(Received for publication, September 19, 1994; and in revised form, November 3, 1994)
From the
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.
The importance of the physiological functions of insulin-like
growth factor I (IGF-I) ()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.
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-1 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 (RSV
-gal) containing the Rous sarcoma virus
promoter and enhancer directing the expression of the
-galactosidase gene was included in the transfection experiments.
Hep3B cells were transfected using the
calcium phosphate coprecipitation technique(18) . Transfections
were performed at 30-40% confluence in 25-cm flasks.
10 µg of promoter-luciferase constructs with or without 1 µg of
the expression vector for mouse HNF-1
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
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
, 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.
-Galactosidase activity in the lysate was measured according to
standard protocols(20) .
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, 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
, 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
g for 15 min at 4
°C. The supernatant was aliquoted and stored 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.
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
10
to 1.7
10
for the thymidine kinase promoter-containing construct.
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.
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-1 (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 DNA
HNF-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-1
(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-1
-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).
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-1. 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-1
expression plasmid is represented by shadedboxes. The values are the average results
± S.D. of two independent duplicate
experiments.
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-1 revealed the clear activating effect of
HNF-1
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) ,
-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
-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-1 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-1. (
)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.