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Endocrinology Vol. 143, No. 6 2268-2276
Copyright © 2002 by The Endocrine Society

GRH-SOMATOSTATIN-GH

Mutational Analysis of the Mouse Somatostatin Receptor Type 5 Gene Promoter

Whitney W. Woodmansee, Rhonda L. Mouser, David F. Gordon, Janet M. Dowding, William M. Wood and E. Chester Ridgway

Division of Endocrinology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Address all correspondence and requests for reprints to: Whitney W. Woodmansee, M.D., Division of Endocrinology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B151, Denver, Colorado 80262. E-mail: . whitney.woodmansee{at}uchsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously characterized the structure of the murine somatostatin receptor type 5 gene (sst5). Initial transient transfection studies in pituitary somatolactotropes (GH3) mapped the promoter activity of this gene to a region 290 bp upstream of the transcription start site. The current study identifies the sst5 promoter region critical for basal activity. A series of deletions was generated, and promoter activity was localized to a region between -83 and -19. Similar promoter deletion patterns were evident in five pituitary cell types. Seven 10-bp transversion mutations encompassing the region between -83 and -19 were generated, and functional activity was assessed. Promoter activity was reduced by the mutations spanning -67 to -47 compared with the wild-type construct. Another mutation between -26 and -17 resulted in promoter activity reduction in GH3 cells, but not TtT-97 thyrotropes. Deoxyribonuclease I protection analysis of the sst5 promoter region between -208/+47 was performed using GH3 and TtT-97 nuclear extracts. The most striking protected regions, located between -61 and -41 and -25 and -3, correlated with functionally important regions identified by transfection studies. In summary, the mouse sst5 gene promoter has been characterized, and functional activity and nuclear factor interactions were mapped to two specific promoter regions. The region between -67 and -47 appears to contain a nucleotide sequence critical for basal transcriptional regulation of the mouse sst5 gene in pituitary cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN IS a neuropeptide widely distributed throughout the central and peripheral nervous systems as well as many other tissues. Since its initial isolation from the hypothalamus, somatostatin has been shown to be an important factor in the regulation of anterior pituitary function. Specifically, somatostatin has been shown to inhibit the secretion of GH, PRL, and TSH from the anterior pituitary (1). Additionally, somatostatin analogs, such as octreotide, have been shown to have antiproliferative and antisecretory effects on a variety of neuroendocrine tumors (reviewed in Refs. 2, 3, 4), including human pituitary adenomas (5, 6, 7, 8). Since 1992, five different somatostatin receptor subtypes have been identified and are thought to mediate the diverse actions of this hormone (reviewed in Refs. 9, 10, 11). Five separate genes (sst1–5) encode the different receptor subtypes. All of these receptors are members of the G protein-coupled receptor family and mediate the actions of somatostatin by complex intracellular signaling pathways (12).

Little is known about the regulation of sst gene expression at the molecular level. Understanding gene regulation requires structural and functional studies of each gene and its promoter region in a variety of cell types. To date, several hormones, including somatostatin itself (13), glucocorticoids (14, 15, 16), sex steroids (17, 18, 19), and thyroid hormone (20, 21, 22) have been shown to alter gene expression of the sst receptors. It is known that somatostatin can alter the expression of its own receptors in vitro. Specifically, experiments using cultured GH3 pituitary cells have shown that incubation with somatostatin up-regulates somatostatin receptors, as assessed by radiolabeled ligand binding. All somatostatin receptor subtypes, including sst5, demonstrated a time-dependent increase in mRNA expression in response to somatostatin (13). Additionally, sex steroids alter the expression of some of the sst receptors, including sst5. Estrogen modulates human sst2 mRNA expression in a time- and dose-dependent manner in breast cancer cell lines (17), and an estrogen-responsive enhancer-like element has been identified in the human sst2 receptor promoter (18). Estrogen has been shown to down-regulate sst5 mRNA in rat pituitaries (19).

Finally, we have previously demonstrated that pharmacological and physiological doses of thyroid hormone up-regulate murine sst5 gene expression in TtT-97 thyrotropes (20, 22). In this model, hypothyroid mice bearing TtT-97 thyrotropic tumors are treated with thyroid hormone. Thyroid hormone induces shrinkage of the thyrotropic tumors in association with a reduction in TSH-staining cells and TSHß mRNA production as well as an increase in sst5 mRNA and protein. Consequently, we have begun to investigate the molecular basis for thyroid hormone stimulation of sst5 gene expression. Before examining hormonal regulation of this gene, we sought to characterize the basic gene structure and function. We have cloned the sst5 gene, identified the transcription start site, and characterized its structure as being comprised of three exons and two introns (23). Initial basal promoter activity has been mapped to a region 290 bp upstream of the transcription start site. In this study we report a detailed analysis of basal promoter activity of the murine sst5 gene in a variety of pituitary cells. Using a deletion and mutation strategy, strong functional promoter activity was mapped to a region between -67 and -47 relative to the transcription start site. Nuclear factor interactions using GH3 pituitary and TtT-97 thyrotrope extracts were also determined and correspond to regions on the promoter shown to be important for basal promoter activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Deletion plasmid constructs of the sst5 gene promoter
Construction of the -290/+47 sst5 promoter fragment upstream of a firefly luciferase reporter has been previously described (23). A series of 5'-deletion constructs between -290 and -19 bp extending to +47 bp of the sst5 promoter relative to the major transcription start site were generated using a PCR strategy. Oligonucleotide primer pairs were used to amplify a progressive series of shorter 5'-deletions (-208, -151, -83, -47, and -19) with identical 3'-ends at position +47 within exon 1 using the -290/+47 pA3Luc construct as a template and were cloned into a pA3Luc promoterless luciferase plasmid. The -290/+47 construct was used as the template for the deletions because this construct was previously shown to have high promoter activity over the promoterless pA3Luc vector in cultured GH3 pituitary cells (~600-fold higher activity) (23). The sense oligonucleotide primer for each of the deletions was designed with a KpnI site at the 5'-end, and all of the deletions employed an antisense oligonucleotide primer that was complementary to a luciferase sequence in the pA3Luc vector. The following five deletions and their sense oligonucleotide primers used in PCR are as follows (KpnI site in bold, sst5 promoter sequence follows, 5' extent of promoter underlined): 1) sst5 -208/+47 oligonucleotide primer, 5'-GCG GGT ACC ACC CAG GCA ACC TGA C-3'; 2) sst5 -151/+47 oligonucleotide primer, 5'-GCG GGT ACC CCC ATT CTT GAC CTG G-3'; 3) sst5 -83/+47 oligonucleotide primer, 5'-GCG GGT ACC GGA GCT CTC GCC CAG C-3'; 4) sst5 -47/+47 oligonucleotide primer, 5'-GCG GGT ACC TCT ATC TCC TCC ACC CT-3'; and 5) sst5 -19/+47 oligonucleotide primer, 5'-GCG GGT ACC TAG CCT GAG GGC GGG C-3'. The sequence of the antisense oligonucleotide primer complementary to the pA3Luc vector was 5'-GCC TTT CTT TAT GTT TTT GGC G-3'. PCR was performed using Taq polymerase with the following cycle parameters: 94 C for 1 min, 52 C for 1 min, and 72 C for 1 min for 25 cycles. After PCR, the fragments were cloned into the pCR 2.1 vector (Invitrogen, Carlsbad, CA). The fragments were then excised by sequential digestion with KpnI and HindIII and ligated into the pA3LUC promoterless luciferase vector to be used in transient transfection assays. All deletion constructs were confirmed by the chain termination method (24) of DNA sequencing using an EXCEL cycle sequencing kit (Epicentre Technologies, Madison, WI). A schematic diagram of the mouse sst5 deletion map and gene structure is shown in Fig. 2Go.



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  		Figure 2. Schematic diagram of mouse sst5 gene structure and deletion constructs. A pictorial representation of the mouse sst5 gene showing location of introns and exons (23 ). The coding region is contained entirely within exon 3. The strongest promoter activity maps to the immediate 290 bp upstream of the transcription start site and is bounded by SalI (-290) and XhoI (+47) restriction enzyme sites. The progressive 5'-deletion constructs were all cloned into a luciferase reporter vector (pA3Luc) as shown. The different shadings represent identical sequences within each of the separate deletion constructs.

 
Mutation plasmid constructs for transfection studies
Based on the results of the deletion analysis, seven 10-bp transversion mutations encompassing the region between -83 and -19 of the sst5 promoter were generated using a multistep PCR protocol with the sst5 -208/+47-pA3Luc construct as a template. The location for mutational analysis was chosen based on functional promoter activity as assessed by deletion analysis. In the first PCR step, two reactions were performed for each mutation to generate two DNA fragments. In one reaction, PCR was performed using a sense oligonucleotide complementary to the -208 region of the promoter and an antisense oligonucleotide containing the respective mutations. In a second reaction, PCR was performed using a sense oligonucleotide for each mutation in combination with an antisense oligonucleotide complementary to the pA3Luc expression vector. These individual fragments were gel-purified, and a second PCR, using the two flanking oligonucleotides, was performed to generate full-length promoter fragments identical to the -208/+47 construct except for the desired mutation. These fragments were cleaved with KpnI and HindIII, gel-purified, and ligated into the pA3Luc vector. The two outer oligonucleotides used were: 1) sense strand sst5 -208/+47 oligonucleotide primer, 5'-GCG GGT ACC ACC CAG GCA ACC TGA C-3'; and 2) antisense strand luciferase primer as specified in the previous section. The specific mutation oligonucleotide sequences employed are as follows with the mutated sequence underlined: 1) mutation 1 (-208/+47 {delta} -87/-78): sense, 5'-CCACCCCACC CCAAAAACAA TTCGCTCTCGCCCAG-3'; antisense, 5'-CTGGGCGAGA GCGAATTGTT TTTGGGGTGGGGTGG-3'; 2) mutation 2 (-208/+47 {delta} -77/-68): sense, 5'-CACCCCACC GGATAGAGAT AAAAGCCTGG CTTAG-3'; antisense, 5'-CTAAGCCAG GCTTTTATCT CTATCCGGTG GGGTGG-3'; 3) mutation 3 (-208/+47 {delta} -67/-57): sense, 5'-GGAGCTCTCG CCCCTAAGTT AGGCGCCACG CCCCCT-3'; antisense, 5'-AGGGGGCGTG GCGCCTAACT TAGGGGCGAG AGCTCC-3'; 4) mutation 4 (-208/+47 {delta} -56/-47): sense, 5'-CAGCCTGG CTTATAACAT AAAACTCTAT CTCCTCC-3'; antisense, 5'-GGAGGAGAT AGAGTTTTAT GTTATAAGCC AGGCTG-3'; 5) mutation 5 (-208/+47 {delta} -46/-37): sense, 5'-TAGCCACG CCCCAGAGCG AGAATCCACC CTCTCC-3'; antisense, 5'-GGAGAGGG TGGATTCTCG CTCTGGGGCG TGGCTA-3'; 6) mutation 6 (-208/+47 {delta} -36/-27): sense, 5'-CCCCCTCTAT CTCCGAACAA AGAGCCCTCC CCCTAG-3'; antisense, 5'C-TAGGGGGAG GGCTCTTTGT TCGGAGATAG AGGGGG-3'; and 7) mutation 7 (-208/+47 {delta} -26/-17): sense, 5'-CCTCCACC CTCTAAAGAA AAAGAGCCTG AGGGCG-3'; antisense, 5'-CGCCCTCA GGCTCTTTTT CTTTAGAGGG TGGAGG-3'. A diagram of the mouse sst5 mutation map is shown in Fig. 4Go. All plasmid constructs were sequenced in their entirety to ensure accuracy.



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  		Figure 4. Mouse sst5 promoter sequence and mutation map. A diagrammatic representation of the -208/+47 sst5 promoter region demonstrating the locations of each of the seven mutations and their locations within the wild-type promoter sequence. The top line illustrates the wild-type sst5 promoter sequence from - 87 to -17. The shaded boxes marked with an X demonstrate the locations of each of the seven mutations within the -208/+47 sst5 promoter. All mutations were generated using the wild-type sst5 -208/+47 construct as a template.

 
Transient transfection and luciferase assays
Five pituitary cell types were used for transfection assays: 1) dispersed primary TtT-97 thyrotropic tumor cells, 2) cultured rat pituitary-derived GH3 mammosomatotrope cells, 3) cultured mouse AtT-20 corticotrope cells, 4) cultured mouse {alpha}T3 gonadotropes, and 5) cultured {alpha}TSH thyrotrope-derived cells. The {alpha}TSH cells were originally derived from thyrotropic cells (25). They secrete the {alpha}-subunit, but have lost the ability to express the TSH ß-subunit (25). All cell types were transiently transfected in a similar manner as previously described (26) with the following modifications. All cells were cotransfected by electroporation (capacitance, 250 µF for all cells, except AtT-20 cells for which capacitance, 750 µF; 220 V using 0.4-cm gap cuvettes) using 20 µg of the respective mouse sst5 promoter-luciferase construct and 2 µg pCMV ß-galactosidase as an internal control for transfection efficiency. TtT-97 thyrotropic cells were obtained from hypothyroid LAF1 mice bearing TtT-97 thyrotropic tumors as previously outlined (26). After cell dispersion and electroporation of 5–10 x 106 thyrotropes, cells were incubated at 37 C in 4 ml DMEM supplemented with 10% FCS (HyClone Laboratories, Inc., Logan, UT), 1% HEPES (Life Technologies, Inc.-Invitrogen, Grand Island, NY), and 0.2% penicillin G (10,000 U/ml)-streptomycin (10,000 µg/ml; Life Technologies, Inc.-Invitrogen) for 24 h. An identical protocol using 2–5 x 106 cells was employed for all cultured pituitary cells. All cells were harvested, subjected to freeze-thaw extraction, and assayed for luciferase and ß-galactosidase activities as previously described (26, 27). Light units were normalized to ß-galactosidase activity and were corrected to the activity of a Rous sarcoma virus-luciferase construct transfected in parallel as an interexperimental control. For all figures, except Fig. 1Go, the data were expressed as the percent activity of the full-length -290/+47 sst5 promoter or the wild-type -208/+47 sst5 promoter. Figure 1Go displays the data as the fold induction over the promoterless pA3luciferase construct to allow comparisons to be made between different cell types. Experiments were performed a minimum of six times in duplicate in the cultured pituitary cells and three times in the dispersed thyrotropic tumor cells.



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  		Figure 1. Comparison of -290/+47 sst5 promoter activity in pituitary cell lines. The -290/+47 construct in a luciferase reporter was transfected into the cells by electroporation. Promoter activity is represented as fold ± SEM over promoterless vector (pA3Luc) alone. All cells were transfected with 20 µg of the mouse sst5 promoter-luciferase construct and 2 µg cytomegalovirus (CMV) promoter ß-galactosidase plasmid. All light unit activity has been normalized to CMV ß-galactosidase activity as an internal control for transfection efficiency and corrected to the activity of a Rous sarcoma virus-luciferase construct transfected in parallel as an interexperimental control. All subsequent transfection data presented in this paper were normalized in an identical manner, and cells were transfected with identical amounts of sst5 and CMV ß-galactosidase plasmids.

 
DNA-protein interactions
Protein-DNA interactions were assessed using a deoxyribonuclease I (DNase I) protection assay as previously described (28). A number of modifications to our published protocol have been made and are summarized below. The sst5 -208/+47 promoter fragment was subcloned into pGEM7zf+ (Promega Corp., Madison, WI) and then reexcised using EcoRI and MluI restriction enzymes to allow for differential end radiolabeling. After gel purification, the fragment was radiolabeled with {alpha}-32P-labeled nucleotides: [{alpha}-32P]dATP and [{alpha}-32P]dTTP (EcoRI end-antisense strand) or [{alpha}-32P]dGTP and [{alpha}-32P]dCTP (MluI end-sense strand), purified, and incubated with TtT-97 and GH3 cell nuclear extracts or BSA as a negative control. The DNase I protection assays were performed in 50 µl using 20,000–40,000 cpm of the probe, 125 mM KCl, and 400 ng sheared salmon sperm DNA. The protein amounts used were 20 µg BSA, 50 µg TtT-97 nuclear extract, and 45 µg GH3 nuclear extract. The protein extracts were incubated with the nonspecific DNA on ice for 15 min. This reaction was incubated with the radiolabeled probe for 20 min at 15 C to allow protein-DNA interactions and was followed by DNase I (Cooper Biomedical, Malvern, PA) digestion at 5 ng for BSA, 750 ng for TtT-97, and 250–400 ng for GH3 extracts for 60 sec at 15 C. The reactions were stopped by adding 10 µl DNase I stop buffer [0.5% SDS, 0.2 M NaOAc (pH 5), and 30 mM EDTA], and 100 µg/ml mussel glycogen (Sigma-Aldrich Corp., St. Louis, MO). Reaction products were separated on a denaturing gel and visualized by autoradiography (28) to identify the areas of DNA-protein interactions. An identical protocol was employed to generate mutant sst5 promoter probes for mutations 3 and 4.

Statistics
All results were analyzed using a repeated measures one-way ANOVA and post hoc Tukey paired comparisons. Transfection data were log-transformed due to nonnormality. Significance was defined as P < 0.05. The main significant results are indicated in the figures by asterisks.

Animal care
All animals were treated in a humane manner in accordance with the NIH Guide for the Care and Use of Laboratory Animals and under protocols approved by the committee on animal care and use of University of Colorado Health Sciences Center.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mapping of murine sst5 promoter activity in pituitary cells
We have previously shown that a high level (600- to 800-fold over promoterless vector) of sst5 promoter activity maps to a region -290 bp upstream of the transcription start site in GH3 and TtT-97 cells (23). An additional 6100 bp upstream of this area did not enhance promoter activity in these cells. In the current study we examined promoter activity of the -290/+47 fragment in a total of five different pituitary cell types. Although somewhat variable in absolute magnitude of response, the sst5 promoter was highly active in all cell types assessed compared with a promoterless pA3Luc vector (Fig. 1Go). The TtT-97, GH3, and AtT-20 cells have the highest promoter activity, ranging from 600- to 900-fold over the promoterless vector. Promoter activity is somewhat lower in {alpha}TSH and {alpha}T3 cells, ranging from 100- to 300-fold over the promoterless vector.

To define promoter sequences critical for basal sst5 gene transcriptional activity, a series of 5'-deletions of the murine sst5 promoter was generated as described in Materials and Methods. Figure 2Go represents a schematic diagram of the structure of the sst5 gene and the location of each of the 5'-deletions. Transient transfection experiments demonstrated no reduction in promoter activity relative to the initial -290/+47 promoter in the largest deletion constructs: -208/+47, -151/+47, and -83/+47 constructs (Fig. 3Go). A significant reduction (P < 0.001) in promoter activity was observed in all pituitary cell types with the -47/+47 and -19/+47 constructs. These two constructs also differed significantly from one another, with the -19/+47 construct demonstrating the lowest promoter activity. The pattern of promoter activity was similar in all pituitary cell types (Fig. 3Go). As an example, luciferase activity was reduced by 78% in the -47/+47 construct and by 98% in the -19/+47 construct compared with the -290/+47 construct in GH3 cells (Fig. 3AGo). A similar promoter activity pattern was observed in transfected primary TtT-97 thyrotropes (Fig. 3BGo) and all of the other pituitary cell types (Fig. 3Go, C–E). In GH3 (Fig. 3AGo) and {alpha}TSH cells (Fig. 3CGo), an increase in activity relative to the -290/+47 construct was noted in the -208/+47 and -151/+47 constructs, suggesting that these deletions led to removal of inhibitory factor-binding sites in these cells. Additionally, the -83/+47 fragment demonstrated significantly higher activity than the -290/+47 fragment in {alpha}TSH cells. These results demonstrate that the critical sequence for basal sst5 promoter activity in pituitary cells is located in the region between -83 and -19 relative to the transcription start site. There may also be cell-specific regions involved in mediating promoter inhibition.



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  		Figure 3. Deletion analysis of the sst5 promoter in pituitary cells. A, Promoter activity of sst5 genomic fragments in GH3 pituitary somatotropes. The fragments depicted in Fig. 2Go were fused to a luciferase reporter and transfected into the cells by electroporation. Promoter activity is shown as a percentage (±SEM) of the fully active -290/+47 promoter construct. Asterisks denote statistically significant differences (P < 0.001) using one-way ANOVA with Tukey post hoc analysis in all panels. The -208/+47 and -151/+47 constructs were significantly different from the -290/+47 fragment (P < 0.001). The smallest constructs, -47/+47 and -19/+47, were significantly (P < 0.001) different from each other and all other transfected constructs. B, Promoter activity of sst5 genomic fragments in TtT-97 thyrotropes. The constructs used and the transfection protocol were identical to those described for GH3 cells. Promoter activity is shown as a percentage (±SEM) of the fully active -208/+47 promoter construct. Again, the -47/+47 and -19/+47 constructs were significantly (P < 0.001) different from each other and all other transfected constructs. C, Promoter activity of sst5 genomic fragments in {alpha}TSH cells. The constructs used and the transfection protocol were identical to those in A. Promoter activity is shown as a percentage (±SEM) of the full-length active -290/+47 promoter construct. The activities of -208/+47, -151/+47, and -83/+47 constructs did not differ from each other, but all had significantly higher promoter activity than the -290/+47 fragment (P < 0.001). Again, the -47/+47 and -19/+47 constructs were significantly (P < 0.001) different from each other and all other transfected constructs. D, Promoter activity of sst5 genomic fragments in AtT-20 corticotrope cells. The constructs used and the transfection protocol were identical to those in A. Promoter activity is shown as a percentage (±SEM) of the full-length active -290/+47 promoter construct. Although the larger constructs did not differ from each other or from the -290/+47 construct, the -47/+47 and -19/+47 constructs were significantly (P < 0.001) different from each other and all other transfected constructs. E, Promoter activity of sst5 genomic fragments in {alpha}T3 gonadotrope cells. The constructs used and the transfection protocol were identical to those in A. Promoter activity is shown as a percentage (±SEM) of the full-length active -290/+47 promoter construct.

 
Mutational analysis of the murine sst5 promoter in pituitary cells
We undertook a mutation strategy to more precisely define the gene sequence(s) important for basal promoter activity of the sst5 gene. A series of seven 10-bp transversion mutations was generated in the context of the fully active -208/+47-pA3Luc promoter construct and is schematically shown in Fig. 4Go. The promoter activity of these mutant constructs was assessed in both GH3 and TtT-97 cells. Mutations 3 and 4 resulted in a significant reduction (P < 0.001) in promoter activity in both cell types (Fig. 5Go, A and B) compared with the wild-type construct. Mutation 3, which involved the mutation of 10 nucleotides between -67 and -57 bp relative to the transcription start site, reduced basal promoter activity by 56% in GH3 cells and by 58% in TtT-97 cells relative to the control promoter construct. Mutation 4 (transversion mutation of the sequence between -56 and -47) had the greatest effect and inhibited promoter activity by 78% and 75% in GH3 and TtT-97 cells, respectively. Finally, the construct with the mutation between -26 and -17 (mutation 7) demonstrated a lesser reduction of 33% (P < 0.001 compared with wild-type) in promoter activity in GH3 cells only, suggesting that this region may have disrupted binding of a somatotrope-restricted factor. None of the other mutations significantly altered sst5 basal promoter activity. These data indicate that sequences between -67 and -47 as well as -26 to -17 are important for basal activity of the murine sst5 promoter in pituitary cells.



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  		Figure 5. Mutational analysis of the sst5 gene promoter in GH3 and TtT-97 pituitary cells. A, Promoter activity of wild-type and mutant sst5 -208/+47 constructs in GH3 cells. Light unit activity is normalized as described in Materials and Methods and in Fig. 2Go. All data are expressed as a percentage of the wild-type -208/+47 construct. Asterisks denote statistically significant differences (P < 0.001) using one-way ANOVA with Tukey post hoc analysis. Promoter activity was significantly (P < 0.001) reduced in mutations 3, 4, and 7 compared with the wild-type construct. B, Promoter activity of mutant sst5 -208/+47 constructs in TtT-97 cells. All data are expressed as a percentage of the wild-type -208/+47 construct. Promoter activity was significantly (P < 0.001) reduced in mutations 3 and 4 compared with the wild-type construct.

 
Analysis of DNA-protein interactions
DNA-protein interactions were investigated using the technique of DNase I protection analysis. A wild-type fragment spanning -208 to +47 as well as the same region containing mutations 3 and 4 were employed in the analysis. Using the sense strand radiolabeled sst5 -208/+47 promoter region as a probe, three protected regions were identified using TtT-97 thyrotrope and GH3 pituitary cell nuclear extracts (Fig. 6Go). Only one protected region was localized to the area identified as important for basal promoter activity. This region between -61 and -41 corresponded to the area shown in the 5'-deletion studies to be functionally important. Moreover, this region spans the area of mutations 3 and 4 and confirms this as a critical site for protein-DNA interactions on the sst5 promoter. Although not located in the area determined by functional analysis to be critical for basal promoter activity, two other more distal protected regions were identified. These areas were located between -206 and -195 and -186 and -170 relative to the transcription start site. These regions may indicate important sites for protein-DNA interactions involved in mediating cell-specific sst5 promoter inhibition, as deletions of the region between -290 and -151 were associated with enhanced promoter activity in GH3 and {alpha}TSH cells. Additionally, two protected regions were localized when the antisense labeled wild-type sst5 -208/+47 construct was incubated with both TtT-97 and GH3 nuclear extracts. One protected area seen in both cell types between -55 and -44 corresponded to that seen with the sense labeled probe and confirmed the importance of the region spanned by mutation 4 in DNA-protein interactions. A second protected region was identified between -25 and -3 in GH3 cells. This sequence overlapped with the sequence of mutation 7, which was shown in the earlier experiment to play a modest role in basal sst5 promoter activity in GH3 pituitary cells. These data suggest that this region is important for binding of a somatotropic transcription factor.



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  		Figure 6. DNase I protection analysis of wild-type sst5 promoter. DNase I protection analysis of wild-type sst5 promoter (-208/+47) single-end 32P-radiolabeled sense and antisense strands incubated with 50 µg TtT-97 thyrotrope or 45 µg GH3 somatotrope nuclear extracts. BSA (20 µg) was included as a control. The locations of protected regions are represented adjacent to the autoradiography. The contents of each lane are designated at the top of each autoradiograph. DNase I protected regions are depicted as black boxes to the right of the autoradiographs, and their locations are shown by negative numbers relative to the sst5 transcription start site.

 
Sequence analysis of the protected regions revealed potential binding sites for several transcription factors. The region between -61 and -41 that corresponded to the functionally important region identified by mutational analysis contains recognition sites for the transcription factors PitX1 (Ptx1) and GATA factors, which have been identified in thyrotropes (29, 30). Additionally, this region contains a GC-rich area that is very similar to the consensus recognition site for the transcription factor Egr-1 (also known as NGFI-A, Krox-24, and Zif 268) that has been shown to be important for gonadotrope function and LHß gene transcription. (31, 32, 33). The footprint region between -25 and -3 contains a putative recognition sequence for the ubiquitous transcription factor Sp1. Sp1 was the first factor identified of a family of transcription factors (34) and has been shown to be an important regulator of multiple genes, including the pituitary genes for GH (35, 36, 37) and LH ß-subunit (38). Additionally, a Sp1 site has recently been shown to be functionally important in the regulation of the rat sst2 promoter (39).

Repetition of the DNase I protection analysis was performed using sense strand radiolabeled sst5 -208/+47 promoter constructs containing either the 10-bp transversion of mutation 3 or mutation 4. When the radiolabeled sense strand containing mutation 3 was used as a probe with TtT-97 and GH3 nuclear extracts, the same footprint region was present, but was reduced in size by approximately five bases at the 5'-end. The protected region with the mutation 3 probe spanned -56 to -41 (Fig. 7Go). It should be noted that mutation 3 contained base transversions in the region between -67 and -57. Therefore, binding of protein from GH3 and TtT-97 nuclear extracts was prevented in the region of mutation 3 only starting at base -57. The same regions of protection were no longer present when the experiment was performed using the sense strand radiolabeled sst5 -208/+47 promoter constructs containing mutation 4 (Fig. 7Go). These results indicated that the region between -61 and -41 is involved in basal promoter activity, presumably by DNA interactions with protein(s) involved in transcriptional regulation of the sst5 gene.



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  		Figure 7. DNase I protection analysis of the mutant sst5 promoter. DNase I protection analysis of the sst5 promoter (-208/+47) radiolabeled sense strand containing either mutation 3 or mutation 4 incubated with TtT-97 thyrotrope and GH3 somatotrope nuclear extracts. The locations of protected regions are represented as shaded boxes adjacent to the autoradiography. The wild-type sequence of the protected region in the sst5 promoter is noted for the mutation 3 probe. The contents of each lane are designated above the autoradiograph. The amounts of BSA and TtT-97 or GH3 nuclear protein extract were identical to those used for the reactions in Fig. 6Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously demonstrated that the -290/+47 region mediates sst5 promoter activity. This area of the promoter is active in all five pituitary cell types assayed. In the present study deletion analysis identified the critical sequences of the promoter to lie between positions -83 and -19. Finer mapping of basal sst5 promoter activity was undertaken using mutational analysis and demonstrated the importance of two regions: -67 to -47 and -26 to -17. Mutations in the region between -67 and -47 resulted in a reduction of sst5 promoter activity in transient transfection assays in both pituitary cell types. GH3 cells, but not thyrotropes, demonstrated alterations in basal promoter activity with mutations in the region between positions -26 and -17, suggesting that this area may be involved in cell-specific sst5 gene regulation. However, it appears that the region between -67 and -47 is critical for basal sst5 promoter activity in both thyrotropes and somatolactotropes. Protein-DNA interactions occur in both of these important regions, and this lends support to the conclusion that these regions are important for sst5 basal promoter activity because they allow the binding of a protein(s) critical for regulation of sst5 gene transcription.

Currently, limited knowledge exists regarding the regulation of any of the somatostatin receptor genes. Initially, a number of laboratories examined the sequence of somatostatin receptor genes immediately upstream of the translation start site (40, 41, 42, 43). Putative transcription factor-binding sites and hormonal response elements were identified by similarity to consensus recognition sequences. Later studies identified upstream gene transcription start sites and the presence of introns in the 5'-untranslated region. All of the somatostatin receptor genes were initially thought to be intronless, and now it has been shown that several mouse (23, 44, 45) and rat (39) genes have introns. We have previously demonstrated the existence of three exons and two introns in the mouse sst5 gene as well as alternative exon splicing in TtT-97 thyrotropes (23). Additionally, similar to the mouse sst5 receptor gene, the mouse sst2 receptor gene has been shown to have three exons and two introns. This gene has also been shown to have three separate promoters, one upstream of each exon, that differentially regulate sst2 gene expression in a tissue-specific manner (45). The rat sst2 gene has also been shown to have two exons and multiple transcription start sites (39).

More recent studies have characterized the gene sequence involved in basal promoter activity. For example, several studies have examined transcriptional regulation of the rat sst1 promoter (46, 47). Baumeister et al. (46) studied the rat sst1 promoter in both pituitary and pancreatic cell lines. Deletion analysis of the rat sst1 promoter mapped basal promoter activity to a region 324 bp upstream of the transcription start site. Deletions proximal to this area reduced rat sst1 promoter activity (46). Interestingly, this fully active promoter region is approximately the same distance from the transcription start site of the basal promoter we have identified for the sst5 gene, suggesting similarities in promoter structure. These investigators also identified a region between -1985 and -324 that appears to contain negative regulatory elements (46). This does not correlate to the location of the potential regions we have identified in the sst5 promoter that may contain sequence important for cell-specific inhibitory elements. Additionally, they identified two regions in the rat sst1 promoter that are differentially regulated by the pituitary-specific transcription factor, Pit-1 (46). In these studies two functional binding sites for Pit-1 were identified with differential functions. The proximal site mediated promoter activation, and the distal site mediated promoter repression in GH3 pituitary cells (46). In contrast, the functionally important region from -83 to -19 described here for the mouse sst5 promoter did not contain a consensus binding sequence for Pit1, which is known to be expressed in both GH3 and TtT-97 cells. These same investigators studied the rat sst1 promoter in a pancreatic insulinoma cell line and mapped basal promoter activity (47). They also showed that a different POU domain transcription factor, known as Tst-1, activated the rat sst1 promoter in pancreatic ß-cells, demonstrating cell-specific activation of the sst1 receptor.

Data are also accumulating regarding promoter regulation of the sst2 receptor. The transcription start site of the human sst2 promoter has been identified, and basal promoter activity characterized by deletion analysis in pituitary cells (15). A 252-bp sequence defined the basal promoter that permitted cell-specific sst2 gene expression. Again, the region approximately 300 bp upstream of the transcription start site appeared to contain elements critical for basal promoter activity. Dorflinger et al. (48) also examined the transcriptional regulation of the human sst2 gene. This group has found that two transcription factors are involved in regulation of sst2 promoter activity: SEF-2, a basic helix-loop-helix factor, and MIBP1 (c-myc intron binding protein), a zinc finger protein. Regulation of the mouse and rat sst2 genes has also been examined. In contrast to what we have previously shown for the mouse sst5 receptor in pituitary cells, the mouse sst2 uses three tissue- and cell-specific promoters: one upstream of exon 1, a second upstream of exon 2, and the third within exon 3 (45). Further studies in AtT-20 and human pancreatic cells have demonstrated that a region 325 bp in front of the ATG translation site in exon 3 confers maximal promoter activity and contains a region that is activated by TGFß (49). Analysis of rat sst2 receptor promoter activity demonstrated highest activity in a region 77 bp upstream of the transcription start site and noted the importance of Sp1 and cAMP response element sites in promoter regulation (39). This group has also shown activation of the sst2 promoter by PitX1 (Ptx1) (39). Our data support the involvement of these transcription factors in the regulation of somatostatin receptor promoter activity, in that we have also demonstrated the presence of Sp1 and PitX1 consensus sequences in regions critical for basal sst5 promoter activity. The sst5 promoter region containing the putative Sp1 site appears to play a more important role in GH3 cells due to the fact that the mutation spanning this area reduced promoter activity in GH3, but not TtT-97, cells. Additionally, a protected region corresponding to this area was clearly noted only in GH3 cells. Interestingly, our data also demonstrated the presence of a sequence resembling the consensus sequence for the transcription factor Egr-1 in this same critical region. Although Egr-1 has not previously been shown to be involved in regulation of any of the sst receptor promoters, Egr-1 has been found in pituitary cells and appears to regulate LHß promoter activity (31, 33). Mutations encompassing both of these identified sites result in alterations in basal sst5 promoter activity in the current study. Future studies will be undertaken to investigate the roles of these pituitary transcription factors in the regulation of the sst5 gene.

In summary, our results identify a sequence of the mouse sst5 promoter that is important for functional activity. Similar to the sst1 and sst2 genes, the region approximately 300 bp upstream of the transcription start site confers basal promoter activity. We have further defined the most critical regions of this promoter by deletion and mutational analyses. Sequence analysis of these critical regions revealed nucleotide homology to binding sites for the known transcription factors Ptx1, GATA, Egr-1, and Sp1. Additionally, these areas appear to be important sites for protein-DNA interactions, as assessed by DNase I protection analysis. Further studies are needed to identify the roles of these transcription factors or others in the regulation of the mouse sst5 gene promoter.


   Acknowledgments
 


   Footnotes
 
This work was supported by NIH Grant K08-DK-02813-02 and an Endocrine Fellowship grant from the American Thyroid Association (to W.W.W.) as well as NIH Grant RO1-CA-47411 (to E.C.R.).

Abbreviations: sst5, Somatostatin receptor type 5 gene.

Received October 25, 2001.

Accepted for publication February 7, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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