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Cloning and Characterization of the 5′-Flanking Region of the Human Cardiotrophin-1 Gene
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
244, 494–497 (1998)
RC988311
Cloning and Characterization of the 5*-Flanking Region of the Human Cardiotrophin-1 Gene1 Jeanette Erdmann, Sabine Hassfeld, Heike Kallisch, Eckart Fleck, and Vera Regitz-Zagrosek Department of Internal Medicine/ Cardiology, Virchow Klinikum of the Humboldt University and Deutsches Herzzentrum Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany
Received February 10, 1998
To enable the analysis of the regulation of the human cardiotrophin-1 gene expression, the 5*-flanking region of the human cardiotrophin-1 gene was cloned and sequenced. Data bank search revealed several cisactive DNA elements (SP1, CREB, C/EBP, AP1 and AP2 like and GATA) in the proximal 1.1 kb region. Six nested 5-*terminal deletion mutants from 01091//39 to 0218//39 were fused to a luciferase reportergene and proved to be functionally active after transfection into COS-7 cells. q 1998 Academic Press Key Words: cardiotrophin-1 (CT-1); promoter; reportergene assay; COS-7 cells.
Myocyte hypertrophy represents an important adaptive response of the heart to injury or to an increased workload (1). The hypertrophic response is characterized by the reactivation of genes normally expressed during fetal heart development and by accumulation of sacromeric proteins in the absence of DNA replication or cell division (2). Reasoning that fetal or embryonic growth factors may mediate the onset of cardiac hypertrophy, Pennica et al. (3) isolated a 21.5-kD protein, cardiotrophin-1 (CT-1), that potently induces cardiac myocyte hypertrophy in vitro. Besides the induction of hypertrophy, CT-1 can induce ANP in cardiac myocytes and is responsible for the organization of MLC2 into sacromeric units (3). CT-1 belongs to the IL-6 family of cytokines, which also includes leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), IL-6 and IL-11 and has a wide range of growth and differentiation activities on many cell types including those from blood, liver and nervous system (4). CT-1 is active in many of these systems as well (5). The members of the IL-6 related cytokines are distantly related with regard to their primary amino acid sequence (14-24% amino acid iden1 The sequence described in this paper was deposited in EMBL data bank as Accession No. AJ002743.
0006-291X/98 $25.00
tity) (6) and are predicted to share a common four ahelix bundle topology (7). Receptor binding studies and functional studies reveal that CT-1 shares the signal transducing receptor components gp130 and LIFR with the previously identified members of the IL-6 cytokine family. Several members of this family stimulate cardiac myocyte growth, suggesting that the gp130 signalling pathway may play a role in cardiac hypertrophy. Cloning of the human CT-1 gene revealed that the DNA sequence encodes a protein of 201 amino acids that is 79% identical with the 203 residue mouse (3) and rat (8) CT-1. The human CT-1 gene is located on chromosome 16p11.1-16p11.2 (9) and is not linked to other members of the IL-6 cytokine family. The human 1.7 kb mRNA encoding CT-1 was found in high levels in RNA from heart, skeletal muscle, prostate and ovary and lower levels were observed in lung, kidney, pancreas, thymus, testis, and small intestine. Little or no expression was detected in the brain, placenta, liver, spleen, colon or in peripheral blood leukocytes (9). In order to examine the regulation of cardiotrophin1 gene expression, we cloned and sequenced the human CT-1 gene promoter and determined the functional activity of six nested 5-*terminal deletion mutants of the promoter in luciferase reportergene assays using COS7 cells. MATERIALS AND METHODS Materials. Cell culture media, horse serum, fetal calf serum, Lglutamine and gentamycin were purchased from Gibco BRL Life Technologies (Eggenstein, Germany). The Luciferase Assay System and the b-Galactosidase Enzyme Assay System was obtained from Promega (Mannheim, Germany). Restriction enzymes were from MBI Fermentas (St. Leon-Rot, Germany) or Biolabs (Schwalbach, Germany). Oligonucleotide primers were synthesized by Invitek (Berlin, Germany). Cloning of the 5*-flanking region of the human CT-1 gene. Using the human GenomeWalker kit (Clontech, Heidelberg, Germany) we cloned 1,1 kb of the 5*-flanking region of the human CT-1 gene. The GenomeWalker Kit contains five premade ‘‘libraries’’ of adaptor-ligated, species-specific genomic DNA fragments. The libraries are used as templates in nested PCR reactions with gene-specific primers (GSPs) and
494
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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BBRC 8311
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694c$$$921
02-26-98 09:14:17
bbrcg
AP: BBRC
Vol. 244, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the adaptor primers provided in each kit. The first set of PCRs used a sense oligonucleotide specific to the adaptor and an anti-sense oligonucleotide (5-*CGCAGGAGTCAGAGGGATT-3*) specific to the 3*-end of exon 1 of the human CT-1 gene as primers and the five libraries as template. These primary PCR reactions were used as templates for the second PCRs that were performed using nested PCR primers specific to the adaptor (sense) and specific to the 5-*end of exon 1 (anti-sense, 5*CGCTGAGGGGCGCAGGAGTCA-3*). PCR was carried out using the AdvantageTM Genomic PCR Kit (Clontech, Heidelberg, Germany) which includes a proofreading DNA polymerase. After the second round of PCR we obtained one specific 1.1 kb PCR-product, which was subcloned in a T/A cloning vector (Invitrogen, NV Leek, Netherlands) and sequenced in both directions using ABI DyeTerminators (Applied Biosystems, Weiterstadt, Germany). Construction of 5* deletion constructs. The sequence data were used to design primers for amplifying 5*-terminal deletion fragments of the human CT-1 promoter (primer sequence and position see Figure 1). We amplified 6 fragments with sizes from 1173 bp to 300 bp (CT1 to CT6). After cloning the PCR products into T/A cloning vector (Invitrogen, NV Leek, Netherlands), we determined the orientation of the inserts by sequencing. The inserts with 5*r3* orientation were recut with KpnI and XhoI and cloned into pGL3-Basic vector (Promega, Mannheim, Germany) using the KpnI/XhoI site (see Figure 2). The pGL3-Basic vector lacks eukaryotic promoter and enhancer sequences. Analysis of promoter constructs in cell culture. For transfection experiments, african green monkey SV40 transformed kidney cell line COS-7 (American Type Culture Collection Number: CRL-1651) was used (RPMI 1640, 10% horse serum, 5% fetal bovine serum, 2 mmol/L L-glutamine, 50 mg/mL gentamycin). Five micrograms of the hCT-1 promoter luciferase reportergene construct, 2 mg of a b-galactosidase control vector (pCMV b-gal) (Stratagene, Heidelberg, Germany), and 23 mg of pBSSK/ plasmid DNA as carrier were transfected into 3 1 106 cos7 cells in 300 mL culture medium by electroporation (cuvette widht 4 mm; 250 V, 1200 mF, Gene-Pulsery, Biorad). The plasmid pBLuc expressing the luciferase gene controlled by the SV40 early promoter was used as positive control. At 48 h posttransfection, cells were lysed in 100 mL of Reporter Lysis Buffer (Luciferase Assay System, Promega, Mannheim, Germany). The luciferase assay was performed on 50 mL of the cleared lysate and 50 mL of luciferase assay reagent. To standardize for transfection efficiency 20 mL of the cell lysate was submitted to a b-galactosidase assay according to the supplier’s instructions (b Galactosidase Enzyme Assay System, Promega, Mannheim, Germany). The luciferase activity of each transfection was normalized to the respective bgalactosidase activity. Statistics. Data are given as means { SEM. Statistical analysis was performed using the unpaired two-tailed Student‘s t-test. Values of põ0.05 were considered significant.
RESULTS AND DISCUSSION Cloning and Sequencing the 5*-Flanking Region of the Human CT-1 Gene We have cloned and sequenced about 1.1 kb of the 5*-flanking region of exon 1 of the human CT-1 gene. To avoid sequencing errors we used a polymerase mix including a proofreading DNA polymerase and we sequenced each clone in both directions. Data bank search using the transcription factor data base TRANSFAC (10) revealed several potential cis-acting elements (Figure 1), but the human CT-1 promoter does not contain any TATA-like element. This feature has been described for genes such as housekeeping
genes and is usually associated with multiple transcription start sites. Potential binding sites for several ubiquitious transcription factors are present. There are two CAATboxes at position 01043 and 01076. The CAAT-box element seems to be related to the level of basal expression and functions in either orientation (11). Interestingly, C/EBPbeta can bind at position -1076. C/EBP is a widespread transcriptional activator in vitro and in vivo, and can stimulate IL-6 expression (12). At position -984 we identified a putative STAT-site. The STATs (signal transducer and activators of transcription) are latent cytoplasmic proteins that, upon activation by cell surface bound polypeptide ligands, move to the nucleus to direct transcription (13). In addition, there is one Sp1-site (at position 0582). The transcription factor Sp1 is a DNA-binding protein which interacts with a variety of gene promoters containing GC-box elements. Sp1 is generally considered a proximal promoter factor that can only stimulate transcription when bound close to the initiation site. However, Courey et al. (14) presented evidence that distally and proximally bound Sp1 can stimulate transcription synergistically. Overall nine AP-2 like binding sites (at positions 0716, 0438, 0266, 0251, 0248, 0207, 0181, 073, 072) were identified in the promoter of the human CT-1 gene. A cAMP response element (CRE) is located at position 0639. The cAMP response element (CRE) mediates diverse transcriptionally regulatory effects. It was first identified as an inducible enhancer of genes that can be transcribed in response to increased cAMP levels. Some growth control genes such as FOS have CRE in their transcriptional regulatory region and their expression is induced by an increase in the intracellular cAMP levels. Additionally, the hCT-1 promoter region harbors a binding site for GATA binding factor at position 0484. The GATA-binding proteins are a group of structurally related transcription factors that control gene expression and differentiation in a variety of cell types. Members of this family of DNA-binding proteins recognize a consensus sequence known as the ’GATA’ motif, which is an important cis-element in the promoters of many genes. All GATA-binding proteins contain 1 or 2 zinc-finger motifs of the distinctive form CXNCX (17)CNXC (15). Promoter and enhancer studies suggested that this factor may regulate genes critical for myocardial differentiation and function, including troponin C, cardiac alpha-myosin heavy chain, and braintype natriuretic factor. Promoter Activity in the 5*-Flanking Region of the Human CT-1 Gene To test for the basal promoter activity of the 5*flanking region of the human CT-1 gene six 5* terminal nested deletion mutants ranging from 01091//39 to
495
AID
BBRC 8311
/
694c$$$921
02-26-98 09:14:17
bbrcg
AP: BBRC
Vol. 244, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Sequence analysis of the 5* flanking region of the hCT-1 gene. The sequence is numbered from the ATG-site in exon 1. PCRprimer for amplifying the 5* nested terminal deletion fragments are underlined. Consensus transcription factor recognition sites identified using the TRANSFAC data bank are labelled. The sequence has been submitted to the EMBL data bank with the accession number AJ002743.
0218//39 were cloned into the promoterless luciferase reportergene vector pGL3 basic. These fragments were obtained by PCR with primers indicated in Figure 1 and contained the first 39 nucleotides of exon 1. In the present study, we document that these six 5*terminal nested deletion fragments of the hCT-1 5*flanking region exhibit significant promoter activity after transfection in COS-7 cells (Figure 2). These constructs were introduced by electroporation into COS-7 cells. A b-galactosidase control vector (pCMV b-gal) was cotransfected in each experiment and luciferase activities were normalized in reference to the respective b-galactosidase activity. All six hCT-1 promoter constructs proved to be functionally active in COS-7
cells as compared to the promoterless construct pGL3basic (nÅ3, põ 0,05). The results are shown in Figure 2. Data are expressed as relative activity with respect to the mean activity of construct pGL3basic which has given a value of 1. Activity values represent the mean { S.E.M. of three independent transfections. Luciferase-reportergene assays revealed the highest promoter activity in fragments CT3 and CT6 and the lowest activity in fragment CT5 (põ0.05), indicating the presence of an enhancer element in the proximity of exon 1 and between position 0557 and 0947 and an putative repressor element between position 0218 and 0377. The enhanced activity of fragment CT3 is maybe due to the SP1 site identified at position 0582.
496
AID
BBRC 8311
/
694c$$$921
02-26-98 09:14:17
bbrcg
AP: BBRC
Vol. 244, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Basal promoter activity of six 5*-terminal nested deletion fragments. COS-7 cells were transiently transfected with constructs containing fragments of the 5*flanking region of the human CT-1 gene (hatched boxes) fused into the upstream region of the Luciferase gene (open boxes). Average relative Luciferase activity (gray bars) was determined by at least three transfections and were normalized in reference to the respective b-galactosidase activity. Data are expressed as relative activity of pGL3basic which was set at 1.
The sequence information obtained in this study, allows for further investigations, including the search for polymorphisms in the promoter region, which may associate with dilated cardiomyopathy or other human phenotypes. Detailed investigations of CT-1 regulation by transcriptional mechanism may contribute to understand the role of CT-1 in cardiac hypertrophy. In conclusion, this first characterization of the human cardiotrophin-1 promoter contributes to elucidate molecular mechanisms in human myocardial diseases. ACKNOWLEDGMENTS This study was supported in part by a grant of the Verbund Klinische Pharmakologie Berlin/Brandenburg.
REFERENCES 1. Chien, K. R. (1993) Science 260, 916–917. 2. Chien, K. R., Knowlton, K. U., Zhu, H., and Chien, S. (1991) FASEB J 5, 3037–3046. 3. Pennica, D., King, K. L., Shaw, K. J., Luis, E., Rullamas, J., Luoh, S. M., Darbonne, W. C., Knutzon, D. S., Yen, R., Chien, K. R., Baker, J. B., and Wood, W. I. (1995) Proc Natl Acad Sci U S A 92, 1142–1146.
4. Akira, S., Taga, T., and Kishimoto, T. (1993) Adv Immunol 54, 1–78 5. Pennica, D., Shaw, K. J., Swanson, T. A., Moore, M. W., Shelton, D. L., Zioncheck, K. A., Rosenthal, A., Taga, T., Paoni, N. F., and Wood, W. I. (1995) J Biol Chem 270, 10915–10922 6. Rose, T. M., and Bruce, A. G. (1991) Proc Natl Acad Sci U S A 88, 8641–8645 7. Robinson, R. C., Grey, L. M., Staunton, D., Vankelecom, H., Vernallis, A. B., Moreau, J. F., Stuart, D. I., Heath, J. K., and Jones, E. Y. (1994) Cell 77, 1101–1116 8. Ishikawa, M., Saito, Y., Miyamoto, Y., Kuwahara, K., Ogawa, E., Nakagawa, O., Harada, M., Masuda, I., and Nakao, K. (1996) Biochem Biophys Res Commun 219, 377–381. 9. Pennica, D., Swanson, T. A., Shaw, K. J., Kuang, W. J., Gray, C. L., Beatty, B. G., and Wood, W. I. (1996) Cytokine 8, 183–189. 10. Wingender, E., Kel, A. E., Kel, O. V., Karas, H., Heinemeyer, T., Dietze, P., Knueppel, R., Romaschenko G., and Kolchanov N. A. (1997) Nucleic Acids Res 25, 265–268 11. Morgan, W. D., Williams, G. T., Morimoto, R. I., Greene, J., Kingston, R. E., and Tjian, R. (1987) Mol Cell Biol 7, 1129–1138. 12. Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., and Kishimoto, T. (1990) EMBO J 6, 1897–1906. 13. Horvath, C. M., and Darnell, J. E. (1997) Curr Opin Cell Biol 2, 233–239. 14. Courey, A. J., Holtzman, D. A., Jackson, S. P., and Tijian, R. (1989) Cell 5, 827–836 15. Evans, T. Reitman, M., and Felsenfeld, G. (1988) Proc Natl Acad Sci U S A 85, 5976–5980.
497
AID
BBRC 8311
/
694c$$$921
02-26-98 09:14:17
bbrcg
AP: BBRC
244, 494–497 (1998)
RC988311
Cloning and Characterization of the 5*-Flanking Region of the Human Cardiotrophin-1 Gene1 Jeanette Erdmann, Sabine Hassfeld, Heike Kallisch, Eckart Fleck, and Vera Regitz-Zagrosek Department of Internal Medicine/ Cardiology, Virchow Klinikum of the Humboldt University and Deutsches Herzzentrum Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany
Received February 10, 1998
To enable the analysis of the regulation of the human cardiotrophin-1 gene expression, the 5*-flanking region of the human cardiotrophin-1 gene was cloned and sequenced. Data bank search revealed several cisactive DNA elements (SP1, CREB, C/EBP, AP1 and AP2 like and GATA) in the proximal 1.1 kb region. Six nested 5-*terminal deletion mutants from 01091//39 to 0218//39 were fused to a luciferase reportergene and proved to be functionally active after transfection into COS-7 cells. q 1998 Academic Press Key Words: cardiotrophin-1 (CT-1); promoter; reportergene assay; COS-7 cells.
Myocyte hypertrophy represents an important adaptive response of the heart to injury or to an increased workload (1). The hypertrophic response is characterized by the reactivation of genes normally expressed during fetal heart development and by accumulation of sacromeric proteins in the absence of DNA replication or cell division (2). Reasoning that fetal or embryonic growth factors may mediate the onset of cardiac hypertrophy, Pennica et al. (3) isolated a 21.5-kD protein, cardiotrophin-1 (CT-1), that potently induces cardiac myocyte hypertrophy in vitro. Besides the induction of hypertrophy, CT-1 can induce ANP in cardiac myocytes and is responsible for the organization of MLC2 into sacromeric units (3). CT-1 belongs to the IL-6 family of cytokines, which also includes leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), IL-6 and IL-11 and has a wide range of growth and differentiation activities on many cell types including those from blood, liver and nervous system (4). CT-1 is active in many of these systems as well (5). The members of the IL-6 related cytokines are distantly related with regard to their primary amino acid sequence (14-24% amino acid iden1 The sequence described in this paper was deposited in EMBL data bank as Accession No. AJ002743.
0006-291X/98 $25.00
tity) (6) and are predicted to share a common four ahelix bundle topology (7). Receptor binding studies and functional studies reveal that CT-1 shares the signal transducing receptor components gp130 and LIFR with the previously identified members of the IL-6 cytokine family. Several members of this family stimulate cardiac myocyte growth, suggesting that the gp130 signalling pathway may play a role in cardiac hypertrophy. Cloning of the human CT-1 gene revealed that the DNA sequence encodes a protein of 201 amino acids that is 79% identical with the 203 residue mouse (3) and rat (8) CT-1. The human CT-1 gene is located on chromosome 16p11.1-16p11.2 (9) and is not linked to other members of the IL-6 cytokine family. The human 1.7 kb mRNA encoding CT-1 was found in high levels in RNA from heart, skeletal muscle, prostate and ovary and lower levels were observed in lung, kidney, pancreas, thymus, testis, and small intestine. Little or no expression was detected in the brain, placenta, liver, spleen, colon or in peripheral blood leukocytes (9). In order to examine the regulation of cardiotrophin1 gene expression, we cloned and sequenced the human CT-1 gene promoter and determined the functional activity of six nested 5-*terminal deletion mutants of the promoter in luciferase reportergene assays using COS7 cells. MATERIALS AND METHODS Materials. Cell culture media, horse serum, fetal calf serum, Lglutamine and gentamycin were purchased from Gibco BRL Life Technologies (Eggenstein, Germany). The Luciferase Assay System and the b-Galactosidase Enzyme Assay System was obtained from Promega (Mannheim, Germany). Restriction enzymes were from MBI Fermentas (St. Leon-Rot, Germany) or Biolabs (Schwalbach, Germany). Oligonucleotide primers were synthesized by Invitek (Berlin, Germany). Cloning of the 5*-flanking region of the human CT-1 gene. Using the human GenomeWalker kit (Clontech, Heidelberg, Germany) we cloned 1,1 kb of the 5*-flanking region of the human CT-1 gene. The GenomeWalker Kit contains five premade ‘‘libraries’’ of adaptor-ligated, species-specific genomic DNA fragments. The libraries are used as templates in nested PCR reactions with gene-specific primers (GSPs) and
494
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8311
/
694c$$$921
02-26-98 09:14:17
bbrcg
AP: BBRC
Vol. 244, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the adaptor primers provided in each kit. The first set of PCRs used a sense oligonucleotide specific to the adaptor and an anti-sense oligonucleotide (5-*CGCAGGAGTCAGAGGGATT-3*) specific to the 3*-end of exon 1 of the human CT-1 gene as primers and the five libraries as template. These primary PCR reactions were used as templates for the second PCRs that were performed using nested PCR primers specific to the adaptor (sense) and specific to the 5-*end of exon 1 (anti-sense, 5*CGCTGAGGGGCGCAGGAGTCA-3*). PCR was carried out using the AdvantageTM Genomic PCR Kit (Clontech, Heidelberg, Germany) which includes a proofreading DNA polymerase. After the second round of PCR we obtained one specific 1.1 kb PCR-product, which was subcloned in a T/A cloning vector (Invitrogen, NV Leek, Netherlands) and sequenced in both directions using ABI DyeTerminators (Applied Biosystems, Weiterstadt, Germany). Construction of 5* deletion constructs. The sequence data were used to design primers for amplifying 5*-terminal deletion fragments of the human CT-1 promoter (primer sequence and position see Figure 1). We amplified 6 fragments with sizes from 1173 bp to 300 bp (CT1 to CT6). After cloning the PCR products into T/A cloning vector (Invitrogen, NV Leek, Netherlands), we determined the orientation of the inserts by sequencing. The inserts with 5*r3* orientation were recut with KpnI and XhoI and cloned into pGL3-Basic vector (Promega, Mannheim, Germany) using the KpnI/XhoI site (see Figure 2). The pGL3-Basic vector lacks eukaryotic promoter and enhancer sequences. Analysis of promoter constructs in cell culture. For transfection experiments, african green monkey SV40 transformed kidney cell line COS-7 (American Type Culture Collection Number: CRL-1651) was used (RPMI 1640, 10% horse serum, 5% fetal bovine serum, 2 mmol/L L-glutamine, 50 mg/mL gentamycin). Five micrograms of the hCT-1 promoter luciferase reportergene construct, 2 mg of a b-galactosidase control vector (pCMV b-gal) (Stratagene, Heidelberg, Germany), and 23 mg of pBSSK/ plasmid DNA as carrier were transfected into 3 1 106 cos7 cells in 300 mL culture medium by electroporation (cuvette widht 4 mm; 250 V, 1200 mF, Gene-Pulsery, Biorad). The plasmid pBLuc expressing the luciferase gene controlled by the SV40 early promoter was used as positive control. At 48 h posttransfection, cells were lysed in 100 mL of Reporter Lysis Buffer (Luciferase Assay System, Promega, Mannheim, Germany). The luciferase assay was performed on 50 mL of the cleared lysate and 50 mL of luciferase assay reagent. To standardize for transfection efficiency 20 mL of the cell lysate was submitted to a b-galactosidase assay according to the supplier’s instructions (b Galactosidase Enzyme Assay System, Promega, Mannheim, Germany). The luciferase activity of each transfection was normalized to the respective bgalactosidase activity. Statistics. Data are given as means { SEM. Statistical analysis was performed using the unpaired two-tailed Student‘s t-test. Values of põ0.05 were considered significant.
RESULTS AND DISCUSSION Cloning and Sequencing the 5*-Flanking Region of the Human CT-1 Gene We have cloned and sequenced about 1.1 kb of the 5*-flanking region of exon 1 of the human CT-1 gene. To avoid sequencing errors we used a polymerase mix including a proofreading DNA polymerase and we sequenced each clone in both directions. Data bank search using the transcription factor data base TRANSFAC (10) revealed several potential cis-acting elements (Figure 1), but the human CT-1 promoter does not contain any TATA-like element. This feature has been described for genes such as housekeeping
genes and is usually associated with multiple transcription start sites. Potential binding sites for several ubiquitious transcription factors are present. There are two CAATboxes at position 01043 and 01076. The CAAT-box element seems to be related to the level of basal expression and functions in either orientation (11). Interestingly, C/EBPbeta can bind at position -1076. C/EBP is a widespread transcriptional activator in vitro and in vivo, and can stimulate IL-6 expression (12). At position -984 we identified a putative STAT-site. The STATs (signal transducer and activators of transcription) are latent cytoplasmic proteins that, upon activation by cell surface bound polypeptide ligands, move to the nucleus to direct transcription (13). In addition, there is one Sp1-site (at position 0582). The transcription factor Sp1 is a DNA-binding protein which interacts with a variety of gene promoters containing GC-box elements. Sp1 is generally considered a proximal promoter factor that can only stimulate transcription when bound close to the initiation site. However, Courey et al. (14) presented evidence that distally and proximally bound Sp1 can stimulate transcription synergistically. Overall nine AP-2 like binding sites (at positions 0716, 0438, 0266, 0251, 0248, 0207, 0181, 073, 072) were identified in the promoter of the human CT-1 gene. A cAMP response element (CRE) is located at position 0639. The cAMP response element (CRE) mediates diverse transcriptionally regulatory effects. It was first identified as an inducible enhancer of genes that can be transcribed in response to increased cAMP levels. Some growth control genes such as FOS have CRE in their transcriptional regulatory region and their expression is induced by an increase in the intracellular cAMP levels. Additionally, the hCT-1 promoter region harbors a binding site for GATA binding factor at position 0484. The GATA-binding proteins are a group of structurally related transcription factors that control gene expression and differentiation in a variety of cell types. Members of this family of DNA-binding proteins recognize a consensus sequence known as the ’GATA’ motif, which is an important cis-element in the promoters of many genes. All GATA-binding proteins contain 1 or 2 zinc-finger motifs of the distinctive form CXNCX (17)CNXC (15). Promoter and enhancer studies suggested that this factor may regulate genes critical for myocardial differentiation and function, including troponin C, cardiac alpha-myosin heavy chain, and braintype natriuretic factor. Promoter Activity in the 5*-Flanking Region of the Human CT-1 Gene To test for the basal promoter activity of the 5*flanking region of the human CT-1 gene six 5* terminal nested deletion mutants ranging from 01091//39 to
495
AID
BBRC 8311
/
694c$$$921
02-26-98 09:14:17
bbrcg
AP: BBRC
Vol. 244, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Sequence analysis of the 5* flanking region of the hCT-1 gene. The sequence is numbered from the ATG-site in exon 1. PCRprimer for amplifying the 5* nested terminal deletion fragments are underlined. Consensus transcription factor recognition sites identified using the TRANSFAC data bank are labelled. The sequence has been submitted to the EMBL data bank with the accession number AJ002743.
0218//39 were cloned into the promoterless luciferase reportergene vector pGL3 basic. These fragments were obtained by PCR with primers indicated in Figure 1 and contained the first 39 nucleotides of exon 1. In the present study, we document that these six 5*terminal nested deletion fragments of the hCT-1 5*flanking region exhibit significant promoter activity after transfection in COS-7 cells (Figure 2). These constructs were introduced by electroporation into COS-7 cells. A b-galactosidase control vector (pCMV b-gal) was cotransfected in each experiment and luciferase activities were normalized in reference to the respective b-galactosidase activity. All six hCT-1 promoter constructs proved to be functionally active in COS-7
cells as compared to the promoterless construct pGL3basic (nÅ3, põ 0,05). The results are shown in Figure 2. Data are expressed as relative activity with respect to the mean activity of construct pGL3basic which has given a value of 1. Activity values represent the mean { S.E.M. of three independent transfections. Luciferase-reportergene assays revealed the highest promoter activity in fragments CT3 and CT6 and the lowest activity in fragment CT5 (põ0.05), indicating the presence of an enhancer element in the proximity of exon 1 and between position 0557 and 0947 and an putative repressor element between position 0218 and 0377. The enhanced activity of fragment CT3 is maybe due to the SP1 site identified at position 0582.
496
AID
BBRC 8311
/
694c$$$921
02-26-98 09:14:17
bbrcg
AP: BBRC
Vol. 244, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Basal promoter activity of six 5*-terminal nested deletion fragments. COS-7 cells were transiently transfected with constructs containing fragments of the 5*flanking region of the human CT-1 gene (hatched boxes) fused into the upstream region of the Luciferase gene (open boxes). Average relative Luciferase activity (gray bars) was determined by at least three transfections and were normalized in reference to the respective b-galactosidase activity. Data are expressed as relative activity of pGL3basic which was set at 1.
The sequence information obtained in this study, allows for further investigations, including the search for polymorphisms in the promoter region, which may associate with dilated cardiomyopathy or other human phenotypes. Detailed investigations of CT-1 regulation by transcriptional mechanism may contribute to understand the role of CT-1 in cardiac hypertrophy. In conclusion, this first characterization of the human cardiotrophin-1 promoter contributes to elucidate molecular mechanisms in human myocardial diseases. ACKNOWLEDGMENTS This study was supported in part by a grant of the Verbund Klinische Pharmakologie Berlin/Brandenburg.
REFERENCES 1. Chien, K. R. (1993) Science 260, 916–917. 2. Chien, K. R., Knowlton, K. U., Zhu, H., and Chien, S. (1991) FASEB J 5, 3037–3046. 3. Pennica, D., King, K. L., Shaw, K. J., Luis, E., Rullamas, J., Luoh, S. M., Darbonne, W. C., Knutzon, D. S., Yen, R., Chien, K. R., Baker, J. B., and Wood, W. I. (1995) Proc Natl Acad Sci U S A 92, 1142–1146.
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