(Received for publication, July 11, 1995 )
From the
Insulin-like growth factor-I (IGF-I) gene transcription is
mediated largely via exon 1. In an initial search for regulatory
regions, rat hepatocytes were transfected with IGF-I constructs. Since
omission of downstream sequences led to reduced expression, we then
used in vitro transcription to evaluate potential metabolic
regulation via downstream regions. With templates including 219 base
pairs of downstream sequence, transcriptional activity was reduced
70-90% with hepatic nuclear extracts from diabetic versus normal rats. However, activity was comparable with templates
lacking downstream sequences. The downstream region contained six DNase
I footprints, and templates with deletion of either region III or V no
longer provided reduced transcriptional activity with nuclear extracts
from diabetic rats. Nuclear protein binding to regions III and V
appeared to be metabolically regulated, as shown by reduced DNase I
protection and activity in gel mobility shift assays with nuclear
extracts from diabetic rats. Southwestern blotting probes corresponding
to regions III and V recognized a 65-kDa nuclear factor present at
reduced levels in diabetic rats. These findings indicate that a
downstream region in exon 1 may be important for both IGF-I expression
and metabolic regulation. Altered concentration or activity of a
transcription factor(s) binding to this region may contribute to
reduced IGF-I gene transcription associated with diabetes mellitus.
The insulin-like growth factors (IGFs) ()are
polypeptides with sequence, structure, and biological actions similar
to those of insulin (1) . Since circulating levels of IGF-I are
more responsive to changes in metabolic status than are levels of
IGF-II(2, 3) , IGF-I is thought to be a more important
regulatory factor during postnatal life. While IGF-I is expressed in
many organs and tissues, consistent with paracrine regulation and a
role as a local growth factor, its expression is 50-100 times
higher in the liver than in other tissues, consistent with hepatic
origin of circulating IGF-I, and a role as an endocrine regulator of
growth(1, 3, 4) . In the liver, IGF-I
expression appears to be regulated
pretranslationally(5, 6, 7) . Modulation at
the level of gene transcription is indicated by findings such as
decreased IGF-I gene transcription in streptozotocin-diabetic animals (7) and the ability of insulin to stimulate IGF-I gene
transcription in hepatocyte primary culture(8) . However,
underlying mechanisms are poorly understood.
The single rIGF-I gene gives rise to a complex family of mRNAs with both size and coding sequence heterogeneity (9, 10, 11) and polypeptides which are products of multiple translational initiation sites. Multiple in-frame initiator codons within 5` sequences specify different amino-terminal signal peptides, and preprolGFs with signal peptides containing 22, 32, or 48 amino acids are synthesized depending on utilization of different AUGs(9, 12) . Initiation of transcription is also complex, as several laboratories have identified multiple transcription initiation sites in exons 1 and 2 of the rat, sheep, and human genes(12, 13, 14, 15, 16) . In the rIGF-I gene, initiation sites extend over 140 bp in exon 1, and 770 bp in exon 2(13) . However, Adamo et al.(13) found that two initiation sites in exon 1 could account for 70-80% of IGF-I gene transcription in adult rat liver. Thus, although rat IGF-I gene transcription is regulated by two distinct promoters, the exon 1 promoter appears to be dominant.
Since relatively little is known about molecular regulation of IGF-I gene transcription, we have focused on the liver; while several laboratories have begun to study the basis of IGF-I gene transcription in different immortal cell lines(14, 17, 18, 19) , there has been little evaluation of underlying mechanisms in the dominant source of IGF-I production(1, 3, 4) . Analysis of findings from several laboratories suggests that downstream regions may play a role in IGF-I gene expression(17, 18, 19, 20) , but such an hypothesis has not been tested in the liver. In the present study, we demonstrate that sequences downstream from the exon 1 major transcription initiation site are important both for hepatic IGF-I expression and metabolic regulation, we characterize nuclear protein binding to downstream sequences, and we identify two regions that may be involved in the decreased IGF-I gene transcription associated with diabetes mellitus.
Figure 1:
Luciferase activity in cultured normal
rat hepatocytes transfected with constructs containing rIGF-I sequences
in a reporter vector (see ``Materials and Methods'').
Sequences are shown relative to the exon 1 major transcription
initiation site. Promoter activity was expressed relative to activity
with p0Luc. Results shown are representative of three experiments.
Separate studies indicated that transfection efficiency (measured
according to expression of CMV--galactosidase) was unaffected by
the presence of IGF-I elements. Mean ± S.E., n =
3.
Because of relatively low expression in transient transfection
studies (possibly due to the difficulty in maintaining IGF-I gene
expression in hepatocytes (30) as well as the difficulty of
transfecting cells in primary culture(31) ), a different model
was used to test the hypothesis that downstream sequences contribute to
metabolic regulation of IGF-I gene transcription. A genomic IGF-I
template containing 471 bp of upstream sequence and 219 bp of
downstream sequence was incubated with nuclear extracts from the livers
of normal and diabetic rats, and in vitro transcriptional
activity was evaluated by primer extension. As shown in Fig. 2A, the dominant transcription initiation site in vitro was identical to that used in vivo. The signal originated
from RNA polymerase II transcripts, since it was sensitive to
-amanitin (not shown). Moreover, no signal was detected when
extracts were incubated in the absence of a DNA template, indicating
that signals originated from transcripts generated in vitro (not shown). Since in vitro transcriptional activity for
the adenovirus major late promoter template driven by nuclear extracts
from the livers of diabetic rats was comparable or greater to that with
extracts from normal rats (Fig. 2B), we concluded that
nuclear extracts from the livers of diabetic rats contained adequate
transcriptional machinery and that changes in IGF-I gene transcription
were likely to be specific. Using templates containing downstream
sequence, in vitro transcriptional activity of nuclear
extracts from the livers of diabetic rats was reduced 90%, compared
with nuclear extracts from the livers of normal rats, (p <
0.05), but transcriptional activity with an IGF-I template lacking
downstream sequence was not significantly decreased with extracts from
diabetic versus normal rats (p > 0.1, Fig. 2C). Since this observation is consistent with our
previous finding(7) , that IGF-I gene transcription rates were
reduced
97% in the livers of diabetic rats as compared with normal
rats, downstream sequences may be important for both IGF-I expression
and metabolic regulation.
Figure 2:
In
vitro transcriptional activity of nuclear extracts from the livers
of normal and diabetic rats. Panel A, genomic IGF-I templates
containing 471 bp of upstream sequence and 219 bp of downstream
sequence were incubated with separate batches of hepatic nuclear
extracts from normal (lanes 4 and 6) and diabetic (lanes 5 and 7) rats, and the in vitro transcripts were quantitated by primer extension; 30 µg of
total liver RNA from normal (lane 2) and diabetic (lane
3) rats were used as positive controls, and yeast tRNA (lane
8) was used as a negative control. f174 DNA digested by HinfI was used as a size marker (lane 1). Panel
B, a genomic IGF-I fragment from -471 to +3 bp was
subcloned to pUC13(CAT) and used as a template in in
vitro transcription assays, and a template for the adenovirus
major late promoter (pML(C
AT)
, AMLP)
was used as an internal control. Panel C, relative
transcriptional activities of normal and diabetic liver nuclear
extracts. Data were obtained from three different pairs of normal and
diabetic extracts; for each pair of extracts, IGF-I transcriptional
activity of normal liver extracts (determined by densitometric scanning
and expressed relative to adenovirus major late promoter activity) was
designated as 100%. Mean ± S.E., * indicates p <
0.05.
Figure 3: Binding of nuclear proteins to the 272-bp AccI/BglII (-54/+219) fragment. End-labeled probe (5000 cpm) was incubated with liver extract at 25 °C for 25 min. Protein-DNA complexes I and II were visualized on a 5% polyacrylamide gel as described under ``Materials and Methods.''
Figure 4: DNase I protection assay. An end-labeled 272-bp AccI/BglII (-53/+219 bp) fragment was incubated with normal or diabetic liver extract at 25 °C for 20 min and then digested with DNase I as described. Protected regions determined according to Maxam-Gilbert sequencing are shown as boxes, with naked DNA digested with DNase I as a negative control (C). pBR322 plasmid DNA digested with HpaII was used as a size marker (M). Panel A, probe labeled on coding strand; Panel B, probe labeled on noncoding strand.
Figure 5: Nucleotide sequence of 272 hp AccI/BglII fragment. Sequence originally determined by Shimatsu and Rotwein (32) was confirmed in our laboratory. Protected regions are underlined. The exon 1 major transcription initiation site is indicated by an arrow and designated +1. The transcription initiation sites identified by Adamo et al.(12, 13) are also indicated.
Figure 6: In vitro transcription assay with deletion mutants as templates. Panel A, summary of deletion mutant constructs. Regions of DNase I protection are indicated, and deleted regions are shown by a thin line. The right side of the panel shows the relative transcriptional activities of diabetic rat liver nuclear extracts as compared with those from normal animals for each template. Panel B, relative transcriptional activities of normal (lanes 3 and 5) and diabetic (lanes 4 and 6) nuclear extracts with both wild type DNA (lanes 3 and 4) and a deletion mutant (lanes 5 and 6) as template. Lane 1 contained 20 pg of liver RNA; lane 2 contained tRNA. In vitro transcripts were quantitated by primer extension as described under ``Materials and Methods.'' Sequencing reactions utilized an oligonucleotide complementary to +141 to 161 bp electrophoresed along with primer extension products on a 6% polyacrylamide-8 M urea gel. Panel C, transcriptional activities of normal (lanes 1, 3, and 5) and diabetic (lanes 2, 4, and 6) nuclear extracts with both wild type DNA (lanes 1 and 2) and mutants lacking region III (lanes 3 and 4) or region V (lanes 5 and 6) as templates. Lane 7 contained 10 µg of liver RNA. pBR322 DNA digested with HpaII was used as a size marker (M). A 23-bp oligonucleotide complementary to +79/+101 (region IV) was used as a primer to quantitate in vitro transcripts. Sequencing reactions were performed using the same primer, but with a construct lacking region III d(42-68) as a template. Both sequencing reactions and primer extension products were electrophoresed on a 10% polyacrylamide-8 M urea gel. IGF-I cDNA is indicated with an arrow. Panel D, transcriptional activities of normal (lanes 3 and 5) and diabetic (lanes 4 and 6) nuclear extracts with both wild type DNA (lanes 3 and 4) and a mutant lacking region IV (lanes 5 and 6) as templates. Liver RNA (10 µg, lane 2) and yeast tRNA (lane 7) were used as positive and negative controls for primer extension. pBR322 plasmid DNA digested with HpaII was used as a size marker (lane 1). A 20-bp oligonucleotide complementary to +42/+61 was used as a primer to quantitate in vitro transcripts. IGF-I cDNA is indicated with an arrow.
Figure 7: Binding of nuclear proteins to oligonucleotides III and V. Each binding reaction contained 6 µg of nuclear protein from normal rat liver, 5000 cpm probe, pBR322 plasmid DNA or/and cold double stranded oligonucleotides as indicated. Protein/DNA complexes were separated on a 6% polyacrylamide gel.
Figure 8: Specific interactions of nuclear proteins with oligonucleotides III, IV, and V. Each binding reaction contained 5,000 cpm probe, and different amounts of nuclear proteins as indicated. Protein-DNA complexes were visualized on a 6% polyacrylamide gel.
Figure 9: Identification of proteins associated with oligonucleotides III and V. Proteins (20 µg/lane) from hepatic nuclear extracts from normal and diabetic rats were electrophoresed, blotted onto nitrocellulose paper, and then hybridized with oligonucleotides III and V as indicated. After washing with binding buffer at 25 °C for 2 h with 2 changes of solution, the filter paper was air dried and subjected to autoradiography. Molecular markers in kDa are shown at left.
The IGF-I promoters analyzed to date have several common features, such as lack of a ``TATA'' box, presence of transcription ``initiator'' sequences(13, 32) , and binding sites for well recognized transcription factors such as Sp1, C/EBP, and HNF-1 located upstream from the major transcription initiation sites(32) . The present studies demonstrate that sequences downstream from the major transcription initiation site in exon 1 are important for both IGF-I gene expression and metabolic regulation. Within the -54/+219 bp region of exon 1, we found six loci of binding with hepatic nuclear factors; protected regions were similar to those described by Thomas et al.(33) . With our model, DNase I footprinting and gel mobility shift assays revealed that nuclear factors in the livers of diabetic rats have reduced interactions with region III (+42/+68) and region V (+129/+152). Transfection studies revealed a 230% increase in expression with a construct containing 180 bp of downstream sequence (including both regions III and V). Our findings are consistent with those of Hall et al.(14) , who observed that the presence of downstream sequence increased IGF-I gene expression when the same constructs were transfected into SK-N-MC cells and Lowe et al.(18) and Adamo and co-workers (19) who found that downstream sequence increased IGF expression when constructs were transfected into C6 glioma cells. Further evidence of biological significance was provided by in vitro studies; specific differences in IGF-I transcriptional activity between normal and diabetic rat liver extracts could be detected only in the presence of downstream sequences.
The two protein-DNA complexes observed in gel mobility shift assays with oligonucleotides III and V likely result from binding of multiple nuclear factors rather than formation of a dimer, since only complex I could be cross-competed with both oligonucleotides III and V. A putative common factor could interact with motifs such as CCTGC(G/C)CA found within both regions III and V. In both gel mobility shift assays and Southwestern blotting studies, the formation of protein-DNA complexes could be competed with unlabeled oligonucleotides but was not blocked with a great excess of pBR322 DNA, indicating that binding was specific.
The DNA-binding protein(s) identified by Southwestern blotting appears to be metabolically regulated, as reduced binding was provided by hepatic nuclear extracts from diabetic as compared with normal rats. While gel mobility shift assays point to the presence of at least two DNA-binding proteins, we do not yet know if other putative factors are metabolically regulated as well. Lack of identification of a second DNA-binding factor by Southwestern blotting may also be attributed to the denaturing conditions used in this procedure, which could interfere with protein-protein interactions that may be stabilized by the caging effect in gel mobility shift analysis.
A number of viral and cellular transcriptional units contain essential sequences which are downstream from transcription initiation sites (34, 35, 36, 37) . Such downstream elements may influence RNA elongation, processing, and translation, in addition to transcription initiation. Promoters commonly associated with housekeeping and growth control genes often require downstream elements to achieve full gene expression (38) . Moreover, intragenic enhancers or activators have been described for numerous extrahepatic genes such as immunoglobulins(39) , adenosine deaminase(40) , and muscle creatine kinase(41) . Thus, the requirement for both upstream and downstream elements to achieve full gene expression is not unique to the IGF-I gene.
There has been
relatively little characterization of tissue-specific and hormone
response elements of the IGF-I gene. Since this manuscript was
submitted, Nolten et al.(42) recently found that
C/EBP and HNF-1 can stimulate hIGF-I gene expression in Hep3B cells
through binding 120 bp upstream from the major exon 1
transcription initiation site. Within regions of interest identified in
the present studies, region III includes the AAATAAA silencer motif
identified in the rat prolactin gene(43) , and the
(T/A)GATA(A/G) binding motif found in the promoters or enhancers of
erythroid-expressed genes(44) , the histone H-5
gene(37) , and immunoglobulin genes(45, 46) .
The nontranscribed strand sequence GGNGCAGGA in region V is similar to
the silencer binding protein motif GGAGCAGGA found in the rat
glutathione transferase P gene(47) . A GenBank/EMBL search
indicates that there is substantial homology between region III and
region V sequences and elements of over 50 eukaryotic and prokaryotic
genes.
The present studies add to understanding of the regulation of IGF-I biosynthesis. Hepatic IGF-I gene transcription is decreased under conditions of reduced provision of essential amino acids or regulatory hormones, due presumably to differences in nuclear factors that either bind directly to the IGF-I gene or interact with other transcription factors involved in the formation of transcription initiation complexes. Our results suggest that a change in the concentration or activity of factors bound to downstream sequences may lower IGF-I gene transcription in conditions of diabetes mellitus. Additional studies are now aimed at characterization of transcription factors involved in modulation of IGF-I promoter activity.