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Endocrinology Vol. 140, No. 2 624-631
Copyright © 1999 by The Endocrine Society

ARTICLES

Gene Expression of the Human Glucagon-Like Peptide-1 Receptor Is Regulated by Sp1 and Sp31

Inka Wildhage, Heidi Trusheim, Burkhard Göke and Brigitte Lankat-Buttgereit

Clinical Research Unit for Gastrointestinal Endocrinology, Department of Internal Medicine, Philipps University of Marburg, D-35033 Marburg, Germany

Address all correspondence and requests for reprints to: Dr. Brigitte Lankat-Buttgereit, Clinical Research Unit for Gastrointestinal Endocrinology, Department of Internal Medicine, Philipps University of Marburg, Baldingerstrasse, D-35033 Marburg, Germany. E-mail: lankatbu{at}mailer.uni-marburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human glucagon-like peptide-1 (GLP-1) receptor mediates the insulinotropic effects of the incretin hormone GLP-1. It is expressed in a cell- and tissue-specific manner. Recently, we cloned the 5'-region of the GLP-1 receptor gene and found that tissue and cell specificity is lost by 5'-deletion to -574. In this region proximal to the main transcription start point three putative binding sites for Sp1 were localized. Now, in vitro binding of Sp1 was shown by deoxyribonuclease footprint analysis with DNA fragments using either recombinant Sp1 or nuclear extracts from HIT cells. To elucidate the roles of the three Sp1-binding sites, we mutated each of the sites individually as well as in different combinations. The activity of each construct was analyzed in comparison to the wild-type promoter. Mutation of two adjacent Sp1-binding sites showed a clear reduction of activity. Contrasting results were obtained after mutation of the third, more distal Sp1-binding site. Here, a clear increase (~150%) revealed a silencing effect of this cis-regulatory element, possibly resembling a Sp3-binding site. Electrophoretic mobility shift analysis revealed binding of Sp1 and Sp3, which was demonstrated by supershifts using specific antibodies. Cotransfection with Sp1 and Sp3 expression vectors in insect cells lacking endogenous Sp factors clearly demonstrated the involvement of Sp1 and Sp3. Therefore, the basal activity of the GLP-1 receptor gene is mediated by two proximal Sp1-binding sites, whereas a more distal site acts as a repressor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCAGON-LIKE peptide 1-(7–36) amide (GLP-1) arises as a posttranslational product of preproglucagon processing in intestinal cells. GLP-1 is released in response to nutrients and stimulates insulin secretion and proinsulin gene expression (for review, see Refs. 1, 2). At the pancreatic ß-cell, its action is mediated by a stimulatory G protein-coupled receptor (GLP-1R) connected to the adenylate cyclase pathway in a glucose-dependent manner (3). The GLP-1R is a protein of 63 kDa containing 463 amino acids (4, 5) and is expressed tissue and cell specifically in rat islets (6), rat lung (7), human insulinoma (8), human islets (9, 10), a human gastric tumor cell line (11), and human brain and heart (12). It belongs to a family of seven transmembrane-spanning receptors, including the receptors for secretin (13), PTH (14), calcitonin (15), and glucagon (16, 17).

As the GLP-1R is target for a new therapeutic approach using the incretin hormone GLP-1 to treat diabetes mellitus (18, 19, 20), the cell- and tissue-specific expression of the GLP-1R is of special interest. Recently, we cloned the 5'-flanking region of the human GLP-1R gene (21) (accession no. U66062). Two transcription start sites were identified, one major site 44 nucleotides upstream the translation initiation site and a minor, more distant site about 360 nucleotides upstream the translation start codon. Sequence analysis revealed no TATA or CAAT box. Transient transfections of three GLP-1 receptor-producing and nonproducing cell lines with different 5'-deletions revealed that cell and tissue specificities are lost by deletion to -574 (in relation to the main transcription start point). In this region three putative Sp1-binding sites proximal to the main transcription start point are located. This was of interest because Sp1 is a well known transcription factor with important regulator effects on various cellular and viral promoters (22). Recently, three related proteins, Sp2, Sp3, and Sp4, have been characterized (23, 24). Sp1 is expressed in most tissue types (25) and seems to facilitate constitutive basal expression. Presumably, G+C-rich and TATA-less promoters bind one or more Sp1 molecules that recruit specific cofactors such as TATA-binding protein-associated factors, which, in turn, bind to transcription factor IID (TF IID) (26, 27, 28, 29). Sp1 and Sp3 bind to DNA with similar specificities and affinities, and it was suggested that Sp3 is an inhibitory member of the family and represses Sp1-mediated transcriptional activation (30, 31, 32). However, in some promoters, Sp1 and Sp3 additively or independently activate transcription (33, 34), indicating that the function of Sp3 is context or cell type dependent. This study examines the role of Sp1-binding sites in regulating the human GLP-1R promoter. We demonstrate that two Sp1-binding sites are essential for activation of transcription, whereas a third acts as a repressor of gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
GLP-1R promoter-reporter gene construction
The wild-type reporter gene construct (WT) was created by cloning 485 bp of the human GLP-1R promoter (-449 to +36) into the promoterless luciferase vector (pGL2 basic, Promega Corp., Madison, WI). For construction of mutated Sp1-binding sites, the WT was digested with restriction enzymes adjacent to each Sp1-binding site and gel purified (SacII-HindIII, 112 bp for Sp1/1, -68 to +37, including part of the multiple cloning site; BssHII-ApaI, 73 bp for Sp1/2, -192 to -119; StuI-PvuII, 56 bp for Sp1/3, -372 to -315). Appropriate oligonucleotides (MWG Biotech, Ebersberg, Germany) were annealed, and the resulting double strands were ligated into the digested WT. Mutation was verified by dideoxy chain termination sequencing (Sequenase, U.S. Biochemical Corp., Cleveland, OH).

Transient transfections
The different promoter reporter gene constructs were transiently cotransfected with pSV-ß-Gal vector (Promega Corp.) into HIT cells (hamster insulinoma tumor cells) using diethylaminoethyl-dextran. After transfection, cells were plated into 35-mm dishes and incubated for 24 h in supplemented medium (RPMI 1640-L-glutamine, 10% FCS, 5% horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin). All media and supplements were obtained from Life Technologies (Eggenstein, Germany). Cells were lysed, and luciferase and ß-galactosidase activities were determined by chemoluminescence in a luminometer (Lumat LB 9051, Berthold, Bad Wildbad, Germany) using the appropriate substrates (Tropix, Bedford, MA). Cell lysates for ß-galactosidase measurements were first incubated for 50 min at 48 C to inactivate endogenous ß-galactosidase. All measurements were taken in duplicate and from at least five independent transfections. Luciferase activity was normalized to ß-galactosidase activity for plate to plate variations in transfection efficiencies.

Schneider cells SL-2 were maintained at 25 C in Schneider medium (Life Technologies) supplemented with 10% FCS and antibiotics. One day before transfection, cells were plated onto 35-mm dishes and transfected with the PerFect Lipid-method (Pfx-4) according to the supplier’s protocol (Invitrogen, San Diego, CA). Each plate received 1 µg promoter reporter gene construct and various amounts of expression plasmids for Sp1 and Sp3 as indicated in Results (pPac, pPacSp1, and pPacSp3, gifts from G. Suske, Institute of Molecular Tumor Biology, Philipps University, Marburg, Germany). DNA amounts of expression plasmids were compensated for with pPac. Transfections were normalized by cotransfection with 1 µg ß-galactosidase expression plasmid under the control of a cytomegalovirus promoter.

Nuclear extracts and deoxyribonuclease I (DNase I) protection experiments
Nuclear extracts from HIT cells were prepared according to the method of Dignam et al. (35). DNA restriction fragments from the WT plasmid of about 176 bp containing Sp1/1 and Sp1/2 (-192 to -16; WT1/2) and of about 500 bp containing Sp1/3 (-449 to +37, including part of the multiple cloning site; WT3) were radioactively end labeled using T4 polynucleotide kinase and digested with an appropriate second restriction enzyme to create a fragment with a single radioactive label. For DNase protection the probe was mixed with either purified recombinant Sp1 [1–3 footprinting unit (fpu), Promega Corp.] or with 10–40 µg nuclear extract and treated according to the supplier’s protocol (Core Footprinting System, Promega Corp.). G and G+A sequencing reactions were performed as described by Maxam and Gilbert (36). The DNA fragments were resolved on a 6% denaturing polyacrylamide gel.

Electrophoretic mobility shift assay
Three restriction fragments, each containing one Sp1 binding site (SacII-HindIII: 112 bp for Sp1/1, -68 to +37 including part of the multiple cloning site; BssHII-SacII: 124 bp for Sp1/2, -192 to -68; StuI-PvuII: 56 bp for Sp1/3, -372 to -315) were radioactively end labeled using T4 polynucleotide kinase. Binding reactions with 2 µg nuclear extracts from HIT cells were performed according to the supplier’s protocol at room temperature (Gel Shift Assay System, Promega Corp.). Polyclonal rabbit antisera against Sp1 and Sp3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added to the binding assay mixture 15 min after the addition of the radioactively labeled probe and incubated at room temperature for another 20 min. Complexes were separated on a nondenaturing 4% polyacrylamide gel.

Western blot analysis
Sixteen micrograms of protein of HIT nuclear extract were separated by 12% SDS-PAGE according to the method of Laemmli (37) and transferred onto nitrocellulose membranes (Schleicher & Schuell, Inc., Dassel, Germany). Membranes were probed with anti-Sp1 and anti-Sp3 polyclonal antibodies followed by a horseradish peroxidase-conjugated secondary antibody. Detection was performed by the ECL light system (Amersham, Braunschweig, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DNase footprint experiments
Sequence analysis of the proximal 5'-flanking region of the human GLP-1R gene revealed three typical Sp1-binding sites (Sp1/1, Sp1/2, and Sp1/3, Fig. 1Go). To delineate binding for Sp proteins, two restriction fragments of 176 bp (-192 to -16) containing either Sp1/1 and Sp1/2 (WT1/2) and of about 500 bp (-449 to +37) containing Sp1/3 (WT3) were used for DNase protection experiments. The DNA fragments were radioactively end labeled and digested with a second restriction enzyme, creating a single end label. Recombinant Sp1 (1 or 3 fpu) or nuclear extract from HIT cells (10–40 µg protein) was incubated with the DNA at room temperature for binding reactions. Subsequent partial DNase treatment yielded DNA fragments that were analyzed on a 6% denaturing polyacrylamide gel (Fig. 2Go). The addition of recombinant Sp1 revealed two distinct protected regions in WT1/2 centered around the putative Sp1-binding sites, although protection of Sp1/1 was weaker with both recombinant Sp1 and nuclear extract (Fig. 2Go). Directly next to the Sp1/1-binding site a CT motif was located, which was protected by recombinant Sp1 and to a lesser extent by nuclear extract. This is in accordance with an earlier report that Sp1 can bind to these elements (38). In WT3, one protected region was found covering the third Sp1-binding site with recombinant Sp1, as was the case with HIT nuclear extract. Further protections with nuclear proteins may be due to binding of transcription factors that have not yet been identified.



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  		Figure 1. Sequence of the promoter fragment from -449 to +36 used for mutational analysis of the putative Sp1-binding sites (boxed). The mutated sequence is underneath; the large arrow indicates the main transcription start point. Small arrows show the restriction sites used for oligo cloning of the mutated sequence. The HindIII site is located in the multiple cloning site of the pGL2 Basic vector.

 


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  		Figure 2. DNase footprint analysis of DNA fragments containing Sp1/1 and Sp1/2 (WT1/2) or Sp1/3 (WT3). The fragments were incubated with recombinant Sp1 or nuclear proteins, partially digested with DNase I, and resolved on a denaturing 6% gel as described in Materials and Methods. Protected regions are indicated with the sequence, and Sp1-binding sites are boxed. A, DNase protection of WT1/2. Lanes 1 and 6 are controls with BSA (2 and 40 µg), lanes 2 and 3 are recombinant Sp1 (1 and 3 fpu), and lanes 4 and 5 are HIT nuclear proteins (10 and 40 µg). B, DNase protection of WT3; numbering of lanes is the same as in A. A clear protection of the three Sp1-binding sites is detected. G+A ladders were run in parallel on the gel, and the sequence was aligned to the footprint.

 
Mutations of the putative Sp1-binding sites and their effects on the activity of the human GLP-1R promoter
To evaluate whether the three Sp1-binding sites represent functional elements, single, double, and triple inactivating mutations (M1, M2, M3, M1+2, M2+3, and M1+2+3) were introduced into the wild-type promoter luciferase reporter gene vector. These constructs were transiently cotransfected together with ß-galactosidase into HIT cells, and the activity of each construct was measured in comparison to that of the WT (Fig. 3Go). Single mutations of either Sp1/1 or Sp1/2 decrease promoter activity to about 30–40%. These data are consistent with the idea that Sp1 trans-activates gene transcription. Mutation of sites 1 and 2 or sites 2 and 3 had no further effect or only a slight reduction, and therefore, no synergistic effect was seen. This was possibly due to binding of Sp1 to site 1 or 2 only. In contrast to these results, an inactivating mutation of Sp1/3 increased promoter activity, pointing to a silencing role of this site and, therefore, binding of Sp3, which is reported to have activating and repressing functions. Mutations of all three sites decreased promoter activity to about 20%, indicating that Sp1-binding sites are necessary for the major part of transcriptional activation. Transient transfections with these mutation constructs into non-GLP-1R-producing cells (INR1G9, hamster pancreatic A cell line and CHL, hamster lung fibroblasts) yielded similar results (data not shown).



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  		Figure 3. Transient transfections of WT and the different mutation constructs into HIT cells. WT was set at 100%. Decreases in promoter activities of the mutated constructs are evident, except for M3, which increases promoter activity. Luciferase activity was normalized by cotransfection with ß-galactosidase. Results represent values from six to eight independent transfections along with the SD. Control transfections with promoterless reporter gene vector are shown by bar Basic.

 
Western blot and electrophoretic mobility shift analysis
Sp1 acts as an activator of transcription, and no silencing effect is known to date, whereas Sp3 can activate and repress gene expression (39, 40). To clarify whether both transcription factors are present in HIT cells, Western blot analysis with nuclear extracts and specific antibodies for Sp1 and Sp3 was performed (Fig. 4Go). Specifically, a single band of 105 kDa was detected with Sp1 antibodies, whereas signals of 97 and doublet bands of 60 kDa appeared with Sp3 antibodies. These results are similar to the pattern previously reported for HeLa cells (30). To prove that these transcription factors are not only present in HIT cells but also can, in fact, bind to the GLP-1R promoter, we used electrophoretic mobility shift analysis. Three DNA restriction fragments, each containing individual Sp1-binding sites, were incubated with HIT nuclear extracts. The resulting complexes were resolved on a nondenaturing 4% polyacrylamide gel (Fig. 5Go). With all three fragments the addition of nuclear extract yielded retarded bands (Sp1 and Sp3 complex) that were displaced in the presence of a 100-fold molar excess of Sp1 consensus oligonucleotide, whereas an excess of AP2 consensus oligonucleotide had only minor effects. The fact that both of the indicated Sp1/3 bands disappeared with Sp1 competitor oligonucleotide is consistent with Sp1 and Sp3 binding, as both factors bind to the same element with equal affinities (24).



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  		Figure 4. Western blot analysis of nuclear proteins of HIT cells. Proteins were separated on denaturing gels, blotted onto nylon membranes, and treated with antibodies against Sp1 or Sp3 as described in Materials and Methods. Anti-Sp1 recognizes a protein of about 105 kDa; anti-Sp3 recognizes two proteins of about 97 and 60 kDa. Arrows indicate marker protein positions.

 


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  		Figure 5. Electrophoretic mobility shift analysis of DNA fragments, each containing one Sp1-binding site. Additions to the free probe are indicated above; arrows on the left side of each gel indicate shifted bands by nuclear proteins; arrows on the right side indicate the positions of supershifted bands. A, DNA fragment containing Sp1/1; B, DNA fragment containing Sp1/2; C, DNA fragment containing Sp1/3. All fragments show three retarded bands by addition of nuclear proteins, which are weakened or disappearing by competition with Sp1 consensus oligonucleotide but not by competition with AP2 consensus oligonucleotide. These bands are supershifted by antiserum against Sp1 or Sp3. NE, HIT nuclear extract; Sp1 cons., Sp1 consensus oligonucleotide; AP2 cons., AP2 consensus oligonucleotide; IgG, rabbit IgG; {alpha} Sp1, antiserum against Sp1 protein; {alpha} Sp3, antiserum against Sp3 protein.

 
Immunological analysis of the differently shifted bands was achieved by addition of Sp1 and Sp3 antiserum, resulting in supershifted complexes, with concomitant weakening or even disappearance of Sp1/3 and Sp3 complexes. Combined addition of both antisera yielded the same results as both antisera used individually. Therefore, it can be deduced that Sp1 and Sp3 are involved in the protein/DNA complexes of the examined GLP-1R promoter fragments.

To prove that, particularly with mutation of Sp1/3, no binding site for another (possibly up-regulating) factor is introduced, a control experiment was performed with a DNA fragment containing the mutated sequence for Sp1/3 (Fig. 6Go). Using the unmutated Sp1/3 sequence, the addition of recombinant Sp1 as well as nuclear extract from HIT cells yielded retarded bands. Using the mutated sequence, no protein-DNA complex formation could be found, and no other transcription factor bound to the DNA.



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  		Figure 6. Electrophoretic mobility shift analysis of two DNA fragments containing the Sp1/3 (I3) or the mutated Sp1/3 sequence (M3). Lanes 1 in each panel show the free DNA fragments; in lanes 2, recombinant Sp1 (1 fpu) was included; in lanes 3, HIT nuclear extracts were used (2 µg). Whereas shifted bands are observed with the unmutated sequence (arrows; I3), no signals are visible with the mutated sequence (M3) by addition of Sp1 or nuclear extracts.

 
Cotransfection with Sp1 and Sp3 expression plasmids into Drosophila melanogaster SL2 cells
To test the roles of Sp1 and Sp3 in transcription of the GLP-1R gene, cotransfection experiments with Sp1 and Sp3 expression vectors were performed in D. melanogaster SL-2 cells lacking endogenous Sp activity. Four promoter/reporter gene constructs were used: the WT promoter, the promoter with mutation of all three Sp1 binding sites (M1+2+3), and promoters with mutation of either Sp1/1 (M1) or Sp1/3 (M3). Reporter constructs were transfected into SL-2 cells along with expression plasmids pPacSp1, pPacSp3, or parental pPac. DNA amounts were kept constant using pPac plasmid. Whereas cotransfection of Sp1 and/or Sp3 had only a small effect on promoter activity with M1+2+3, a clear induction was observed with WT and, to a lesser extent, with M1 and M3 (Fig. 7Go). These data are in accordance with our results from transfections of HIT cells. Sp3 can induce the promoter activity of WT, M1, and M3, but only to about half that with Sp1. Combination of Sp1 and Sp3 increased promoter activity further as well as overexpression of Sp3 compared to Sp1. A 6-fold overexpression of Sp3 showed no further effect, which may be due to saturation. Transfections with promoter/reporter gene constructs M2 (mutated Sp1/2 site) or M1+2 (mutations of both activating Sp1-binding sites) yielded essentially the same results as those produced with M1 (data not shown). These data are in accordance with the results obtained by transfections of HIT cells.



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  		Figure 7. Cotransfection experiments with expression plasmids for Sp1 and Sp3. SL2 cells were transfected as described in Materials and Methods. The promoter/reporter gene constructs used were the WT promoter, the promoter with mutations in all three Sp1 binding sites (M1+2+3), and promoter constructs with mutations in Sp1/1 (M1) or Sp1/3 (M3). The amounts of expression plasmids for Sp1 and Sp3 are indicated. Values represent the averages of three independent transfections after normalization with control ß-galactosidase along with the SD. Overexpression of Sp1 or Sp3 results in a clear increase in promoter activity if unmutated Sp1 binding sites are present.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previously, we have shown that the tissue and cell specificity of the human GLP-1R promoter is lost by 5'-deletion to -574 and that a basal activity is driven in GLP-1R-producing and nonproducing cells by these 600 bp proximal of the main transcription start point (21). In this region, three putative Sp1-binding sites were identified by sequence analysis. This study clearly demonstrates that interaction of Sp1 or Sp3 with the human GLP-1R promoter is required for basal transcription of the GLP-1R gene. Sp1 is a sequence-specific, ubiquitously present, nuclear factor essential for basal expression of a variety of eukaryotic genes (22, 25). Although Sp1 activates transcription, Sp3 was first reported to repress Sp1 mediated trans-activation of several genes (30, 31, 32, 41). However, recent studies have shown that Sp3 can trans-activate genes such as c-fos, c-myc, transforming growth factor-ß (42), and platelet-derived growth factor (33). These data suggest that the different effects of Sp3 may be context dependent.

In the last years, the promoter regions of several other G protein-coupled receptors were cloned: PTH/PTH-related peptide receptor (43, 44), calcitonin receptor (45), vasoactive intestinal polypeptide receptor (46, 47), and glucagon receptor (48, 49). All of these promoters, including the GLP-1R promoter, share the feature that they do not posses the TATA box motif, but are very G+C rich and contain several putative Sp1-binding sites. However, only for the vasoactive intestinal polypeptide receptor was it shown that Sp1 can bind in vitro employing DNase footprint analysis with recombinant Sp1 and gel mobility shift with whole cell extract. To date, no detailed analysis has been available about the functionality of the Sp1-binding sites. In this study we have shown for the first time that not only Sp1, but also Sp3, are involved in the regulation of GLP-1R transcription, which may be a common feature for related receptors. Evidence to support this idea comes from specific binding of recombinant Sp1 and nuclear extract proteins to the three Sp1-binding sites and a marked change in promoter activity by inactivating mutations at these sites.

DNase footprint analysis identified three protected regions using recombinant Sp1 as well as HIT nuclear proteins corresponding to the Sp1 cis-regulatory elements. Furthermore, a CT motif is protected, representing another Sp1-binding site that may be an in vitro artifact or may be responsible for residual activity of the promoter in transient transfections. Electrophoretic mobility shift analysis of three DNA fragments, each containing one Sp1-binding site with HIT nuclear proteins, revealed several shifted bands; three of them were displaced or weakened by the addition of Sp1 consensus oligonucleotide. A DNA fragment containing the mutated Sp1/3 sequence showed no binding of other, possibly up-regulating, transcription factors.

Our experiments further demonstrate that proteins immunologically related to both Sp1 and Sp3 comprise these complexes. Western blot analysis of nuclear proteins using anti-Sp1 and anti-Sp3 identified bands indicating apparent molecular masses for these complexes of about 105 kDa (Sp1) or 97 kDa and 60 kDa (Sp3). This was consistent with earlier findings analyzing other nuclear extracts. These experiments demonstrated in vitro binding of Sp1 and Sp3 to the three Sp1-binding sites.

To evaluate the functionality of Sp1/1, Sp1/2, and Sp1/3 in vivo, we mutated each of the sites and tested promoter activity by transient transfections in HIT cells. By inactivating mutations of single sites, we show that Sp1/3 represses transcription in contrast to the other two sites. For Sp1, only trans-activating functions were reported, but Sp3 can act as an activator or repressor. Therefore, we conclude that Sp1/3 is a binding site not only for Sp1 but also for Sp3 in competition to Sp1, which may depend on the availability of Sp proteins or promoter context. Sp1 can bind to Sp1/1 and Sp1/2. A synergistic action was not shown. Possibly, this means that only one site can be occupied. Mutation of two sites (1 and 2, or 2 and 3) shows only a slight decrease compared to a single mutation of 1 or 2. At a glance, this seems unreasonable for M1+2, as both activating sites are mutated, and only the repressing Sp1/3 is unaffected. There are, however, several reports that positive or negative function of Sp3 may depend on the number of functional Sp1-binding sites (39, 50). Promoters containing multiple GC boxes display Sp3-mediated repression, whereas a single GC box is not responsive to repression by Sp3. Our data would support this idea. Of course, one cannot exclude the possibility that Sp1 can occupy only Sp1/1 or Sp1/2 and that the observed activity of M1+2 is due to Sp1 or Sp3 trans-activation by binding to Sp1/3. Mutation of all three Sp1-binding sites leads to a decrease in promoter activity to about 20% that of WT. This residual activity is possibly conferred by other activating proteins that have not yet been identified or by binding of Sp1 to the CT box motif next to the Sp1/1-binding site, which is protected in DNase footprint experiments by Sp1.

To further clarify the roles of Sp1 and Sp3 in GLP-1R gene transcription, we performed cotransfection experiments with expression plasmids for Sp1 and Sp3 into D. melanogaster SL-2 cells, which lack endogenous Sp activity. A clear induction of promoter activity occurred by expression of Sp1 and, to a lesser extent, Sp3. We were not able to show a repression of promoter activity by overexpressed Sp3, but there are data indicating that the repression domain of Sp3 cannot function in insect cells and that Sp3 acts as a weak activator of reporter genes containing either a single or multiple Sp1-binding sites in SL-2 cells (39). Sp3 function is dependent on the context of DNA-binding sites and on cellular background. Our transfection results of mammalian cells clearly point to a silencing effect of Sp1/3.

In summary, it was shown for the first time that Sp1 and Sp3 are responsible for the major part of GLP-1R promoter activation and that a repressing function can be exerted by Sp3. Despite the possibility that specific regulation of gene expression may be possible by varying ratios of Sp1 to Sp3 or the phosphorylation state of Sp1 in different cells, our data point to a cell and tissue specificity through negatively acting cis-regulatory elements upstream from the Sp1-binding sites. This idea is supported by the observation that mutations in the Sp1-binding sites of the 485-bp promoter fragment have the same effects on promoter activity in transient transfections of non-GLP-1R-producing cells as in HIT cells. A 3000-bp promoter/reporter gene construct containing cell- and tissue-specific elements showed no promoter activity in non-GLP-1R-producing cells, but the activity was decreased in HIT cells by deletion of Sp1/1, thereby underlining the importance of the Sp1-binding sites (21). The emerging picture of promoter action is one of basal activity driven by Sp1 and Sp3 and intricate interactions of multiple negative control elements. The similarity of promoter structure of related receptors makes it likely that within this receptor family, gene expression is regulated in the same manner.


   Footnotes
 
1 This work was supported by grants from the Deutsche Forschungsgemeinschaft. The presented results are part of the M.D. thesis prepared by I.W. Back

Received March 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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