexamer sequence, “AATCTT” [16], but it does not exist in chicken pepsinogen genes. Moreover, three oligonucleotide sequences commonly found in promoter regions of human and rat pepsinogen C genes [16] do not exist in cPgC gene. Together, this demonstrates that the promoter regions of avian and mammalian pepsinogens are not conserved, suggesting that promoter regions have changed after avian and mammalian pepsinogens diverged and this may have caused different temporal expression patterns of vertebrate pepsinogens. Some putative transcription factor binding sites of GATA, Sox and HNF-3b were found in 59-flanking regions of cPgA and cPgC genes (Fig. 5). Chicken GATA-5 is strongly expressed in chicken proventricular epithelium (Sakamoto, unpublished data), suggesting that it may in effect regulate the expression of cPgA and/or cPgC genes. Chicken GATA-5 may act as general regulator of chicken pepsinogen, since its four putative binding site was also found within 1.1kbp upstream of transcription start site of ECPg gene (Fig. 5) which is known to be enough to regulate proper expression of ECPg in the proventriculus [20]. In the chicken, Sox-2, a member of the Sox transcription factor family, expressed in the proventricular epithelium, ceases to be expressed specifically in glandular epithelium after gland formation occurs [21]. This suggests
that Sox-2 may suppress formation of gland and/or expression of pepsinogen. HNF-3b is also a candidate for regulator of chicken pepsinogen genes since it is expressed in gut epithelium in mouse [22] and chicken (our unpublished data). So far, the introns of mammalian pepsinogen genes have not been analyzed thoroughly in relation to their importance in the regulation of expression. However, data showing that intronic enhancers act as important cis-regulatory elements in many genes have recently been reported [23-25]. In this study, we found seven binding motifs human MZF-1 (myeloid zinc finger-1) [26], which is expressed in human myeloid cells and is necessary to granulopoiesis [27, 28] (Fig. 6), in intron 8 of cPgA gene. However, we could not find putative binding motif for MZF-1 in cPgC gene. A MZF-1-like factor might regulate the expression of cPgA, although
FIG. 5. Genomic structures and cis-elements in 59-upstream sequences of the cPgA gene (A), the cPgC gene (B), and the ECPg gene (C). The exons are indicated by solid boxes. Putative transcription factor-binding sites in 59-upstream sequences are indicated as follows: GATA, open ellipse; Sox, striped ellipse; HNF-3b, solid ellipse. The restriction enzyme recognition sites are: B, BamHI; E, EcoRI and H, HindIII.
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FIG. 6. Sequence of intron 8 of the cPgA gene. Seven putative MZF-1 binding motifs, “(A/G)TGGGGA,” are underlined. Sequences of exons 8 and 9 are boxed.
we do not know whether a chicken homolog of MZF-1 is expressed in chicken gut epithelium. Further experiments in which genes encoding the putative transcription regulating factors mentioned above, GATA, Sox, HNF-3b and MZF-1, are introduced into pepsinogen-nonproducing or -producing epithelial cells, will clarify the mechanisms of spatial and temporal regulation of expression patterns of pepsinogen genes among vertebrates. Such experiments are currently being carried out in our laboratory. ACKNOWLEDGMENTS We thank Dr. Paul J. Scotting for his manuscript. This work was supported in Scientific Research Grant from The Japan and by Grants-in-Aid from the Ministry of Culture of Japan to S.Y.
critical reading of the part by the Sasakawa Science Society to N.S. Education, Science and
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