©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Promoter of the Salmon Insulin-like Growth Factor I Gene Is Activated by Hepatocyte Nuclear Factor 1 (*)

(Received for publication, September 19, 1994; and in revised form, November 3, 1994)

Vladimir P. Kulik (1) Vadim M. Kavsan (1) Frederik M. A. van Schaik Linda A. Nolten Paul H. Steenbergh (§) John S. Sussenbach

From the Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, Ukrainian Academy of Sciences, Kiev 252627, Ukraine and the Laboratory for Physiological Chemistry, University of Utrecht, P. O. Box 80042, 3508 TA Utrecht, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Hepatocyte nuclear factor 1 (HNF-1) was found to have a potent stimulatory action on the activity of the promoter of the salmon insulin-like growth factor I (IGF-I) gene in transient transfection experiments. This liver-enriched transcription factor was shown to bind to an element in the proximal region of the promoter with distinct nucleotide sequence homology to the HNF-1 consensus binding sequence. Mutating this sequence to a variant no longer capable of HNF-1 binding resulted in the loss of the stimulatory effect. Since the sequence of the HNF-1 binding site is conserved in all mammalian, avian, and amphibian species from which the IGF-I promoter sequences have been derived to date, we propose that HNF-1 may be an important regulator of IGF-I gene expression in all of these species.


INTRODUCTION

The importance of the physiological functions of insulin-like growth factor I (IGF-I) (^1)is underscored by the high degree of conservation of this peptide in different species. The degree of homology is well over 90% when different mammalian species are compared(1) . More distant species still show a high level of conservation; only 8 out of 70 residues of chicken IGF-I differ from the human IGF-I sequence(2) , and even Xenopus laevis (3) and salmon (4) possess IGF-I peptides with 80% sequence homology to their human counterpart. Consequently, it is not surprising that a high degree of evolutionary conservation has been found for the IGF-I-encoding exons in the genes of various species analyzed to date. The sequences encoding the entire IGF-I precursor peptides in different mammals (human, porcine, bovine, ovine, rat, and mouse) are 90% homologous(1) ; the human and chicken sequences are identical in 80% of their residues(2) , and the salmon cDNA sequence shows 70% homology to the human sequence(4) . However, the structure of the mammalian IGF-I gene is much more complex than those of the avian and fish genes. Whereas the mammalian genes are contained within a stretch of 90 kb of chromosomal DNA(5) , the chicken gene is 50 kb long(6) , and the salmon gene has been shown to comprise only 20 kb of genomic DNA(7) . Alternatively used leader exons, exons 1 and 2, are present in the mammalian IGF-I genes(8) , whereas only the counterpart of exon 1 has been identified in the chicken (6) and salmon (7) genes. Combination of alternatively used 3`-exons and poly(A) addition signals in the mammalian IGF-I genes results in the production of multiple IGF-I mRNA species(1) . In salmon (4) and chicken(2) , only one prominent mRNA is detected on Northern blots, with lengths of 3.9 and 2.6 kb, respectively.

The salmon IGF-I gene has been analyzed in detail(7) . It contains five exons, of which exons 2 and 3 encode mature IGF-I, as is the case for the mammalian and chicken genes. Multiple transcription initiation sites have been detected in the salmon IGF-I gene, the three most prominent of which are located within the first 250 nucleotides upstream of the ATG start codon. The 385-bp fragment preceding the ATG start codon has been shown to possess promoter activity(9) . No TATA or CAAT box-like elements are present in this region. Such basal promoter elements are absent from all IGF-I promoters presently analyzed, causing the heterogeneous initiation of transcription and the low basal activity of the promoters.

The liver is considered to be the only organ producing IGF-I of endocrine nature. It seems very likely that the uniquely high level of expression of the IGF-I gene in liver is caused by a liver-specific or at least liver-enriched transcription factor. As has been shown by a number of authors, liver IGF-I peptide and mRNA levels in various species become elevated considerably after administration of growth hormone (GH)(10, 11) . Moreover, the observed induction was clearly demonstrated to be caused by activation of transcription of the IGF-I gene(12) . GH functions as a transcriptional activator for many genes. In spite of the recent progress in the unraveling of the GH signal transduction pathway, no direct interaction of the components involved in this pathway and the IGF-I gene has been demonstrated so far. Conceivably, the effect of GH on the rate of transcription of the IGF-I gene is indirect. It may involve up-regulation of trans-acting factors capable of stimulating IGF-I expression, especially in the liver as the main producer of IGF-I.

The aim of this study is to investigate whether liver-specific transcription factors are capable of enhancing the activity of the promoter of the salmon IGF-I gene. This would be a first step in the elucidation of the complex tissue- and developmental stage-specific regulation of the IGF-I gene.


EXPERIMENTAL PROCEDURES

Plasmids

The salmon IGF-I promoter constructs p4000D and p385D have previously been described(9) . Promoter constructs p488M and p105M were made by cloning the promoter fragments SspI-MstI and PvuII-MstI isolated from p4000D into the promoterless luciferase gene containing plasmid pFLASH (13) after digestion by PvuII and NcoI. The pFLASH vector without the PvuII-NcoI polylinker fragment was used as a promoterless control plasmid (pFLdPN).

The construct carrying a mutated HNF-1 recognition site was made by polymerase chain reaction-directed mutagenesis. A 68-mer (5`-CAACAGGAAACAGCTGGGGCAGCATTTGCCTTCTCCTCATCCTAAATTATTCCATATGGATTTTGGGC-3`) containing three mutations (underlined) in the HNF-1 binding sequence, extending from positions -119 to -52 and encompassing the PvuII site, was synthesized. The second polymerase chain reaction oligonucleotide (5`-GCAACTGGATCCGTGCACTGTACTAAACACTC-3`) corresponds to the sequence between positions -488 and -469 and contains a 12-nucleotide nonhybridizing 5`-terminal part. The polymerase chain reaction product was digested with PvuII and SspI and inserted into promoter construct p105M after digestion by PvuII.

The mouse HNF-1alpha expression vector used in cotransfection experiments and for preparation of HNF-1-enriched extract from COS-7 cells was kindly provided by Dr. G. R. Crabtree (Stanford University) and has been described previously(14) . As an internal control for transfection efficiency, a plasmid (RSVbeta-gal) containing the Rous sarcoma virus promoter and enhancer directing the expression of the beta-galactosidase gene was included in the transfection experiments.

Cell Culture and Transfections

All cells used were grown at 37 °C in 5% CO(2). The human hepatoma cell line Hep3B (ATCC HB 8064) (15, 16) was cultured in alpha-modified minimum essential medium. Monkey kidney-derived COS-7 cells (ATCC CRL 1651) (17) were cultured in Dulbecco's modified Eagle's medium. The media were supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 300 µg/ml glutamine.

Hep3B cells were transfected using the calcium phosphate coprecipitation technique(18) . Transfections were performed at 30-40% confluence in 25-cm^2 flasks. 10 µg of promoter-luciferase constructs with or without 1 µg of the expression vector for mouse HNF-1alpha were introduced into the cells. 0.5 µg of RSV-LacZ plasmid was included in each transfection as internal control for transfection efficiency. The total amount of DNA added per transfection was kept constant at 12 µg by the addition of carrier DNA. Precipitates were left on the cells for 4 h. Subsequently, the cells were shocked in 10% Me(2)SO in Serum-free medium for 2 min, fresh medium was added, and the cells were harvested after 18-22 h. Cells were washed in phosphate-buffered saline and incubated in 500 µl of lysis buffer (100 mM potassium phosphate buffer, pH 7.8, 8 mM MgCl(2), 1 mM dithiothreitol, 15% glycerol, 1% Triton X-100). Luciferase activity in 100 µl of cell lysate was measured. Reactions were performed as described(19) . The peak light emission was recorded on a LUMAC/3M M2010A Biocounter. beta-Galactosidase activity in the lysate was measured according to standard protocols(20) .

Electroporation of COS-7 Cells and Preparation of Nuclear Extracts

COS-7 cells were transfected with the HNF-1 expression plasmid by electroporation as described (21) with one modification. To reach higher transfection efficiencies and cell viability, cells were incubated for 10 min at 37 °C immediately after the pulse(22) .

Nuclear extracts were prepared from COS-7 cells and from COS-7 cells overexpressing HNF-1 using a published protocol (23) with the following modifications. Cells were harvested 72 h after electroporation and washed two times in phosphate-buffered saline and once in 0.5 ml of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl(2), 10 mM KCl, 0.5 mM dithiothreitol), followed by incubation of the cells for 10 min on ice in 0.5 ml of buffer A. Lysis was achieved by passing the cell suspension eight times through a 28-gauge needle. Nuclei were checked by light microscopy and collected by brief centrifugation. Nuclei were suspended in an equal volume of buffer C (20 mM HEPES, pH 7.9, 1.5 mM MgCl(2), 420 mM KCl, 0.5 mM dithiothreitol, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 25% glycerol) at 4 °C for 30 min while rotating the tubes. An equal amount of buffer D (20 mM HEPES, pH 7.9, 0.5 mM dithiothreitol, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride) was added. Nuclear debris was removed by centrifugation at 14,000 times g for 15 min at 4 °C. The supernatant was aliquoted and stored at -80 °C.

Electrophoretic Mobility Shift Assay

Nuclear extracts prepared from COS-7 cells and from COS-7 cells overexpressing HNF-1 were used in band shift assays performed essentially as described(24) . Synthetic double-stranded oligonucleotides were labeled 5`-terminally using T4 polynucleotide kinase and [-P]ATP (Amersham Corp.). 20 µl of reaction mixture contained 1 µg of double-stranded poly(dI-dC) competitor DNA, 10^4 cpm probe, and 0.5-1.0 µl of nuclear extract. DNA-protein complexes were formed by incubation of the reaction mixture on ice for 1 h. In the competition experiments, an excess of specific competitor and labeled probe were added to the incubation mixture simultaneously. After incubation, the samples were loaded onto 5% polyacrylamide gels (37.5:1 (w/w) acrylamide/bisacrylamide). After electrophoresis, gels were dried and autoradiographed at -80 °C.

Three double-stranded oligonucleotides were obtained by annealing the corresponding single-stranded oligonucleotides, synthesized on a Pharmacia Biotech Gene Assembler Plus and purified by Sephadex G-50 chromatography. The sequences of the double-stranded oligonucleotides are as follows.


RESULTS

Basal Activity of the Salmon IGF-I Promoter in Human Hepatoma Cells

The basal activity of the salmon IGF-I (sIGF-I) promoter was determined using four luciferase reporter constructs in transient transfection experiments. These constructs contain fragments of 4 kb (p4000D), 488 bp (p488M), 385 bp (p385D), and 105 bp (p105M) of the sIGF-I promoter region (Fig. 1a). p4000D and p385D have been used in previous experiments in Chinese hamster ovary cells(9) . The fragments present in both these constructs were shown to possess promoter activity, with the 385-bp fragment-containing construct being about twice as active as the construct containing the 4-kb fragment in these cells. The p488M and p105M constructs were cloned in a different luciferase gene-containing vector, pFLASH (see ``Experimental Procedures''). The 3`-terminal sequence between the MstI and DsaI sites (Fig. 1a) was left out of these constructs since ATG sequences that might function as out-of-frame start codons are present between these sites. The most downstream transcription start site is still retained within these constructs. The shortest construct, p105M, lacks the two upstream transcription initiation sites.


Figure 1: Structure and activity of the salmon IGF-I promoter-luciferase reporter constructs. a, schematic representation of the promoter-luciferase constructs. The top part represents the genomic organization of the 5`-untranslated region and first exon of the sIGF-I gene. Transcription start sites are indicated by arrows. The lengths of the promoter fragments fused to the luciferase reporter gene and relevant restriction enzyme recognition sites are shown. b, basal activity of the salmon IGF-I promoter in Hep3B cells. The activity of the p4000D construct in transient transfection experiments was set at 100%. The values represent the means ± S.D. of at least three independent duplicate experiments. As an internal control for variations in transfection efficiencies, RSV-LacZ was used. c, transactivation of the salmon IGF-I promoter by HNF-1 in cotransfection experiments in Hep3B cells. Basal activities of the reporter constructs were set at 1 and are shown as blackboxes. The average inductions ± S.D. by HNF-1 observed in at least four independent duplicate experiments are represented by shadedboxes. pFLdPN is the promoterless luciferase vector (see ``Experimental Procedures'').



Hep3B cells were transiently transfected with the four promoter-luciferase constructs using the calcium phosphate precipitation method(18) . All four fragments have weak but measurable promoter activity at least 10-fold higher than the background value obtained with the promoterless reporter construct. The activity of the p385D construct is 1% of the activity of the basal human thymidine kinase promoter cloned in the pFLASH vector. The remaining constructs show a 2-3 times lower activity than p385D (Fig. 1b). Absolute luminometer values ranged from 20 to 30 for the promoterless control, from 200 to 1300 for the sIGF-I constructs, and from 6 times 10^4 to 1.7 times 10^5 for the thymidine kinase promoter-containing construct.

A Putative HNF-1 Binding Site Is Present in the sIGF-I Promoter

Between residues -109 and -97 relative to the most downstream transcription start site of the sIGF-I gene, the sequence 5`-CTTAATGAATAAT-3` is present(7) , showing a high degree of homology to the consensus binding sequence 5`-GTTAATNATTAAC-3` of HNF-1. This consensus is based on HNF-1 recognition sequences identified in the promoters of liver-specific genes of mammalian, avian, and amphibian species(25) . The location of this putative HNF-1 binding site at 100 bp upstream of the most downstream transcription initiation sites is in good agreement with the positions of functional HNF-1 binding elements in other genes. Although HNF-1 recognition sequences in salmon have not been described, its evolutionary conservation, the high degree of homology of the salmon sequence to the consensus sequence, and its distance from the transcription initiation sites prompted us to investigate whether HNF-1 might be involved in the tissue-specific regulation of the salmon IGF-I gene.

HNF-1 Activates the Salmon IGF-I Promoter in Cotransfection Experiments

Two of the promoter-luciferase constructs described above were introduced into Hep3B cells with or without the simultaneous introduction of an expression vector for mouse HNF-1alpha(14) . The p488M construct containing the putative HNF-1 recognition sequence and p105M, lacking this site but otherwise containing the same 3`-terminal promoter and luciferase vector sequences, were used in these experiments.

As can be seen in Fig. 1c, HNF-1 expression in Hep3B cells causes a clear induction of 20-fold in the activity of the p488M construct. The variation in inducibility is considerable; values of between 16- and 34-fold induction were obtained in seven independent duplicate experiments. This variation seems to be caused by slight differences in cell densities and growing rates of the cells at the time of transfection. The values measured for the p105M construct suggest a small but not significant activation of promoter activity by HNF-1. A similar induction of the background activity of the promoterless luciferase vector pFLdPN by HNF-1 was observed. This may be caused by sequences in the vector immediately preceding the luciferase gene, as has also been observed in cotransfection experiments using other transcription factors(26, 27) . These results strongly suggest that HNF-1 is capable of activating the sIGF-I promoter and that sequences located between the SspI and PvuII sites (Fig. 1a) are involved in this induction.

HNF-1 Recognizes the Putative Binding Site in the sIGF-I Promoter

A synthetic 30-mer double-stranded oligonucleotide, SIH, corresponding to the promoter sequence from positions -115 to -86, encompassing the putative HNF-1 binding site, was used as a probe in band shift assays. Nuclear extracts were prepared from COS-7 cells and from transfected COS-7 cells overexpressing HNF-1alpha (see ``Experimental Procedures''). After incubation of radiolabeled SIH with the extracts, DNA-protein complexes were separated on polyacrylamide gel. As a positive control, the 36-mer double-stranded oligonucleotide ATH, containing the well established HNF-1 binding site in the promoter of the human alpha(1)-antitrypsin gene, was used. The results of this assay are shown in Fig. 2a. Clearly visible DNA-protein complex bands were formed with both ATH and SIH when extract from HNF-1-overexpressing COS-7 cells was used. Complexes with identical mobility were not formed when extract from nontransfected COS-7 cells was used. A number of complexes with higher mobility were observed that originate from the binding of proteins present in both transfected and nontransfected COS-7 cells; the formation of these complexes is not dependent on the presence of HNF-1. The results of the band shift assay clearly show that HNF-1 binds to the -115 to -86 region of the promoter of the sIGF-I gene.


Figure 2: Band shift assays using the double-stranded oligonucleotides ATH, SIH, and SIHmut. a, ATH (lanes 1-5) and SIH (lanes 6-10) were labeled and incubated with nuclear extracts from COS-7 cells (0.5 (lanes1 and 6) and 1.0 (lanes2 and 7) µl of extract) and from COS-7 cells overexpressing HNF-1alpha (0.5 (lanes3 and 8) and 1.0 (lanes4 and 9) of extract). In lanes5 and 10, no extract was added. The position of the DNAbulletHNF-1 complex is indicated. b, competition assays were performed by incubation of radiolabeled oligonucleotides ATH (lanes 1-8) and SIH (lanes 9-16) with increasing amounts (0-, 5-, 10-, and 20-fold molar excess) of the unlabeled oligonucleotides. Competition by unlabeled ATH is shown in lanes 1-4 and 9-12, and competition by SIH in lanes 5-8 and 13-16. 1.0 µl of extract of HNF-1-overexpressing COS-7 cells was used in each incubation. c, labeled SIHmut was incubated with nuclear extracts from COS-7 cells (1.0 µl of extract (lane 1)) and from COS-7 cells overexpressing HNF-1alpha (1.0 µl of extract (lane 2)). Lanes3 and 4, same as lanes1 and 2, using wild-type SIH instead of SIHmut; lanes 5-8, labeled SIH and increasing amounts (0-, 1-, 10-, and 20-fold molar excess) of unlabeled SIHmut incubated with 1.0 µl/incubation of nuclear extract from HNF-1alpha-overexpressing COS-7 cells. P, probe; C, competitor.



To unambiguously show that the same protein is binding to SIH and ATH and that this binding is specific and to estimate the relative affinities of the binding to the two sites, unlabeled SIH or ATH was included in the band shift assays as specific competitor. Increasing amounts of unlabeled SIH or ATH were added to the nuclear extracts simultaneously with constant amounts of the radiolabeled double-stranded oligonucleotide probes. The results of these assays are shown in Fig. 2b. Unlabeled ATH competes in complex formation with both labeled ATH and SIH. Reversely, unlabeled SIH also competes in complex formation with both of the double-stranded oligonucleotides. ATH shows a higher binding affinity than SIH, by roughly a factor of 2. A 10-fold molar excess of ATH competes with approximately the same efficiency as a 20-fold molar excess of unlabeled SIH (Fig. 2b, lanes3 and 8 and lanes11 and 16). Again, the DNA-protein complex with higher electrophoretic mobility also formed with extract from untransfected COS-7 cells is clearly visible. SIH has a much higher affinity than ATH for this protein, and competition by unlabeled SIH (Fig. 2b, lanes 13-16) is stronger than competition by ATH (lanes 9-12).

Mutations in the Binding Site Abolish Both Binding and Transactivation by HNF-1

The HNF-1 binding element 5`-CTTAATGAATAAT-3` was mutated to 5`-CATATGGAATAAT-3`. These mutations in 3 highly conserved residues of the HNF-1 consensus binding sequence were expected to prevent the interaction of HNF-1 with this site. The result of the band shift experiment represented in Fig. 2c indeed shows that HNF-1 does not bind to the 30-mer double-stranded oligonucleotide SIHmut, containing the mutated sequence (lane2). Furthermore, the mutated oligonucleotide SIHmut does not compete with SIH for HNF-1 binding, but can substitute for SIH in the higher mobility complexes formed with extracts from both nontransfected and HNF-1-expressing COS-7 cells (Fig. 2c, lanes 5-8).

The same mutations present in SIHmut were introduced into the promoter-luciferase reporter construct p488M by oligonucleotide-directed mutagenesis. The resulting construct, p488Mmut, and its wild-type counterpart, p488M, were subsequently used in cotransfection experiments in Hep3B cells, and the inducibility of their activities by HNF-1 was measured. In Fig. 3, the results obtained for the two constructs are represented. As a consequence of the three substitutions in p488Mmut, otherwise completely identical to the wild-type construct p488M, the transactivating effect of HNF-1 is completely abolished. From this we conclude that HNF-1 is able to enhance the transcriptional activity of the sIGF-I promoter by specific binding to the element between positions -109 and -97 relative to the most downstream transcription initiation site.


Figure 3: Transactivation of wild-type and mutant constructs by HNF-1alpha. Hep3B cells were transfected with the constructs harboring the wild-type or mutant -109 to -97 sequence. The activities of the promoter constructs were set at 1 (blackboxes). The -fold induction upon cotransfection of the HNF-1alpha expression plasmid is represented by shadedboxes. The values are the average results ± S.D. of two independent duplicate experiments.




DISCUSSION

As in mammalian, avian, and amphibian species, the liver has a unique role in IGF-I production in fish species. During the process of transformation from parr to smolt, a sharp rise in IGF-I mRNA levels has been observed in the liver(11) , while the IGF-I mRNA levels in muscle, brain, and ovary remained constant. Injection of bovine GH into salmon also leads to a large increase in the hepatic IGF-I mRNA levels (4) . Interestingly, the peak in IGF-I mRNA synthesis during smoltification is shortly preceded by the onset of GH production. This suggests the involvement of some liver-specific, or at least liver-enriched, factor playing an intermediary role in the transduction of the GH signal onto the IGF-I gene. In the proximal promoter of the salmon IGF-I gene, a putative HNF-1 recognition sequence was detected comprising residues -109 to -97. To test the ability of HNF-1 to induce the activity of the sIGF-I promoter, transfection studies were carried out in the human cell line Hep3B. Although liver-derived, these extensively dedifferentiated cells do not produce liver-specific transcription factors in significant amounts, and the endogenous IGF-I gene is not expressed. Introduction of a vector expressing HNF-1 reconstitutes the synthesis of a liver-specific factor in the Hep3B cells. Simultaneous introduction of sIGF-I reporter constructs into these well transfectable cells allows the comparison of the activities of the promoter constructs in the presence and absence of HNF-1.

Cotransfection experiments using two salmon IGF-I promoter constructs, p488M and p105M, and the expression vector for HNF-1alpha revealed the clear activating effect of HNF-1alpha on the longer construct. Subsequently, we have shown that HNF-1 can indeed bind to the -109 to -97 sequence in the sIGF-I promoter. The activating effect of HNF-1 was demonstrated to be caused by interaction with this element since mutations in the sequence prohibiting HNF-1 binding also resulted in the loss of inducibility of promoter activity.

HNF-1 is a well known transcriptional regulator containing both a homeodomain and sequence motifs similar to the POU proteins that are responsible for DNA binding(28, 29) . Unlike other homeodomain-containing proteins, to which it is only distantly related, HNF-1 must dimerize to bind to DNA(29, 30) . The homodimer recognizes a sequence with dyad symmetry(31) . Binding to this sequence is essential for the liver-specific expression of many genes, e.g. albumin(32, 33) , alpha(1)-antitrypsin(34, 35) , and fibrinogen (31) . Although termed hepatocyte nuclear factor due to its initial apparent restriction in expression to hepatocytes, it has now been demonstrated that this protein is also expressed, albeit at lower levels, in a few extrahepatic tissues, e.g. kidney and intestine(14, 31) . The same holds true for IGF-I expression. Although the liver is still looked upon as the main source and only endocrine producer of IGF-I, significant production has also been documented in various extrahepatic tissues, of which the kidney is a prominent one (36) . Thus, HNF-1 may not only be implicated in the process leading to high IGF-I expression in liver, but also in renal expression of IGF-I.

A consensus binding sequence for HNF-1 has emerged from the studies on a variety of genes(25) . The 13-nucleotide-long consensus binding site 5`-GTTAATNATTAAC-3` is not very strict, however. Not one of the 13 residues is invariant in 26 established HNF-1 binding sites. The sequence between positions -109 and -97 in the sIGF-I promoter differs at three positions from this consensus binding sequence, yet was shown to bind HNF-1 with only 2-fold lower affinity than the well established binding site in the alpha(1)-antitrypsin gene (34, 35) . The introduction of three mutations in this sequence, specifically designed to disrupt the dyad symmetry in the element, prevented the binding of HNF-1.

In this study, mouse HNF-1alpha is shown to interact with an element in the salmon IGF-I gene promoter. This raises the question as to whether the salmon counterpart of mammalian HNF-1 exists and whether it recognizes a homologous sequence. In evolution, HNF-1 seems to be a highly conserved protein. It has been found in species as distantly related as chicken (37) and X. laevis (38). An overall 64% homology exists between rat and Xenopus HNF-1, and an even higher percentage, 90%, was found in the two most important domains, the homeodomain (96%) and the POU-related domain (83%), both involved in DNA binding. A similar homology has been reported for chicken HNF-1. The HNF-1 binding site in the X. laevis albumin promoter has been shown to be fully functional in mammalian systems(32) . In view of this high structural and functional conservation among amphibian, avian, and mammalian species, the existence of HNF-1 in fish is very plausible. Moreover, it probably recognizes a very similar binding sequence since chicken and even Xenopus HNF-1 have been shown to bind to the consensus sequence derived for mammalian species. For a related POU/homeodomain-containing transcription factor, Pit-1, the above interspecies functional and structural interchangeability between mammals and fish has been documented. The POU-specific domain and the homeodomain show 88 and 83% sequence conservation, respectively, between rat and salmon(39) . The consensus binding sequences for mammalian and fish Pit-1 are essentially the same(40) . As a consequence, it could be shown that Pit-1 is conserved structurally as well as functionally in fish and mammals, exhibiting little or no species specificity in transfected heterologous cells(41) . In our view, this strengthens the argument that the same degree of structural and functional conservation may exist for the related factor, HNF-1, in mammalian and fish species.

In relation to the above, it is of interest to note that the HNF-1 binding site in the sIGF-I promoter has been well conserved during evolution. A comparison of the homologous sequences in the IGF-I genes of different species is presented in Table 1. Of the sequences listed, the salmon sequence has been proven to be bound by HNF-1 in the present report, and also the human sequence has been tested in this respect in a band shift assay and was seen to form a complex with mouse HNF-1alpha. (^2)From this we surmise that the sequences present in the IGF-I promoters of the remaining species will also be bound by HNF-1, and thus, HNF-1 may very well play an important role in the tissue-specific expression of the IGF-I gene in all of these species. Initiation sites of transcription have been identified at 109-124 bp downstream of the binding site in salmon(7) , human(8, 42) , rat(43) , and sheep(44) . In the chicken gene, no transcription initiation site has been found at the corresponding position; however, transcription initiation sites are present at 30 bp downstream of the putative HNF-1 site in this gene (6) .



In the salmon genome, a second nonallelic IGF-I gene has recently been discovered(45) . Nonallelic duplicated genes for insulin and melanin-concentrating hormone in salmon have also been described. This phenomenon is explained as a result of tetraploidization of the salmon genome. In evolutionary terms, the salmon is regarded to be a recently diploidized autotetraploid, and structural and functional diversification of the pairs of duplicated genes is now evident. However, the proximal promoter sequences of the two IGF-I genes, termed sIGF-I-1 and sIGF-I-2, are almost 100% homologous, and no essential differences in position of transcription initiation sites or location of cis-acting elements are expected to exist. In Table 1, the sequence of the HNF-1 binding site in the salmon promoter (sIGF-I-1) is compared with the homologous sequence in the sIGF-I-2 gene. One variant residue is present in the second gene (C instead of T at position -97), but since the same residue is also present in many of the established HNF-1 binding sites, this mutation is not likely to result in a lower affinity for HNF-1. Hence, the promoter of the sIGF-I-2 gene is expected to interact with HNF-1 in the same way as described for the sIGF-I-1 gene.


FOOTNOTES

*
This work was supported by International Science Foundation Grant U4H000 (to V. P. K. and V. M. K.), the Netherlands Foundation for the Advancement of Pure Research, and NATO Linkage Grant 931886. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 31-30-538986; Fax: 31-30-539035.

(^1)
The abbreviations used are: IGF-I, insulin-like growth factor I; sIGF-I, salmon IGF-I; HNF-1, hepatocyte nuclear factor 1; kb, kilobase(s); bp, base pair(s); GH, growth hormone.

(^2)
L. A. Nolten, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. G. R. Crabtree for making the HNF-1 expression plasmid available. V. P. K. also gratefully acknowledges Dr. A. P. Koval for continuous support and advice.


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