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Endocrinology Vol. 141, No. 3 1118-1126
Copyright © 2000 by The Endocrine Society

ARTICLES

Two Putative GATA Motifs in the Proximal Exon 1 Promoter of the Rat Insulin-Like Growth Factor I Gene Regulate Basal Promoter Activity1

Lai Wang, Xia Wang and Martin L. Adamo

Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7760

Address all correspondence and requests for reprints to: Dr. Martin L. Adamo, Department of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760. E-mail: adamo{at}biochem.uthscsa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The insulin-like growth factor I gene is transcribed from two promoters, which direct synthesis of alternative first exons (exon 1 and exon 2) in insulin-like growth factor I messenger RNAs (mRNAs). An exon 1 promoter construct extending from +75 to +192 (the most upstream exon 1 start site was designated as +1) showed significant promoter activity in C6, OVCAR-3, and SK-N-MC cells. Within the +75 to +192 region, there are two perfect matches to the consensus binding site for GATA transcription factor, at +108 (GATA-A) and at +183 (GATA-B). Mutations of the GATA-A or GATA-B sequences resulted in slight (or no) effect on exon 1 promoter activity in both C6 and OVCAR-3 cells. However, mutation of the GATA-A sequence stimulated exon 1 promoter activity by 68% in SK-N-MC cells. Mutation of the GATA-B sequence inhibited exon 1 promoter activity by 4.4-fold in SK-N-MC cells. Electrophoretic mobility shift assays showed that there were nuclear proteins in SK-N-MC cells capable of specifically binding to the GATA-A and GATA-B elements and that this binding was GATA-sequence specific. GATA-2, GATA-3, and GATA-4 are the only GATA proteins that have been reported to be expressed in neurons. None of the antibodies against these three GATA proteins were capable of inhibiting or supershifting the bands formed by the nuclear proteins and oligonucleotides containing GATA-A or GATA-B elements. A GATA-1 expression vector was used to perform cotransfection experiments. The GATA-A mutation abolished the stimulatory effect of the GATA-1 factor on promoter activity. In contrast, the GATA-B mutation enhanced the stimulatory effect of GATA-1 protein. Anti-GATA-1 antibody was also incapable of inhibiting or supershifting the bands formed by the nuclear proteins and oligonucleotides containing the GATA-A or GATA-B elements. In conclusion, the GATA-A element seems to bind an inhibitory endogenous factor(s) in SK-N-MC cells, whereas the GATA-B element may bind a stimulatory factor(s). These factors seem to be related to GATA transcription factors but are immunologically distinct from GATA-2, GATA-3, or GATA-4. GATA-1 has the potential to transactivate the exon 1 promoter through the GATA-A element but is unlikely to be the endogenous protein binding to the GATA-A or the GATA-B motifs in SK-N-MC cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS NOW well established that many physiological regulators of insulin-like growth factor I (IGF-I) biosynthesis act, at least in part, by stimulating IGF-I gene transcription in a tissue-specific manner (reviewed in Ref. 1). Attention has thus been focused on elucidating the cis-acting elements and trans-acting factors responsible for the regulation of IGF-I promoter activity. The IGF-I gene is transcribed from two promoters, which direct synthesis of alternative first exons (exon 1 and exon 2) in IGF-I messenger RNAs (mRNAs). The upstream promoter directs transcription of exon 1 from multiple sites extending from approximately 380 to approximately 35 bp upstream of the 3' end of exon 1 (Fig. 1Go). The majority of transcription occurs from two sites, termed start sites 2 and 3, which initiate transcription at approximately 345 and approximately 245 bp upstream of the 3' end of exon 1, respectively.



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  		Figure 1. Proximal IGF-I exon 1 promoter region including all of exon 1 showing transcription start sites (tss) (arrows), translation start site (ATG), and the location and sequences of putative GATA elements. Numbers relative to tss1 (+1). The white bar represents 5'-UTR and the black bar represents coding sequence.

 
IGF-I mRNAs transcribed from the exon 1 start sites are ubiquitous and predominant over those initiated from the exon 2 promoter. Thus, much attention is focused on characterizing the exon 1 promoter region and determining whether it contains elements that mediate regulated IGF-I transcription. Although the importance of the initial part of exon 1 in regulating basal exon 1 transcription in C6 cells and in SK-N-MC cells has been reported (2, 3), deletion studies indicate that regions downstream of +50 are also important for promoter activity (start site 1 is designated as +1). Four to five regions of DNA-protein binding have been observed in the region between +134 and +320 using rat liver nuclear extract (4, 5). Four of these are part of a developmentally induced hypersensitive site in rat liver (6), and at least two of these footprints may be important in mediating the reduction in liver IGF-I transcription during insulinopenic diabetes (5). Moreover, the HS3D site at approximately +192 has been clearly shown to mediate the stimulation of IGF-I transcription by PGE2 in osteoblasts (7).

Recently, our lab reported that an exon 1 promoter construct extending from +75 to +362, which includes start sites 3 and 4, could function as a promoter independently of the sequence upstream of +75 (8). In searching this region for potential transcription factor binding sites, we have noted perfect matches to the consensus GATA transcription factor binding site at approximately + 108 and at approximately + 183. The GATA transcription factors are characterized by two unique zinc-finger DNA-binding domains that recognize a common DNA-binding site, (A/T) GATA (A/G) (reviewed in Ref. 9). They are designated as GATA-1/Eryf1, GATA-2, GATA-3, GATA-4, GATA-5, and GATA-6.

The six GATA family members show distinct but overlapping expression patterns. GATA-1 is expressed at high levels in hematopoietic cell lineages and is also present in a nonhematopoietic tissue, the testis (10). More widely expressed GATA-2 is found in erythroid cells, endothelial cells, and embryonic brain cells (11). GATA-3 is expressed in many tissues, including embryonic brain, T cells, mast cells, and kidney (12).

The newly identified family members GATA-4/5/6 play a role in expression of lineage-specific genes in a variety tissues derived from mesoderm, including heart and gut (13). GATA-4 was also recently reported to be expressed in neuronal cell lines (14). In this study, we test the hypothesis that the putative GATA elements in exon 1 of the IGF-I gene are important for basal exon 1 promoter activity. We also have attempted to characterize the proteins that bind to these sites.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The human neuroblastoma cell line SK-N-MC, rat glioma cell line C6, and human ovarian carcinoma cell line OVCAR-3 were obtained from the American Type Culture Collection (Manassas, VA). Cells were seeded onto 60-mm tissue culture plates and were grown to 60–80% confluence before transfection. SK-N-MC cells were grown in DMEM with 4.5 g/liter glucose, 4 mM glutamine, and 10% FBS. C6 cells were grown in DMEM with 4.5 g/liter glucose, 4 mM glutamine, and 10% newborn calf serum. OVCAR-3 cells were grown in RPMI with 4 mM glutamine and 10% FBS. All media contained 100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were maintained in a humidified incubator at 37 C and 5% CO2.

Plasmids and site-directed mutagenesis
All IGF-I exon 1 promoter constructs were cloned upstream of the firefly luciferase structural gene in the pGL2-Basic vector (Promega Corp., Madison, WI). A vector consisting of the SV40 promoter/enhancer directing luciferase transcription (pGL2-Control, Promega Corp.) was used as a positive control. The -1500/+319, -1000/+319, -500/+319, -500/+282, -500/+192, -250/+319, -250/+282, -250/+192, +75/+319, +75/+282, and +75/+192 constructs were generated by PCR-amplification of these regions using a sense primer with a KpnI restriction site and an antisense primer with a HindIII restriction site. Primers were synthesized at the Center for Advanced DNA technologies (University of Texas Health Science Center at San Antonio). The PCR products were ligated into the same sites of pGL2-Basic.

Two perfect matches to the consensus GATA transcription factor binding site are located in the sense strand of exon 1 at +108 and at +183, respectively (Fig. 1Go). The sequence at +108 is 5'-AGATAA-3' (GATA-A), and the sequence at +183 is 5'-AGATAG-3' (GATA-B). The consensus GATA sequence is 5'-(A/T)GATA(A/G)-3' (9). Each of these sequences was mutated to 5'-CTCGCC-3' in the +75/+192 construct by using a mutagenic sense primer for the GATA-A element and mutagenic antisense primer for the GATA-B element. The GATA-A/GATA-B double mutation was generated using a similar strategy. In addition, a second GATA-A mutation was generated by mutating the sequence to 5'-GAGCGG-3'. The identities of the mutations in the +75/+192 constructs were confirmed by DNA sequence analysis.

A GATA-1 transcription factor expression plasmid under control of the SV40 enhancer/promoter (pXM-GATA-1) was a kind gift of Drs. Andrew Perkins and Stuart Orkin, Harvard Medical School. For cotransfection studies, the pXM plasmid lacking the GATA-1 structural gene insert was used as a baseline control.

Transfections
Transient transfection was performed using 2 µg of each DNA and the lipofectamine plus system in Opti-MEM medium (Life Technologies/BRL, Gaithersburg, MD). Three hours after transfection, Opti-MEM medium was replaced with 5 ml complete medium. After 24-h incubation, lysates were prepared and assayed for luciferase enzyme activity using the reagents and protocol supplied by Promega Corp., with chemiluminescence measurements performed on a model IL-A911 semiautomatic luminometer from Tropix (Bedford, MA). Protein concentration was assayed on the lysates using the method of Bradford (15).

Preparation of nuclear extracts and oligonucleotide labeling
Nuclear extracts were prepared from SK-N-MC cells using the high salt extraction method (16). Protein concentrations were determined by the method of Bradford (15). Oligonucleotides (sense and antisense strands) corresponding to the sequences of the wild-type and mutant GATA-A and GATA-B elements at +108 and +183 (see Table 1Go) were annealed and end-labeled with {gamma}-32P-ATP and T4 DNA kinase and separated from unincorporated nucleotides using the method described in Adamo et al. (17).


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  		Table 1. Sequences of oligomers used in EMSAs

 
Electrophoretic mobility shift assays (EMSAs) and supershift analysis
EMSAs were performed using the Bandshift kit (Pharmacia & Upjohn, Piscataway, NJ). Briefly, for 32P-labeled GATA-A probe, binding assays were carried out in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM dithiothreitol, 10% glycerol, 0.05% Nonidet P-40, and 3 µg poly(dI-dC). For 32P-labeled GATA-B probe, binding assays were carried out in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM dithiothreitol, 10% glycerol, 0.05% Nonidet P-40, 5 mM MgCl2, and 1 µg poly(dI-dC). For each reaction, 0.25 µg of SK-N-MC nuclear extracts were preincubated in the absence of 32P-labeled probe for 5 min in room temperature. Then, about 1 ng 32P-labeled probe was added into the reaction system, and the binding reactions were carried out for another 20 min at room temperature. For gel supershift analysis, 4 µg of GATA-2, GATA-3, or GATA-4 gel supershift antibodies (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA) were added to each reaction. Reactions were electrophoresed on 4% native polyacrylamide gels.

Statistical analysis
All data are mean ± SEM for the indicated number of observations. Statistical differences between means were determined using one-way ANOVA in the SIMSTAT 3 package (Normand Peladeau, Provalis Research, Montréal, Canada).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Defining the minimal exon 1 promoter
We first tested the promoter activities of exon 1 promoter constructs that contained various amounts of 5'-flanking sequence or exon 1 sequence in SK-N-MC cells. Constructs containing 1500 bp of 5'-flanking sequence and 319 bp of exon 1 sequence stimulated luciferase activity by approximately 21-fold in SK-N-MC cells (Fig. 2AGo). Deletion of 5'-flanking/5'-UTR sequence to -1000, -500, or to +75 resulted in higher levels of luciferase activity (Fig. 2AGo). In the constructs containing 500 or 250 bp of 5'-flanking sequence, deletion of 3' exon 1 sequence to +282 or +192 did not alter the promoter activity (Fig. 2BGo). However, when the 5' end of the promoter was deleted to +75, 3'-deletion to +192 lowered the promoter activity, but still resulted in a significant promoter activity over pGL2-Basic (Fig. 2BGo). A promoter construct containing exon 1 sequence from +75 to +192 also significantly stimulated luciferase activity, compared with pGL2-Basic in C6 and OVCAR-3 cells (Fig. 3Go, A and B). This is consistent with the data from SK-N-MC cells. Thus, a minimal IGF-I exon 1 promoter region is located between +75 and +192, a region that includes start site 3 but not start sites 1 or 2.



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  		Figure 2. IGF-I exon 1 basal promoter activity in SK-N-MC cells. The promoter activity of pGL2-Control is shown at 0.01 x to fit in the same graph. The arrows indicate location of presumed tss. Numbering of constructs is relative to tss1 at +1. Data are mean ± SEM for three separate transfections.

 


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  		Figure 3. The effect of GATA mutations on IGF-I exon 1 promoter activity. Wild-type represents IGF-I exon 1 promoter construct +75/+192. AM represents GATA-A mutation. BM represents GATA-B mutation. See text for sequences of the mutations. The promoter activity of pGL2-Control is shown at 0.01 x to fit in the same graph. Data are mean ± SEM for three separate transfections.

 
The GATA-A and GATA-B elements exert a cell-type specific effect on promoter activity
Within the +75 to +192 region, there are two perfect matches to the consensus binding site for the GATA transcription factor, at +108 (GATA-A) and at +183 (GATA-B). To determine whether these elements contributed to exon 1 promoter activity, they were mutated in the +75 to +192 promoter construct. Wild-type and mutant constructs were transfected into C6 cells, OVCAR-3 cells, and SK-N-MC cells. As seen in Fig. 3Go, A and B, single or double mutations of the GATA-A and GATA-B elements had slight or no effect on promoter activity in both C6 and OVCAR-3 cells. However, in SK-N-MC cells, where the wild-type +75/+192 construct stimulated luciferase activity by 19-fold, mutation of the GATA-A element increased luciferase activity to 32-fold above pGL2-Basic, i.e. a 68% stimulation of wild-type promoter activity (Fig. 3CGo). In contrast, mutation of the GATA-B element resulted in luciferase activity that was only 4.3-fold over pGL2-Basic, i.e. a 4.4-fold inhibition of wild-type promoter activity (Fig. 3CGo). The GATA-A/GATA-B double mutation also resulted in luciferase activity that was only 6.5-fold over pGL2-Basic, i.e. a 2.9-fold inhibition of wild-type promoter activity (Fig. 3CGo).

To distinguish between the possibilities that the GATA-A element binds an inhibitory transcription factor(s) or that this GATA-A mutation introduces a positive transcription factor binding site, a second mutation, GATA-AM (2), was made in the GATA-A element and was assessed in SK-N-MC cells. This mutation resulted in an increase of luciferase activity to 110-fold over pGL2-Basic (Fig. 4Go). These data suggest that the GATA-A element is a binding site for an endogenous inhibitory factor(s) in SK-N-MC cells. The GATA-B sequence seems to be a binding site for an endogenous stimulatory factor(s) in SK-N-MC cells.



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  		Figure 4. The effect of the GATA-A mutation 2 on IGF-I exon 1 promoter activity. AM (2 ) represents GATA-A mutation 2. See text for response of the mutation. The promoter activity of pGL2-Control is shown at 0.01 x to fit in the same graph. Data are mean ± SEM for three separate transfections.

 
Both GATA elements bind endogenous nuclear proteins from SK-N-MC cells
EMSAs were performed using double-stranded oligonucleotides encompassing the GATA sequences (see Table 1Go for sequence). Incubation of nuclear extracts with a labeled oligomer containing the wild-type GATA-A sequence resulted in two bands of reduced mobility, compared with free probe (Fig. 5Go). The intensity of these bands was abolished when 30-fold to 300-fold molar excess of unlabeled wild-type GATA-A oligomer was used but was not altered by 100-fold molar excess of an Oct-1 factor binding site (Fig. 5Go). An oligomer containing a mutation of the GATA-A sequence, which stimulated promoter activity, or one containing a mutant GATA consensus sequence, did not compete for binding at 30-fold and 100-fold molar excess but were weakly competitive at 300-fold molar excess (Fig. 5Go). Oligomers containing the GATA-B sequence or a GATA consensus sequence were capable of competing for binding at 30-fold molar excess and abolished the binding at 300-fold molar excess (Fig. 5Go).



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  		Figure 5. EMSA of the GATA-A element. All reactions contained 0.25 µg nuclear protein and 1 ng of 32P-labeled GATA-A double-stranded oligomer, except lane C-, which contains probe alone. Oct-1: Oct-1 oligomer; GATA-A WT: GATA-A wild-type oligomer; GATA-AM: GATA-A mutant oligomer; GATA-B WT: GATA-B wild-type oligomer; GATA WT; GATA consensus oligomer; GATA M: GATA mutant oligomer. The sequences of oligomers are shown in Table 1Go. The numbers above the lanes represent the fold molar excess of unlabeled oligomer.

 
When a labeled oligomer containing the GATA-B sequence was incubated with SK-N-MC cell nuclear extracts, a band of reduced mobility, compared with free probe, was also observed (Fig. 6Go). The intensity of this band was abolished when 30-fold to 300-fold molar excess of unlabeled wild-type GATA-B oligomer was used, but it was not altered by 100-fold molar excess of an Oct-1 factor binding site (Fig. 6Go). Oligomers containing either a mutation of the GATA-B sequence or a mutant GATA consensus sequence did not compete for binding at 30-fold and 100-fold molar excess but were weakly competitive at 300-fold molar excess (Fig. 6Go). Oligomers containing the GATA-A site or the GATA consensus sequences were capable of competing for binding at 30-fold molar excess and abolished the binding at 300-fold molar excess (Fig. 6Go). These data suggest that there are distinct endogenous nuclear proteins in SK-N-MC cells binding to the GATA-A and GATA-B elements, and this binding is GATA sequence-specific. In contrast, no bands with reduced mobility were detected in EMSAs using nuclear extracts from C6 or OVCAR-3 cells and double-stranded oligonucleotides containing GATA-A element or GATA-B element (data not shown).



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  		Figure 6. EMSA of the GATA-B element. All reactions contained 0.25 µg nuclear protein and 1 ng of 32P-labeled GATA-B double-stranded oligomer, except lane C-, which contains probe alone. Oct-1: Oct-1 oligomer; GATA-B WT: GATA-B wild-type oligomer; GATA-B M: GATA-B mutant oligomer; GATA-A WT: GATA-A wild-type oligomer; GATA WT; GATA consensus oligomer; GATA M: GATA mutant oligomer. The sequences of oligomers are shown in Table 1Go. The numbers above the lanes represent the fold molar excess of unlabeled oligomer.

 
Known neuronal cell-expressed GATA proteins are incapable of binding to these GATA elements
It has been reported that GATA-2, GATA-3, and GATA-4 are present in neuronal cells (13). To determine whether the GATA-2, GATA-3, or GATA-4 transcription factors bind to the GATA-A and GATA-B elements, gel supershift analysis was performed. As shown in Fig. 7Go, none of the antibodies against these three GATA proteins were capable of inhibiting or supershifting the bands formed by GATA-A or GATA-B oligomers and nuclear extract from SK-N-MC cells. In contrast, these antibodies were capable of supershifting the complexes formed by 32P-labeled double-stranded oligonucleotides containing consensus GATA binding site and cell extracts from two hematopoietic cell lines, BB88 and ATL-16T (data not shown).



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  		Figure 7. Gel supershift analysis of GATA elements. Labeled GATA-A oligomer was used in reactions shown in A and labeled GATA-B oligomer was used in reactions shown in B. Lanes 1 and 2 represent control without or with nuclear protein; lanes 3, 4, and 5 represent addition of antibody against GATA-2, GATA-3, or GATA-4, respectively.

 
GATA-1 protein has the potential to stimulate IGF-I promoter activity through the GATA-A site
To determine whether a GATA transcription factor could activate IGF-I exon 1 promoter activity, cotransfection experiments were performed using pXM-GATA-1, a GATA-1 factor expression vector, and its parent vector pXM plasmid. When pGL2-Basic was cotransfected with pXM-GATA-1, there was about 4-fold stimulation of luciferase activity, compared with cotransfection with pXM plasmid (Fig. 8AGo). This suggests that there is a GATA-binding site(s) in the presumed cryptic pGL2-Basic promoter region.



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  		Figure 8. GATA-1 protein has the potential to stimulate IGF-I promoter activity through the GATA-A site. A, IGF-I exon 1 promoter constructs and pGL2-Basic were transfected alone (black bar), or cotransfected with 1 µg of pXM-GATA-1 (gray bar), or 1 µg of pXM plasmid (white bar) into SK-N-MC cells. Luciferase activity is shown as fold over that of pGL2-Basic or IGF-I exon 1 promoter constructs transfected alone. Data are mean ± SEM for three separate transfections. B, Gel supershift analysis of GATA elements with anti-GATA-1 antibody. Labeled GATA-A oligomer was used in reactions shown in lanes 1–3 and labeled GATA-B oligomer was used in reactions shown in lane 4–6. Lanes 1 and 4 represent control without nuclear protein; lanes 2 and 5 represent control with nuclear protein; lanes 3 and 6 represent addition of antibody against GATA-1.

 
Cotransfection of wild-type exon 1 promoter construct (+75/+192) with pXM-GATA-1 resulted in 6.4-fold stimulation of luciferase activity, compared with cotransfection with pXM plasmid (Fig. 8AGo). GATA-A mutation abolished the stimulatory effect of the GATA-1 protein on promoter activity. In contrast, when the +75/+192 construct containing the GATA-B mutation was cotransfected with pXM-GATA-1, luciferase activity was stimulated by 15-fold, compared with cotransfection with pXM plasmid. Cotransfection of the +75/+192 construct containing the GATA-A/GATA-B double mutation with pXM-GATA-1 resulted in only 2.9-fold stimulation of luciferase activity compared with cotransfection with pXM plasmid. As shown in Fig. 8BGo, the anti-GATA-1 antibody was unable to inhibit or supershift the bands formed by GATA-A or GATA-B oligomers and nuclear extract from SK-N-MC cells. However, it was capable of supershifting the complexes formed by 32P-labeled double-stranded oligonucleotides containing consensus GATA binding site and cell extracts from BB88 cells (data not shown). These data suggest that GATA-1 protein is capable of stimulating IGF-I exon 1 promoter activity through the GATA-A element but is unlikely to be the endogenous protein binding to the GATA-A and GATA-B sites in SK-N-MC cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A previous study had shown that in C6 and OVCAR-3 cells, exon 1 promoter constructs including promoter sequences from +75 to +362 or from -133 to +192 retained significant promoter activity (8). In this study, the promoter construct +75/+192 was generated and showed a significant promoter activity in C6 and OVCAR-3 cells. We also determined the activities of IGF-I promoter constructs in SK-N-MC cells. Deletion of 5'-flanking region from -1500 to -1000 increased promoter activity by 2-fold in this cell line, suggesting that silencer(s) may occur between -1500 and -1000. Five-prime deletion to +75 and 3' deletion to +192 still resulted in significant promoter activity over pGL2-Basic in SK-N-MC cells. These data suggest that the +75 to +192 region contains basal promoter activity in SK-N-MC cells, as it does in other cell lines.

Hall et al. (18) showed that the rat IGF-I exon 1 promoter required approximately 1700 to approximately 800 nucleotides of 5'-flanking region and 319 nucleotides of exon 1 sequence for maximum promoter activity in SK-N-MC cells. Deletion to only 131 bp of 5'-flanking region abolished the promoter activity. The same group observed similar results for the human exon 1 promoter: maximum promoter activity was detected in the construct -1633/+319 and 5'deletion to -238 led to loss of promoter activity (19). However, Jansen et al. (20) reported that, in the context of 192 bp of exon 1 sequence, only 553 bp of 5'-flanking region was required for the greatest promoter activity, and a construct containing only 212 bp of 5'-flanking sequence still contained significant promoter activity. The major difference between the results obtained by these two laboratories and our laboratory seems to be in how much 5'-flanking sequence is required for significant promoter activity. We observed significant promoter activity in the -250/+192 construct, which is consistent with the results of Jansen et al. (20). Furthermore, we detected significant promoter activity in the +75 to +192 region in SK-N-MC cells. It is not clear why the -131/+319 construct was inactive in the study of Hall et al. (18). One possibility would be the use of different clones of SK-N-MC cells.

In this study, we have characterized the function of two potential GATA elements in the IGF-I exon 1 promoter. These elements are located at +108 and +183, i.e. within the +75 to +192 region of exon 1. Mutations of these two elements had a cell-type specific effect on promoter activity. Minor effects were observed in both C6 and OVCAR-3 cells. Although the GATA-A and GATA-B elements are both perfect matches to the GATA consensus sequence, their mutants resulted in opposite effects on promoter activity in SK-N-MC cells. Mutation of the GATA-A sequence stimulated promoter activity, whereas mutation of the GATA-B sequence inhibited promoter activity. In gel mobility shift assays, we showed that unlabeled oligonucleotides containing the consensus GATA binding site competed with the 32P-labeled GATA-A and GATA-B oligonucleotides for binding by nuclear extracts from SK-N-MC cells. These data indicated that the proteins binding to the GATA elements in the IGF-I promoter region are probably related to the GATA family. Only three GATA family proteins, GATA-2, GATA-3, and GATA-4, have been reported to be expressed in neuronal cells (13). However, our supershift assays suggested that none of these three GATA family proteins bound to either the GATA-A or GATA-B sites. Therefore, it is possible that new GATA family proteins are binding to these GATA elements.

Cotransfection experiments suggested that GATA-1 protein had the ability to stimulate IGF-I promoter activity through the GATA-A element. However, luciferase activity resulting from pGL2-Basic was also elevated when it was cotransfected with the GATA-1 expression vector. Searching the sequence upstream of the luciferase gene in the pGL2-Basic plasmid, we found several perfect matches to the GATA consensus sequence. Thus, we would hypothesize that when GATA-1 protein is overexpressed in SK-N-MC cells, it is capable of binding to the GATA-A and the GATA-B elements in the IGF-I exon 1 promoter, and the cryptic GATA-binding site(s) in pGL2 plasmid backbone. Furthermore, GATA-1 protein can only stimulate promoter activity by binding to the GATA-A site and the cryptic GATA-binding site(s) in the vector. The affinity for GATA-1 protein binding is presumably different among these sites. Binding of GATA-1 factor to the GATA-A and GATA-B elements is much more favorable than binding to the cryptic GATA binding site(s)in pGL2 plasmid backbone. Thus, when expressed, GATA-1 transactivates IGF-I exon 1 promoter through the wild-type GATA-A sequence. Moreover, when the GATA-B site is mutated, there is more GATA-1 available to transactivate IGF-I exon 1 promoter through the GATA-A site. However, the possibility that GATA-1 protein stimulates IGF-I exon 1 promoter activity in an indirect manner, which may involve increased expression or stimulation of other transcription factors, should not be excluded.

Site-directed mutagenesis suggests that the GATA-A site is a binding site for an inhibitory factor(s). It has not been reported that GATA-1 protein is expressed in neuronal cells, so it is unlikely that GATA-1 protein is the transcription factor that binds to the GATA-A site in intact SK-N-MC cells. However, this study suggests that GATA-1 protein has the potential to stimulate IGF-I exon 1 promoter activity through the GATA-A site in those cell lines and tissues in which GATA-1 protein is expressed.

Site-directed mutagenesis and DNA-protein binding assays indicate that sequences between about -10 and +50, including IGFI-FP1 at -3, are important for basal activity in C6 and SK-N-MC cells (2, 3). C/EBP {delta} is the best characterized transcription factor involved in the regulation of IGF-I promoter activity (7). This mechanism involves the PGE2 stimulation of cAMP levels, followed by a protein kinase A-dependent increase in the amount of nuclear C/EBP {delta} protein that then binds to a sequence within footprint HS3D located at approximately +192, resulting in increased exon 1 promoter activity. C/EBP {alpha}, LAP, and HNF-1 {alpha} can stimulate IGF-I exon 1 promoter activity by binding to cis-acting elements located from +18 to +30 of the IGF-I exon 1 sequence in Hep3B cells (21, 22). In addition, HNF-1 {alpha} can act through another site, located at approximately -145.

HNF-3ß has a strong stimulatory effect on the IGF-I exon 1 promoter in Hep3B cells (23). The locations of the two binding sites for HNF-3ß are between +105 to +114 and +142 to +151. The upstream binding site overlaps with the GATA-A element. Our study suggests that the GATA-A element may function as a stimulatory factor-binding site in hematopoietic cell lineages in which GATA-1 protein is expressed, although this hypothesis remains to be tested. It is possible that a multiprotein complex is formed between GATA protein(s), HNF-3ß and/or other transcription factors in the region around +108. The GATA-B sequence is included in region III detected by DNase I footprinting using rat liver nuclear extract (5), which is metabolically regulated during insulinopenic diabetes. The GATA-B sequence also overlaps the beginning portion of footprint HS3D (4). However, it does not overlap with the C/EBP {delta} binding site within HS3D. Identification of the transcription factors that bind to these GATA elements will be a future goal.

In summary, a minimal IGF-I exon 1 promoter region is located from +75 to +192. The GATA-A and GATA-B elements located within this minimal promoter region have a cell-type specific effect on promoter activity. Two distinct GATA transcription factor family-related proteins or protein complexes, apparently other than GATA-2, GATA-3, and GATA-4, probably bind to the GATA-A and GATA-B sites in SK-N-MC cells. They inhibit promoter activity through the GATA-A motif and stimulate promoter activity through the GATA-B motif. GATA-1 protein has the potential to stimulate IGF-I exon 1 promoter activity through the GATA-A site, but it is unlikely to be the endogenous protein binding to these GATA elements in SK-N-MC cells.


   Acknowledgments
 
We thank Drs. Andrew Perkins and Stuart Orkin for providing GATA-1 expression vector and its parental plasmid, Dr. Jim Karras (Isis Pharmaceuticals, Inc.) for providing ATL-16T cell line, and Mr. Jose Talamantez for technical assistance.


   Footnotes
 
1 These studies were supported by Grant AQ-1385 from the Robert A. Welch Foundation and Grant DK-47357 from NIH (to M.L.A.). Back

Received June 4, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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