©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the Rat Insulin-like Growth Factor-I Gene Involves Metabolism-dependent Binding of Nuclear Proteins to a Downstream Region (*)

(Received for publication, July 11, 1995 )

Ching-I Pao Juan-li Zhu David G. Robertson Kai-wei M. Lin Paul K. Farmer Svijetlana Begovic Guang-jer Wu (1) Lawrence S. Phillips (§)

From the Division of Endocrinology and Metabolism, Department of Medicine Department of Microbiology/Immunology, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The insulin-like growth factors (IGFs) (^1)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.


MATERIALS AND METHODS

Chemicals

Restriction endonucleases and DNA modifying enzymes were obtained from New England BioLabs (Beverly, MA); streptozotocin from Pfanstiehl (Waukegan, IL); [-P]ATP (6000 Ci/mmol), [alpha-P]dATP, and [alpha-P]dGTP (800 Ci/mmol) were from Amersham Corp. Oligonucleotides were from Operon Technology (Alameda, CA); luciferase and luciferin were from Boehringer Mannheim. Superscript RNase H reverse transcriptase was from Life Technologies, Inc., and DNase I was from Pharmacia Biotech Inc. All other chemicals (of molecular biology grade) were purchased from Sigma.

Animals

Male Sprague-Dawley rats (Charles River, Lexington, MA), weighing approximately 120-160 g, were fed ad libitum. Chronic diabetes was produced through tail vein injection of streptozotocin 140 mg/kg. Animals were sacrificed by cervical dislocation 5-7 days later, and livers were used for nuclear extract preparation immediately. Streptozotocin at 250 mg/kg was used to produce acute diabetes, with sacrifice of animals two days after injection.

Transfection

Constructs with IGF-I sequences cloned into a luciferase reporter (p0Luc) were generously provided by Dr. Peter Rotwein from Washington University, as reported previously(14) . Relative to the rIGF-I exon 1 major transcription initiation site (181 bp upstream from the leader sequence(7, 13) ), constructs used for transfection extended from -4398 to -32 bp; -1859 to -32 bp; -1262 to -32 bp; -1859 to +55 bp; and -1859 to +180 bp. Hepatocytes were isolated from 150-200-g male Sprague-Dawley rats using a modification of the collagenase perfusion method of Seglen(21) , as reported previously(5, 8) . Cells were transfected with 6 µg of supercoiled DNA calcium-phosphate precipitate in a volume of 0.3 ml/plate 5 h after plating(22) . Following overnight exposure to the DNA, the cells were rinsed twice with 4 ml of phosphate-buffered saline and placed in 3 ml of serum-free defined medium containing 10M insulin and 500 ng/ml human growth hormone, since both insulin and growth hormone increase IGF-I secretion in primary cultures of hepatocytes(23) . After 24 h, the cells were rinsed twice with 4 ml of phosphate-buffered saline and scraped into 0.7 ml of 50 mM Tris-MES, pH 7.8, containing 1 mM DTT and 1% Triton X-100. Following centrifugation, 50 µl of the supernatant was assayed for luciferase activity in a 200-µl reaction containing 50 mM Tris-MES, pH 7.8, 10 mM magnesium acetate, 2 mM ATP, and 0.5 mM luciferin. Standard curves were performed with purified luciferase to ensure linearity. In all experiments, transfections were performed in triplicate plates for each condition.

Rat Liver Nuclear Extract Preparation

Liver nuclear extracts were prepared as described by Gorski et al.(24) and Triezenberg et al.(25) . Briefly, 15 g of tissue were homogenized in 120 ml of buffer containing 10 mM Hepes, pH 7.6, 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 10% glycerol, and 2.0 M sucrose. The homogenate was layered onto a 2 M sucrose cushion and centrifuged at 27,000 rpm in an SW28 rotor at 4 °C for 1 h. The nuclear pellet was resuspended in lysing buffer containing 10 mM Hepes, pH 7.6, 100 mM KCl, 3 mM MgCl(2), 0.1 mM EDTA, and 10% glycerol at a concentration of 10 A/ml. Nuclei were lysed by adding one-tenth of a volume of 4 M (NH(4))(2)SO(4), and chromatin was removed by centrifugation at 39,000 rpm in an SW40 rotor for 2 h. Nuclear proteins were concentrated by (NH(4))2SO(4) precipitation (0.33 g/ml) and dialyzed against buffer containing 25 mM Hepes, pH 7.6, 100 mM KCl, 0.1 mM EDTA, and 10% glycerol for 4 h. Proteinase inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) were added to buffers just before use. Extracts prepared from animals with either acute or chronic diabetes had comparable activity with in vitro transcription assays.

Construction of 3` Deletion Mutants

For mutants with deletion of +84 to +152 bp, the wild type template was linearized with BstXI, treated with Bal 31 nuclease, and then religated. Other mutants were constructed by polymerase chain reaction, similar to methods described by Higuchi et al.(26) . Primers used are in Table 1. In each case, a -309/+373-bp polymerase chain reaction product with specific internal deletions was digested with BanII and BglII, gel purified, and then subcloned into a wild type template. All mutants were sequenced.



In Vitro Transcription Assay

Transcription reactions (30 µl) contained 1.0 µg of linear DNA template, 50 ng of pAML(C(2)AT), 60 µg of liver extract, 50 mM KC1, 6 mM MgCl(2), 0.5 mM ATP and CTP, 35 µM UTP, 10 µCi of [alpha-P]UTP (400 Ci/mmol), 0.1 mM 3`-O-methyl GTP, 10% glycerol, I u/µl RNasin, 0.05 mM EDTA, and 1 mM DTT. The DNA template and extract were incubated on ice for 20 min. Transcription was then initiated by the addition of nucleotides and carried out for at 30 °C for 45 min. The transcripts were purified by phenol/chloroform extraction, ethanol precipitated, separated on an 8 M urea, 6% polyacrylamide gel, and visualized by autoradiography. When transcription assays were performed in the presence of 0.5 mM of ATP, UTP, GTP, and CTP, the DNA template was degraded by 50 units of RNase-free DNase I as described previously (7) , and the RNA synthesized in vitro was quantitated by primer extension.

Primer Extension

Oligonucleotides complementary to the sequence from +42 to +61 or +79 to +101 were end-labeled by polynucleotide kinase to a specific activity of 10^8 cpm/µg. Probe (50 times 10^4 cpm) was annealed to RNA synthesized in vitro, to 10 µg of yeast tRNA, or to 10-30 µg of liver RNA at 42 °C for 3 h. The cDNA was synthesized as described by Boorstein and Craig (27) at 42 °C for 1 h, ethanol precipitated, and separated on an 8 M urea, 6% polyacrylamide gel.

DNA Probes

Plasmid DNA containing an 0.9-kilobase pair SacI/PstI rIGF-I genomic DNA fragment was linearized with BglII or AccI, and end-labeled. After digestion with AccI or BglII, the 272-bp fragment, -54 to +219 bp relative to the exon 1 major transcription initiation site(7, 13) , was purified from a polyacrylamide gel as described previously(28) . Pairs of oligonucleotides, corresponding to +42/+68 bp (oligo III), +79/+101 bp (oligo IV), and +129/+152 bp (oligo V) downstream from the exon 1 major transcription initiation site, were annealed, labeled, and gel purified.

Gel Mobility Shift Assay

End-labeled 272-bp AccI/BglII fragments were incubated with 0.5-15 µg of nuclear extract in 25 µl of binding buffer containing 10 mM Tris, pH 7.5, 50 mM KCl, 1 mM EDTA, 0.5 mM DTT, 0.2% Nonidet P-40, 20 µg of bovine serum albumin, 4 µg of poly(dI-dC), and 10% glycerol at 25 °C for 25 min. Protein-DNA complexes were separated from free probe at 4 °C on a 5% polyacrylamide gel in 0.5 times TBE (45 mM Tris, pH 8.0, 45 mM boric acid, 1 mM EDTA) at 11 V/cm for 2-3 h, and visualized by autoradiography. For double-stranded oligonucleotides, the probe was incubated with 5-15 µg of extract in same buffer, except that 200 µg/ml of salmon sperm DNA and 24 µg/ml of pBR322 were included to reduce nonspecific binding.

DNase I Protection Assay

End-labeled 272 bp AccI/BglII fragments were incubated with 4-24 µg of nuclear extract in 25 µl of binding buffer containing 10 mM Hepes, pH 7.9, 50 mM KCl, 1 mM EDTA, 0.5 mM DTT, 1 µg of poly(dI-dC), and 10% glycerol at 25 °C for 25 min. An equal volume of binding buffer containing 10 mM MgCl(2), 2 mM CaCl(2), and 10 units/ml of DNase I was added, and the sample was incubated at 25 °C for 2 min. The reaction was stopped with buffer containing 40 mM EDTA and 10 µg/ml tRNA and deproteinized with plenol. DNA was precipitated with ethanol, electrophoresed on an 8 M urea, 6% polyacrylamide gel, and visualized by autoradiography.

Southwestern Blotting

20 µg of extract were mixed with an equal volume of loading buffer containing 5 mM Tris, pH 6.8, 200 mM DTT, 5% SDS, 20% glycerol, and 0.05% pyromin Y. Proteins were denatured at 100 °C for 3 min, separated on a 10% SDS-polyacrylamide gel, and then blotted onto nitrocellulose paper(29) . The blots were incubated with buffer containing 10 mM Hepes, and 5% nonfat dry milk at 25 °C for 1 h, and then incubated with binding buffer containing 10 mM Hepes, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.25% nonfat dry milk, 5 µg/ml salmon sperm DNA, and 1 times 10^6 cpm/ml probe at 25 °C for 1 h. After washing with 2 changes of binding buffer containing 300 mM NaCl at 25 °C for 2 h, the filter paper was air dried and subjected to autoradiography.


RESULTS

Sequences Downstream from Exon 1 Transcription Initiation Sites Are Important for IGF-I Gene Expression and Metabolic Regulation

Constructs with IGF-I 5`-flanking sequences were transfected into rat hepatocytes in primary culture, as summarized in Fig. 1. Relative to expression of a promoterless luciferase reporter vector (p0LUC), expression was decreased 30% by the presence of IGF-I sequence extending from -4 kilobase pairs to -32 bp relative to the exon 1 major transcription initiation site(7, 13, 14) . Expression was increased 40 and 113% with constructs containing 1.86 and 1.26 kilobase pairs of 5` sequence, respectively, and the same 3` terminus. Compared with expression of p(-1859/-32)LUC (40% above that of p0LUC), increased expression was obtained with constructs containing additional downstream sequences. Expression was increased 126% above p0LUC with a construct containing 1.86 kilobase pairs of 5` sequence and 3` sequence terminating at +55 bp, and maximum expression, 230% above p0LUC, was obtained with a construct containing the same 5` sequence and 3` sequence extending to +180 bp; expression was significantly greater than that of the construct with similar 5` sequence but lacking downstream sequence (p < 0.005). Thus, these findings suggested that downstream sequences enhanced IGF-I gene expression.


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-beta-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 alpha-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(C(2)AT) and used as a template in in vitro transcription assays, and a template for the adenovirus major late promoter (pML(C(2)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.



Nuclear Protein(s) Binding to Downstream Sequences

To search for regions that might be involved in gene regulation, the binding of hepatic nuclear factors to the 272-bp AccI/BglII fragment (-54/+219 bp) was first studied by gel mobility shift analysis, as shown in Fig. 3. Densitometric scanning revealed that the intensity of shifted protein-DNA complexes was reduced 30-60% with extracts from streptozotocin-diabetic as compared with normal rats. Binding was specific, since formation of DNA-protein complexes could be competed with an unlabeled 272-bp fragment but not with pBR322 DNA (not shown). All protein-DNA binding studies were repeated with at least three different batches of extracts, and extract activity was monitored by in vitro transcription assays.


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.''



Protein Binding Sites Assessed by DNase I Footprinting

Protein binding sites were determined on both coding and noncoding strands, and results are shown in Fig. 4. Region I corresponded to the major transcription initiation site in exon 1. Five additional protected regions were observed consistently, with footprints at +17/+25 (region II), +42/+68 (region III), +79/+101 (region IV), +129/+152 (region V), and +155/+169 (region VI). Binding of factors in nuclear extracts from the livers of diabetic as compared with normal rats was reduced in both region III (especially with the noncoding strand) and region V (especially with the coding strand) (lanes 4, 5, and 6 versus lanes 1, 2, and 3 in panels A and B; differences in binding were not consistent in other regions. Nucleotide sequences and protected regions are summarized in Fig. 5.


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.



Regions III and V Are Necessary for the Diabetes-associated Reduction in IGF-I Gene Transcription

The importance of downstream regions in metabolic regulation was evaluated with in vitro transcription assays using deletion mutants as templates, as shown in Fig. 6A. A template lacking regions IV and V (+84/+152) no longer provided reduced transcriptional activity with nuclear extracts from diabetic versus normal rats (panel B). Similar results were also obtained with templates lacking regions III (+42/+68) or V (+129/+152) (panel C). In contrast, a template with deletion of region IV continued to exhibit decreased transcriptional activity with nuclear extracts from diabetic versus normal rats (panel D). While the potential involvement of other regions is still being investigated in our laboratory, these data were reproducible with different batches of extracts, and the transcriptional activities of normal and diabetic extracts remained comparable as determined with the adenovirus major late promoter as template.


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.



Nuclear Factors Associated with Regions III and V Are Reduced by Streptozotocin-induced Diabetes

Double-stranded oligonucleotides corresponding to regions III (+42/+68 bp) and V (+129/+152 bp) were used in gel mobility shift analyses to examine DNA-protein interactions, as shown in Fig. 7. Addition of pBR322 DNA was necessary to decrease nonspecific binding (lanes 2 and 10 versus lanes 4 and 12). The association of nuclear factors with region V was stronger than that with region III, especially in formation of complex II (lanes 3 and 4 versus lanes 11 and 12). While formation of both complexes I and II could be competed with unlabeled oligonucleotides (lane 3 versus lanes 5 and 6 and lane 11 versus lanes 13 and 14), cross-competition was incomplete (lane 3 versus lanes 7 and 8 and lane 11 versus lanes 15 and 16). Thus, binding of nuclear factors to regions III and V was relatively specific, particularly for complex II. Activities of nuclear extracts from the livers of normal and diabetic rats are shown in Fig. 8. Nuclear proteins associated with region IV showed similar affinity with normal and diabetic rat liver extracts. In contrast, nuclear protein binding to regions III and V was reduced with extracts from diabetic animals, typically 30-50% of that of extracts from normal rats.


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.



Identification of Protein Factors Associated with Regions III and V

To characterize the size and relative abundance of proteins associated with regions III and V, hepatic nuclear extracts from normal and diabetic rats were subjected to polyacrylamide gel electrophoresis and then blotted to nitrocellulose and probed with corresponding oligonucleotides, as shown in Fig. 9. Proteins with apparent molecular weight of 65 kDa were associated with both regions III and V and were present in extracts from both normal and diabetic animals. However, apparent abundance of the 65-kDa protein in extracts from diabetic animals was 75% of normal with region III and 50% of normal with region V, as determined by densitometric scanning.


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.




DISCUSSION

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.


FOOTNOTES

*
This is article XXXIV in a series entitled ``Nutrition and Somatomedin.'' Portions of these data were presented in the 3rd International Symposium on Insulin-like Growth Factors, on February 6-10 in Sydney, Australia(20) . This work was supported in part by research awards from the Emory Medical Care Foundation (to D. G. R., and C.-I. P.), the American Diabetes Association (to L. S. P.), Biomedical Research Supporting Grant SO7-RR05364, American Cancer Society Cancer Center Seed Money Fund, and Emory University Research Committee (to G.-J. W.) and by National Institutes of Health Grants GM-37261 (to G.-J. W.), DK-01872 (to D. G. R.), and DK-33475 and DK-48124 (to L. S. P.). 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 and reprint requests should be addressed: Div. of Endocrinology and Metabolism, Dept. of Medicine, Emory University School of Medicine, P. O. Drawer AH, Atlanta, GA 30322. Tel.: 404-727-1390; Fax: 404-727-1300.

(^1)
The abbreviations used are: IGF, insulin-like growth factor; bp, base pair(s); MES, 2-(N-morpholino)ethanesulfonic acid; DTT, dithiothreitol.


ACKNOWLEDGEMENTS

We thank Sharon DePeaza and Mary Lou Mojonnier for assistance in the preparation of this manuscript.


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