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Molecular Endocrinology 18 (2): 471-483
Copyright © 2004 by The Endocrine Society

CpG Methylation and Transcription Factors Sp1 and Sp3 Regulate the Expression of the Human Secretin Receptor Gene

Ronald Ting-Kai Pang, Leo Tsz-On Lee, Samuel Sai-Ming Ng, Wing-Ho Yung and Billy Kwok-Chong Chow

Department of Zoology (R.T.-K.P., L.T.-O.L., S.S.-M.N., B.K.-C.C.), University of Hong Kong, People’s Republic of China; and Department of Physiology (W.-H.Y.), The Chinese University of Hong Kong, People’s Republic of China

Address all correspondence and requests for reprints to: Billy Kwok-Chong Chow, Department of Zoology, University of Hong Kong, Special Administrate Region, People’s Republic of China. E-mail: bkcc{at}hkusua.hku.hk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human secretin receptor (hSR) is an important glycoprotein receptor for regulating the secretion of pancreatic bicarbonate, water, and electrolytes. In this study we investigated the transcriptional regulation of the hSR gene. A minimal 106-bp promoter was identified, and it contains two GC boxes (GC box-A, -240 to -226; and GC box-B, -203 to -194, from the translation start site). EMSA and supershift analyses showed that both GC boxes interact with Sp1 and Sp3 transcription factors. Transient transfection in pancreas-derived human pancreatic ductule carcinoma (PANC)-1 and bovine pancreatic duct-1 cells showed that mutation of either GC box-A or -B reduced the promoter strength by 56–67%, whereas mutation of both GC boxes caused more than 90% reduction of promoter activity. Cotransfections of the hSR promoter with Sp1 and Sp3 expression vectors in Sp-deficient Drosophila SL-2 Schneider cells further demonstrated that the ratio of Sp1 to Sp3 is the key mechanism to modulate hSR gene expression. The methylation statuses of 27 CpG sites within the promoter region (-400 to -151 bp) were assessed in various human pancreas and liver cell lines. The hSR promoter is unmethylated (CAPAN-1, human pancreatic adenocarcinoma) or partially methylated (PANC-1 and HPAC, human pancreatic adenocarcinoma) in hSR-expressing cell lines but is completely methylated in hSR nonexpressing HepG2 cells. Methyltransferase inhibitor 5-aza-2'deoxycytidine increased hSR gene expression level in PANC-1 cells and induced hSR gene expression in HepG2 cells. Together, our study shows that, in addition to Sp1 and Sp3, promoter methylation also plays a role in the regulation of hSR gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PRIMARY FUNCTION of secretin in the gastrointestinal system is to stimulate bicarbonate, electrolytes, and volume release from the pancreatic ductule cells. Apart from the pancreas, secretin has numerous physiological and pharmacological effects in our body with effector tissues such as the stomach, intestine, heart (1), and kidney (2). The discovery of the potential use of secretin as a drug for treating autistic patients (3), together with several conflicting reports (4, 5, 6), has raised a lot of research interest in the function of secretin as a neuropeptide. Recently, our laboratory has shown the expression of secretin and secretin receptor in distinct neuronal populations; and secretin has a direct neuroactive function in the rat cerebellum (7, 8). These data indicate that secretin, in addition to being a classical gastrointestinal hormone, is also a neuromodulator in the central nervous system.

Secretin elicits its biological effects by interacting with specific cell surface receptors that exhibit high affinity for secretin and low affinity for vasoactive intestinal polypeptide. The human secretin receptor (hSR) cDNA was cloned (9, 10, 11) and the gene was mapped to 2q14.1 by fluorescence in situ hybridization (12). The hSR gene consists of 13 exons and 12 introns, and it spans more than 65 kb of genomic sequences (13). In an initial study, a 3-kb fragment containing the 5'-flanking region of the hSR gene was sequenced and functionally tested by transiently transfecting various promoter-reporter gene constructs into several cell lines. A promoter element (-408 to -158, relative to the ATG start codon) was identified that supports reporter gene expression in pancreatic ductule-derived cells, human pancreatic ductule carcinoma (PANC)-1 and bovine pancreatic duct (BPD)-1, but not in stomach-derived cells, Hs 262.St and Hs 746 St, or pituitary gonadotrope-derived cells, {alpha}T3–1 (13). This promoter fragment does not contain putative TATA and CCAAT boxes. In fact, typical TATA and CCAAT boxes are also absent in other G protein-coupled receptor genes, including the human {alpha}1b-adrenergic receptor (14), the mouse GnRH receptor (15), the rat VPAC-1 receptor (16), the human glucagon receptor (17), the human glucagon-like peptide 1 receptor (18), and the human GHRH receptor (19) genes.

Our initial study provided some information with respect to the general properties of the hSR core promoter, but the transcriptional regulation of the hSR gene is still poorly understood. Within the promoter, there are two GC boxes that are putative binding sites for the Sp family of transcription factors (20). In addition to its role on transcription activation, Sp1 appears to play a critical role for the maintenance of the hypomethylation status of CpG islands (21, 22). The genes encoding leukosialin (CD43) (23, 24), cyclin D1 (25), and lung epithelial T1{alpha} (26) are silenced by methylation of the critical Sp1 sites within the promoter region. It is possible that methylation of the Sp1 binding sites is a relatively common mechanism that regulates gene expression.

Pancreatic cancer has the poorest likelihood of survival among all of the major malignancies. There were findings suggesting that secretin receptor was transcriptionally down-regulated during pancreatic neoplastic development (27, 28). The understanding of the regulation of hSR gene expression should provide insights into the molecular mechanisms of pancreatic cancer. In this study, we investigated the interactions of GC motifs within the core promoter region of the hSR gene with Sp1 and Sp3. In addition, the connections between the Sp proteins, cytosine methylation, and methylation imprinting were also studied to elucidate the epigenetic regulation of expression in the hSR gene.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Functional Analysis of the hSR Core Promoter Element -263 to -158
The hSR gene with its promoter was previously cloned (13) and was mapped to chromosome 2q14.1 (12). The promoter is cell specific, and it can drive reporter gene expression in pancreas-derived cells but not in stomach- or pituitary-derived cells (13). The promoter fragment, -408 to -158 relative to the translation start site, is responsible for 12.6 ± 1.2-fold of promoter activity. By progressive deletion coupled to transient promoter assays (data not shown), we have identified that the region -263 to -158, a 106-bp fragment, functions as a core promoter element (Fig. 1Go: PANC-1 12.2 and BPD-1 15.8 fold, respectively). This promoter fragment is functionally bidirectional as it can drive reporter gene expression in both forward and reverse orientations in PANC-1 and BPD-1 cells (Fig. 1Go).



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  		Fig. 1. Functional Properties of the hSR Promoter Fragment (-263 to -158) in PANC-1 and BPD-1 Cells

The hSR promoter fragment was fused with the luciferase reporter gene in the pGL-2 basic vector in both forward (p263/158luc) and reverse (p263/158R luc) orientations. Luciferase activities are normalized by ß-galactosidase expression and are shown as the fold increases in relative promoter activities compared with that in the control (pGL-2 basic). Values reported in the figure represent the mean ± SEM of three independent experiments, each in triplicate. Bars bearing an asterisk are statistically different from the control (P < 0.05).

 
To identify the cis-acting motifs within the core promoter, 8-bp scanning mutations were introduced (Fig. 2Go). Significant reductions in luciferase activities were observed in some of the mutants, and the largest reductions were observed in M3 (GC box-A, -240 to -226 from the ATG start codon) and M6 (GC box-B, -203 to -194). Mutation of GC box-A led to 58% and 56% reduction of promoter activities in PANC-1 and BPD-1 cells, respectively. Similarly, mutation of GC box-B resulted in 64% and 67% loss in promoter strength in these cells. The trends in the increase/decrease of promoter activities of most of the mutants in these cells are similar except in M5. A larger reduction in reporter gene activity was observed in PANC-1 cells than in BPD-1 cells (55% in PANC-1 and 40% in BPD-1), and the significance of this observation is unclear. To confirm the functional importance of the GC boxes, we performed fine mapping of the M6 region by introducing 3-bp mutation across the M6 region (Fig. 3Go, mutants M6-1 to M6-4). We observed a dramatic loss (<=85%) of activity particularly when the 5'-region of the GC box was mutated (M6-1 and M6-2). A major difference between the two cell types is observed in M6-3: we found 20% and 60% loss in PANC-1 and BPD-1 cells, respectively. Again, the importance of this observation is unclear; it is possible that there are transcription factors acting cooperatively with Sp1 to compensate for the loss of promoter activity in PANC-1 cells. In summary, our data clearly show the functional importance of GC box-B. Figure 4Go shows the combined effects of various mutations within the promoter, double mutants with M6 and M1 to M9 were constructed and were functionally tested. Mutations of both GC boxes (M3 and M6) resulted in an almost total loss of function, i.e. less than 10% of promoter strength remained when compared with the wild-type construct. These results therefore strongly indicate that GC box A and B together are playing a pivotal role in determining the transcriptional activities of the characterized hSR promoter.



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  		Fig. 2. Scanning Mutation Studies of the hSR Gene Promoter in PANC-1 and BPD-1 Cells

A family of 8-bp block replacement mutants were constructed and used for transient promoter assays. The upper panel is the DNA sequence of the promoter, and the underlined sequences are the sites of the NotI scanning mutations (M1–M9). Luciferase activities are normalized by ß-galactosidase expression and are shown as the fold increases in relative promoter activities compared with that in the control (pGL-2 basic). Values reported in the figure represent the mean ± SEM of three independent experiments, each in triplicate. Asterisks indicate significant difference (P < 0.05) from the p263/158 luc.

 


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  		Fig. 3. Scanning Mutation Studies of the GC Box-B Region in PANC-1 and BPD-1 Cells

A family of 3-bp block replacement mutants were constructed and used for transient promoter assays. The upper panel is the DNA sequence of the promoter, and the underlined sequences are the putative GC box-B and the scanning mutants (M6-1 to M6-4). Luciferase activities are normalized by ß-galactosidase expression and are shown as the fold increases in relative promoter activities compared with that in the control (pGL-2 basic). Values reported in the figure represent the mean ± SEM of three independent experiments, each performed in triplicate. Asterisks indicate significant difference (P < 0.05) from the control (p263/158 luc).

 


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  		Fig. 4. Combined Effects of the M6 with M1–M9 Double Mutations in PANC-1 and BPD-1 Cells

A family of double mutants with M6 and M1–M9 8-bp block replacement mutants (DM1–6 to DM 5–6 and DM 6–7 to DM 6–9) was constructed and used for transient promoter assays. Luciferase activities are normalized by ß-galactosidase expression and are shown as the fold increases in relative promoter activities compared with that in the control (pGL2-basic). Values reported in the figure represent the mean ± SEM of three independent experiments, each in triplicate. Asterisks indicate statistical difference (P < 0.05) from the control (p263/158 luc).

 
Identification of the Transcription Factors Interacting with the hSR Promoter Element
To initially characterize the transcription factors that bind to the two functional GC boxes, GC box A (-244 to -224, Table 1Go) and GC box B (-212 to -191, Table 1Go) were used as probes in EMSAs. Figure 5Go shows the results of the EMSA, when GC box probe B was used, three DNA-protein complexes (labeled as I, II, and III) were revealed. These complexes could be eliminated only when competitions were carried out using 100-fold molar excess of the wild-type unlabeled oligonucleotide or consensus Sp1 probe but not with nonspecific oligonucleotides. Mithramycin is a well known GC-specific DNA binding antibiotic that inhibits the initiation of RNA synthesis (29, 30). It can inhibit the binding of transcription factors to GC-rich sequences by interacting with the GC-rich region itself. Figure 5CGo shows the results when various mithramycin concentrations (1, 10, and 100 nM) were added to the EMSA reactions containing the GC box probe B and PANC-1 (Fig. 5CGo, lanes 2–4) or BPD-1 (lanes 5–7) nuclear extract. The intensities of the DNA-protein complexes decreased with increasing concentrations of mithramycin. These observations indicate that the interaction of the proteins with GC box-B is specific. As a control to demonstrate the specificity of mithramycin to GC-rich promoters, pRC-CMV with the cytomegalovirus (CMV) promoter driving the reporter gene was transfected into PANC-1 cells cultured in medium with or without 300 nM mithramycin. We found that drug treatment has no significant effect on the strength of a non-GC-rich promoter activities (data not shown).


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  		Table 1. A List of PCR Primers and Gelshift Oligonucleotides

 


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  		Fig. 5. Formation of DNA-Protein Complexes Using GC box-B as a Probe and 10 µg Nuclear Extract Prepared from (A) PANC-1 and (B) BPD-1 Cells

Competitive gel shift assay was done in the presence of 100-fold molar excess of unlabeled double-stranded oligonucleotides corresponding to the wild-type (WT), nonspecific (NS), and Sp1 consensus motif (Sp1). The arrows (I, II, and III) indicate specific binding, whereas the asterisks represent nonspecific binding. C, Inhibition of binding with increasing concentrations of mithramycin (1, 10, and 100 nM) using PANC-1 nuclear extract (lanes 2–4) or BPD-1 nuclear extract (lanes 5–7).

 
To identify the proteins in DNA-protein complexes, supershift assays using antibodies specific for Sp1 and Sp3 were performed (Fig. 6Go). Addition of anti-Sp1 antibody shifted complex I, indicating that Sp1 is responsible for forming this complex (Fig. 6AGo). Formation of complexes II and III was specifically inhibited when an anti-Sp3 antibody was included in the reactions (Fig. 6Go), showing that complexes II and III were formed by binding with various forms of the Sp3 protein. When both anti-Sp1 and anti-Sp3 antibodies were included in a single reaction, complex I was supershifted, and simultaneously the intensities of complexes II and III were much reduced (Fig. 6Go, lane 4 of panel A and B). Similar gel-shift and supershift data were observed in a parallel study using GC box-A as a probe (data not shown) and also in previous studies (31, 32, 33).



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  		Fig. 6. Identification of Proteins in Complexes I, II, and III by Supershift Assays

Antibodies (2 µg) for Sp1 and/or Sp3 were preincubated with the (A) PANC-1 and (B) BPD-1 nuclear extract before the addition of the GC box-B probe. BSA (2 µg) was used as a control. The supershifted Sp1 bands were labeled with open arrows.

 
Functional Analysis of the hSR Promoter in Drosophila SL-2 Cells
Sp1 and related transcription factors are virtually expressed in all mammalian cells, and these endogenous factors seriously hampered our ability to interpret the data from Sp1 and Sp3 cotransfection studies. For this reason, we have used an Sp-protein-deficient cell line, Drosophila SL-2 (34), in our study (Fig. 7Go). The expression constructs for Sp1 or Sp3 (pPacSp1 and pPacSp3) (35, 36), together with the wild-type or mutant promoter (-263 to -158), were cotransfected into SL-2 cells. The pPacO vector containing the Drosophila actin promoter that would not be affected by the expression of mammalian Sp1 or Sp3 (35) was used as a control. The expression of Sp1 and Sp3 in the transfected SL-2 cells were confirmed by Northern blot analysis; transcripts corresponding to the sizes of Sp1 and Sp3 were detected (data not shown). In the presence of Sp3, the promoter (-263 to -158) alone was unable to activate reporter gene expression in SL-2 cells. On the other hand, Sp1 enhanced hSR promoter activity in a dose-dependent manner (18- to 31-fold) (Fig. 7AGo). As Sp1 and Sp3 are functionally different and are competing for the same binding sites, we investigated the potential function of Sp3 as a repressor of the hSR promoter. Various amounts (0–4 µg) of pPacSp1 and pPacSp3 vectors were introduced with the hSR promoter construct into SL-2 cells. We found that the promoter activities decreased in a dose-dependent manner from 18-fold to 5.5-fold with increasing amounts of pPacSp3 (Fig. 7BGo). When we cotransfected the promoter construct with 1 µg of pPacSp3 and increasing amounts of pPacSp1 (0–4 µg), the reporter gene activities increased in a dose-dependent manner (Fig. 7BGo). These data clearly indicate that Sp1 and Sp3 are competing for the same GC boxes in the hSR promoter; Sp1 activates transcription whereas Sp3 inhibits transcription by competing with Sp1 for the same binding sites.



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  		Fig. 7. Effects of Overexpressing Sp1 and Sp3 on hSR Promoter (-263 to -158) in Sp-Deficient SL-2 Cells

A, p263/158luc was cotransfected with various amounts of Sp1 (pPacSp1) or Sp3 (pPacSp3) expression vectors. pPacO was used as a control. B, Various ratios of Sp1/Sp3 expression vectors were used to cotransfect with p263/158luc into SL-2 cells. The amount of plasmid DNA used in each transfection study is indicated in Table 1Go. Luciferase activities are normalized by protein concentrations and are shown as the fold increases in relative promoter activities compared with that in the control (pGL2-basic). Values reported in the figure represent the mean ± SEM of three independent experiments, each in triplicate. Asterisks indicate significant differenence (P < 0.05) from control.

 
To determine the functions of the GC boxes and their relationships with the Sp-proteins, the hSR core promoter, the single mutants M3 and M6, and the double mutant DM3–6 were cotransfected with the Sp vectors (Fig. 8AGo). When the wild type or the mutants were cotransfected with pPacSp3, there were no significant increases in reporter gene activities. These results confirm that Sp3 alone cannot drive expression of the core promoter (Fig. 8AGo). Cotransfection of pPacSp1 with the wild-type promoter increased the reporter gene activity by 18-fold whereas mutation of either GC box-A (M3) or -B (M6) only led to 10.6-fold or 8.8-fold increase in reporter gene activities, respectively (Fig. 8AGo). Similar to the mammalian expression system, the double mutant (DM3–6) showed a minimal reporter gene activity. In the presence of both Sp1 and Sp3, the promoter activities were always lowered as compared with the presence of Sp1 alone, again indicating the repressor activity of Sp3.



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  		Fig. 8. Effects of Overexpressing Sp1 and Sp3 on M3, M6, and DM3–6 Mutants in the Sp-Deficient SL-2 Cells

The mutant promoter constructs were cotransfected with pPacSp1 (A), pPacSp3 (B), or both pPacSp1 and pPacSp3 (C). D, The wild-type promoter construct was cotransfected with the control expression vector as a control. Luciferase activities are normalized by protein concentrations and are shown as the fold increases in relative promoter activities compared with that in the control (pGL2-basic). B, Effect of overexpression of anti-sense Sp3. PANC-1 cells were transfected with p263–153 luc and an increasing concentration of pCDNA anti-Sp3 vector. Promoter activities are expressed as % of the control (p263–153 luc). Values reported in the figure represent the mean ± SEM of three independent experiments, each performed in triplicate. Bars bearing an asterisk are statistically different from the control (P < 0.05). C, Western blot analysis to show the reductions in the levels of intracellular Sp-3 protein with increasing concentrations of pCDNA anti-Sp3 vector transfected into the PANC-1 cell.

 
To test whether the levels of endogenous expressions of Sp1 and Sp3 in PANC-1 will change the promoter activity, we have used an antisense approach. In this experiment, an anti-Sp3 overexpression vector was cotransfected with the promoter into PANC-1 cells (Fig. 8BGo). Consistently, our results demonstrated that the anti-Sp3 vector increased promoter activities in a dose-dependent manner. This is likely the result of reductions in intracellular Sp3 protein levels as confirmed by Western blot analysis (Fig. 8CGo).

DNA Methylation Patterns of the hSR Promoter in hSR-Expressing and hSR-Nonexpressing Cells
Computer analysis using the CpGPlot program (European Bioinformatics Institute, Cambridge, UK) revealed that the hSR gene 5'-flanking region contains a putative CG island (-400 to -153, relative to the ATG codon) (data not shown). Within this region, there are 27 CpG dinucleotide sequences, and five of them are present within GC box-A and GC box-B. By methylation-specific PCR (for primer sequences; see Table 1Go) coupled to DNA sequence analysis, the methylation status of individual CpG site was examined. Bisulfite modification of DNA changes all the unmethylated cytosines to uracils, and the uracils are amplified as thymidines in PCR. On the other hand, the methylated cytosines will not be modified and hence will remain as cytosines in subsequent amplification in PCR. The methylation statuses of the hSR promoter in hSR-expressing [PANC-1, human pancreatic adenocarcinoma cell (CAPAN-1), and human pancreatic adenocarcinoma (HPAC)] and hSR-nonexpressing cells (HepG2) were compared (Fig. 9Go).



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  		Fig. 9. Methylation-Specific PCR

A, Using bisulfite-modified genomic DNA prepared from various cells and the primer set U, positive amplification signals were obtained from CAPAN-1, PANC-1, and HPAC cells. Primer set U is designed to amplify unmethylated DNA (see Materials and Methods). B, Positive amplification signal was obtained from modified genomic DNA prepared from HepG2 cells using the primer set M. These primers are designed to amplify CpG-methylated DNA.

 
Positive signals were obtained by amplification of the modified DNA prepared from CAPAN1, PANC-1, and HPAC cells using the primer set U (designed to amplify unmethylated sequence; Table 1Go) and HepG2 cells using the primer set M (designed to amplify methylated sequence; Table 1Go) (Fig. 9Go, A and B). The methylation patterns of the hSR promoter in these cell lines were found to differ: they ranged from unmethylated in CAPAN-1, partially methylated in PANC-1 and HPAC, to completely methylated in HepG2. Because Sp1 to Sp3 ratio plays a very important role in the regulation of the hSR gene expression, we next sought to investigate the methylation status of all the CpG dinucleotides with a focus on the key Sp1/Sp3 binding sites (Fig. 10Go). In CAPAN-1 cells, the Sp1/Sp3 binding sites as well as the regions nearby are unmethylated. In PANC-1 cells, GC box-A is methylated whereas GC box-B is unmethylated. In HPAC cells, most of the CpG sites are methylated whereas in HepG2 cells, all the CpG sites are methylated. Our study clearly indicates that the methylation status of the promoter region, particularly the GC boxes (both A and B), is directly related to the expression of the hSR gene.



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  		Fig. 10. Methylation Status of all the CpG Dinucleotides Present in the Promoter Region of the hSR Gene, from -400 to -158 bp Upstream of the ATG Site

CpG sites are numbered from 1–27 with their relative locations as shown on the dark bar. Methylated and unmethylated CpG sites are indicated with ({blacksquare}) and (U), respectively. Numbers in bold (16–18 and 22–23) are the locations of the GC box-A and -B.

 
Treatment of Cell Lines with 5-Aza-2'Deoxycytidine (5-azaC) Induces hSR Expression
The effect of methylation on the expression of the hSR gene was investigated by using a demethylating agent, 5-azaC (Fig. 11Go). hSR expression in PANC-1 cells and HepG2 cells was compared before and after 5-azaC treatment. The expression levels of the hSR gene were detected by semiquantitative RT-PCR and Southern blot analyses (Fig. 11Go, A and B). After drug treatment, expression of the hSR gene in PANC-1 and HepG2 cells was increased and induced, respectively. Control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR (Fig. 11CGo) was performed to show the quality and quantity of the template DNA used in the study.



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  		Fig. 11. Expression of hSR Transcripts in 5-azaC-treated HepG2 and PANC-1 Cells

A, A semiquantitative RT-PCR analysis to show the relative concentrations of PCR products with respect to the amount of first-strand cDNA template used. The first strand cDNA was synthesized from the mRNAs isolated from the hSR-expressing CAPAN-1 cells. The PCR primers were designed to amplify the N terminus of the hSR cDNA (Table 1Go). B, RT-PCR coupled to Southern blot analysis to show the relative expression of hSR in 5-azaC-treated and control cells. Lanes 1 and 3 show the expression of hSR in untreated HepG2 and PANC-1 cells, respectively. Lanes 2 and 4 show the expression of hSR in 5-azaC-treated HepG2 and PANC-1 cells, respectively. hSR transcript was not detected in untreated HepG2 cells but was detected after 5-azaC treatment. The expression level of hSR in PANC-1 cells was increased after 5-azaC treatment. C, A control GAPDH PCR to show the quality of template cDNA used in the study.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our initial study (13) provided some information with respect to the general properties of the hSR promoter; it does not contain any putative TATA or CCAAT box, but it has two GC boxes that are putative binding sites for the Sp family of transcription factors (20). Sp1 is a sequence-specific and ubiquitously expressed zinc finger transcription factor that supports constitutive basal expression of a variety of eukaryotic genes that lack a functional TATA box (37, 38). It has been proposed that many TATA-less and GC-rich promoters bind one or more Sp1 molecules to recruit specific cofactors such as TATA-binding protein-associated factors, which subsequently interact with transcription factor IID (39, 40, 41, 42) to initiate gene transcription. The Sp family of proteins are some of the most studied transcriptional factors. At least five Sp1-related proteins, Sp2–Sp6, have been described (43). These proteins, like Sp1, contain zinc fingers as well as glutamine and serine/threonine-rich amino acid stretches. Sp1 and Sp4 are transcription activators, whereas Sp3 often acts as a repressor (36, 44).

By site-directed mutagenesis and promoter assays, we found that both GC boxes are crucial to the function of the hSR gene promoter, and their effects are additive in nature. The EMSA and mithramycin inhibition studies show that Sp1 and Sp3 are capable of interacting specifically with the GC boxes in the hSR core promoter. In these studies, complex I was identified to contain Sp1 whereas the proteins in complexes II and III were identified to be Sp3. Sp3 has three isoforms (full-length and the truncated M1 and M2) that are generated by differential usage of translation start sites, and all of these isoforms have the ability to bind to the Sp1 binding site (33). Therefore, it is likely that the proteins in complexes II and III are full-length and truncated forms of Sp3, respectively.

To determine the functions of Sp1 and Sp3 in the hSR promoter, overexpression studies were carried out in a non-Sp protein-expressing cell line, the Drosophila Schneider SL-2 cell (34). We demonstrate that Sp1 activates hSR promoter but Sp3, even in high concentrations, does not drive reporter gene expression. This is further confirmed by the antisense experiment in which we show that overexpression of the anti-Sp3 transcript, and hence reduction in Sp3 protein, leads to an increased promoter function. The zinc fingers of Sp1 and Sp3 are highly conserved, and they have similar DNA binding affinities and specificities (36). When the Sp1 to Sp3 ratio is high, there will be a high level of hSR gene expression. On the contrary, when the level of Sp3 is higher than that of Sp1, hSR gene expression will be inhibited. Because the levels of Sp1 and Sp3 are different in various cells and tissues, we propose here that the relative ratio of Sp1 to Sp3 is one of the critical factors controlling the tissue-specific and stage-specific expressions of the hSR gene.

Genes could be inactivated by an epigenetic mechanism (without altering gene sequence) whereby a gene undergoes transcriptional silencing by hypermethylation of CpG islands in the promoter region. CpG islands are clusters of genomic DNA fragments that contain a high frequency of CpG dinucleotide that is normally underrepresented in the genome. CpG methylation is well known to be important in gene imprinting (45, 46), cell cycle control, tumor suppression, embryonic development, and the regulation of some normal differentiation genes (47). The hSR promoter is GC rich (77% GC content with 27 CpGs within -263 to -158) and has many putative CpG methylation sites. There are several reports showing that DNA methylation interferes with the binding of Sp1 to DNA (23, 24, 25, 26). CpG methylation can inhibit Sp1 binding by two mechanisms. In the retinoblastoma gene, methylation of the CpG island directly inhibits the binding of Sp1 (48). In leukosialin gene, MeCP2 competes with Sp1 for the same binding sites (49). For these reasons, we looked into the relationships between the DNA methylation status of the hSR promoter and gene expression. We found that the hSR promoter region of the CAPAN-1 cell is unmethylated and of PANC1 and HPAC cells are partially methylated. Consistently, the hSR promoter in the hSR-negative liver-derived cell line, HepG2, is completely methylated. These results indicate that CpG methylation of the hSR promoter is likely related to hSR gene expression in these transformed cell lines. If this is true, demethylating agent should be able to activate hSR gene expression in hSR-negative cells, and this idea was subsequently confirmed by 5-azaC treatment; hSR expression was increased and induced in PANC-1 and HepG2 cells, respectively. The effects of 5-azaC could be indirect. For example, treatment of MCF-7L breast and GEO colon cell lines with 5-azaC can induce expression of the TGFß receptor by increasing the activator Sp1 stability and down-regulating the expression of the repressor Sp3 (50). Moreover, demethylation may increase/decrease the expression and/or function of some transcription factors; we cannot exclude the possibility that 5-azaC may indirectly affect the expression of the hSR gene.

Secretin and secretin receptors are potential disease markers of pancreatic cancer (27, 28). The expression of secretin receptor is affected by the ratio of Sp1 to Sp3 and by methylation of the promoter region. There are several possible mechanisms to down-regulate the expression of the hSR gene: 1) The hSR promoter is methylated. As a result, key Sp family transcription factors are unable to bind to the GC boxes and fail to initiate transcription. 2) Transcriptional repressor MeCP-1 and/or MeCP-2 compete with the transcriptional activator Sp1 for methylated DNA binding sites and inhibit transcription. 3) Methylation changes the expression patterns of Sp-like transcription factors; for example, hSR gene expression is down-regulated by changing the relative Sp1 and Sp3 levels. 4) Down-regulation of Sp1 protein, which is needed to drive the expression of the hSR gene and/or to protect the CpG island hSR gene promoter from methylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Construction
Escherichia coli DH5{alpha} was used as the host strain for subcloning and sequencing. Molecular cloning of the hSR promoter region has been described previously (13). The promoter fragment p263/158luc (-263 to -158 relative to the ATG start codon) was generated by exonuclease III/S1 nuclease digestion (Amersham Pharmacia Biotech, Arlington Heights, IL) and subcloned into the MluI/XhoI sites of the pGL-2 basic vector (Promega Corp., Madison, WI). The -263 to -158 promoter fragment was also subcloned in reverse orientation into the SmaI sites of pGL-2 basic vector (Promega Corp.) to form the p263/158Rluc construct. DNA sequences and orientations of all the clones were verified by DNA sequencing using SP-F and SP-R primers (Table 1Go). The expression vector pPacSp1, which expresses human Sp1 driven by the Drosophila actin promoter, and the control vector pPacO, containing only the Drosophila actin promoter, were generously provided by Dr. Robert Tjian (Howard Hughes Medical Institute, Department of Biochemistry, University of California). The expression vector pPacSp3, which expresses the human transcription factor Sp3 driven by the Drosophila actin promoter, was a generous gift from Dr. Guntram Suske (Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Marburg, Germany). The Sp3 anti-sense expression vector was constructed by inserting the NotI-EcoRV fragment from pRC-CMV-Sp3 into pcDNA3.1(+).

PCR-Linker Scanning Mutagenesis
Site-directed mutagenesis were carried out by a three-step PCR (51), using mutagenic primers M1–M9, M6-1 to M6-4, or double mutagenic primers DM and universal primers T7, MP, and MP-B (Table 1Go). The -263/158 promoter fragment was subcloned into the MluI and XhoI sites of the pBluescript-KS+ vector (Stratagene, La Jolla, CA), and was used as a template for PCR amplification. Mutations were confirmed by DNA sequencing. Double mutants were generated by using the M6 mutant as a template.

Cell Culture and Transfection
PANC-1 (ATCC, Manassas, VA), HepG2 (human hepatocellular carcinoma, ATCC), and BPD-1 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). CAPAN-1 cells (human pancreatic adenocarcinoma, ATCC) were grown in INSERM medium supplemented with 15% FBS. HPAC cells (human pancreatic adenocarcinoma, ATCC) were grown in F12/DMEM medium supplemented with 10% FBS. The above media were supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, San Diego, CA). All these cell lines were cultured at 37 C with 5% CO2. Drosophila Schneider line 2 cells (Drosophila melanogaster embryo, SL-2) were purchased from ATCC and were grown in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% FBS at 25 C without CO2.

For transfection, cells were plated at a density of 1.5 x 105 for PANC-1 and 2 x 105 for BPD-1 onto 35-mm six-well plates (Costar, San Diego, CA), and transfection were carried out 48 h later. For each transfection, a cocktail containing 1 µg promoter-luciferase construct, 0.5 µg pRSV-ß-gal, 5 µl lipofectamine (Invitrogen), and 200 µl serum-free medium was prepared. The transfection mixture was added to the cells for 7 h and followed by 1 ml DMEM containing 20% FBS and antibiotics. The transfected cells were incubated for 40 h and used for luciferase and ß-galactosidase assays (Promega Corp.). Luciferase assay was performed using a luminometer (Lumat LB9507, EG&G Berthold, Bad Wildbad, Germany). The ß-galactosidase activity was used to normalize the luciferase activity.

For the SL-2 cells, 2.5 x 105 cells were seeded onto 35-mm six-well plates (Costar) 1 d before transfection. For each transfection, 1 µg promoter construct was cotransfected with pPacSp1 and/or pPacSp3 using lipofectamine reagent (Invitrogen). A blank vector, pBluescript-KS+ (Stratagene, La Jolla, CA) was added to keep the total amount of DNA used in each transfection the same. The cells were incubated with the lipofectamine/DNA mix in a serum-free medium for 5 h, and the transfection cocktail was replaced by fresh medium. The cells were further incubated for 40 h before being tested for promoter activities. Luciferase activities were normalized by the concentrations of the protein extracts. The protein concentrations of the protein extracts were measured using a Bradford protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA).

For 5-azaC treatment, the cells were treated for 4 d with the culture medium supplemented with 2 µM, 5 µM, 0.1 µM, or 0.5 µM 5-azaC. After 4 d, the remaining cells were washed twice and incubated in fresh, drug-free medium for an additional 4 d. In the mithramycim control experiment, the cells were transfected with 1 µg pRC-CMV-luciferase vector with 0.5 µg pCMV ß-gal and cultured in medium with or without 300 nM mithramycin. In the Sp1 and Sp3 antisense experiments, anti-Sp1 or anti-Sp3 constructs was cotransfected with p263-158 luc and 0.5 µg pRSV-ß-gal into PANC-1 cells. pRSV-ß-gal was included as an internal control for normalization.

EMSA and Supershift Assays
Nuclear proteins were extracted as described earlier (52). The double-stranded probes corresponding to GC box-A and GC box-B (Table 1Go) were end labeled using the Ready-To-Go T4 polynucleotide kinase labeling kit (Amersham Pharmacia Biotech, Arlington Heights, IL) with [{gamma}-32P]ATP (5000 Ci/nmol) (Amersham Pharmacia Biotech). Unlabeled nucleotides were removed by passing the sample through a microspin column G-25 (Amersham Pharmacia Biotech) at 3000 x g. Binding reactions were performed by incubating the nuclear extracts with the binding buffer (100 mM Tris-HCl, pH 7.5; 500 mM NaCl, 25 mM MgCl2, and 5 mM dithiothreitol), 1 µg poly(dI-dC), and 0.2 pmol (200,000 cpm) labeled probe for 20 min at room temperature. For competition assays, 100-fold molar excess of unlabeled oligonucleotides (specific, nonspecific, or Sp1 specific) (Table 1Go) were included in the binding reaction. For supershift assays, 2 µg anti-Sp1 and/or anti-Sp3 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were incubated with the nuclear extracts for 1 h at room temperature before the addition of the probe. In control reactions, BSA (2 µg) was added instead of the antibodies. For mithramycin treatment, the oligonucleotide probes were preincubated with various amounts of mithramycin (Sigma Chemical Co., St. Louis, MO) for 1 h at room temperature before the addition of the nuclear extracts. After binding, the samples were separated in a 5% nondenaturing polyacrylamide gel in 0.5x TBE for 2 h at 4 C. The gels were dried and autoradiographed (Biomax MR film; Eastman Kodak Co., Rochester, NY) at -70 C with intensifiers (Amersham Pharmacia Biotech).

Western Blotting
Western blotting was performed essentially according to a protocol as described earlier (53). Transfected PANC-1 cells were lysed and were resolved by SDS-PAGE and transferred onto the Hybond-C extra membrane (Amersham Biosciences). The blot was incubated with the rabbit anti-Sp3 primary antibody (1:1000, Santa Cruz Biotechnology, Inc.) and subsequently the horseradish peroxidase-conjugated secondary antibody (1:3000). The presence of Sp3 in the cell lysate was visualized by the enhanced chemiluminescent system (Amersham Biosciences, Piscataway, NJ).

Bisulfite Modification of Genomic DNA and Methylation-Specific PCR
Genomic DNAs were extracted from the control and 5-azaC-treated cells using the QIAGEN Genomic DNA kit (QIAGEN, Chatsworth, CA). Bisulfite modification of the genomics DNAs was subsequently carried by the CpGenome DNA modification kit (Intergen, Purchase, NY). Methylation-specific PCR was performed using 50 ng modified DNA. The primers for PCR were designed to amplify the region between -400 to -151 (primer set W: for unmodified DNA; primer set M: for methylated cytosine that resists bisulfite modification; primer set U for unmethylated cytosine). The PCR fragments were purified by the High Pure PCR Product Purification Kit (Roche Diagnostics Corp., Indianapolis, IN) and sequenced by the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reactions Kit (Applied Biosystems, Foster City, CA) and the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

RNA Extraction and RT-PCR Analysis
RNAs from 5-azaC-treated cells and the control cells were extracted by TriPure Isolation Reagent (Roche Clinical Laboratories, Indianapolis, IN). mRNA was purified from total RNA by the PolyATract mRNA isolation system (Promega Corp.). Residue genomic DNA was removed by 1 U DNase I (Invitrogen Life Technologies) at room temperature for 20 min, and first-strand cDNA was synthesized using 200 U of MMLV reverse transcriptase (Invitrogen Life Technologies), 40 U of RNase inhibitor (Invitrogen Life Technologies), 200 ng of random hexamers, 0.3 nM deoxynucleotide triphosphates, and 10 mM dithiothreitol in the first-strand buffer provided by the manufacturer (Invitrogen Life Technologies) at 37 C for 90 min. The PCR primers HSR5' and HSR3' (Table 1Go) that specifically amplify the N terminus of the hSR were used to detect the expression levels of hSR in the cell lines. PCR was performed in the following conditions: templates were denatured at 94 C for 5 min and followed by 28 cycles of 1 min each at 94 C, 55 C, and 72 C. PCR products were fractionated by electrophoresis in a 1% agarose gel and followed by transblotting and UV cross-linking onto a Hybond N+ membrane (Amersham Pharmacia Biotech). A control GAPDH PCR was performed in the following conditions: 1 min at 94 C, 1 min at 55 C, and 1 min 30 sec at 72 C for 28 cycles using the primers GAPDH-F and GAPDH-R (Table 1Go). Southern blotting was carried out using a partial hSR cDNA fragment (1–426 bp) as a probe.

Statistical Analysis
The promoter-luciferase constructs were tested by at least three independent transfection experiments within each study, and the study was repeated for two to three times. When appropriate and unless otherwise stated, the transfection data were analyzed by either one- or two-way ANOVA followed by Tukey’s test.


   ACKNOWLEDGMENTS
 
We thank Professor R. Tjian, Howard Hughes Medical Institute, for the pPacSp1 and pPac0 clones; and Professor G. Suske, Institut für Molekularbiologie and Tumorforschung, for the pPacSp3 clone.


   FOOTNOTES
 
This work was supported by Research Grant University of Hong Kong 7219/02M from the Research Grants Council to B.K.-C.C.

Abbreviations: 5-azaC, 5-Aza-2'Deoxycytidine; BPD, bovine pancreatic duct; CMV, cytomegalovirus; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPAC, human pancreatic adenocarcinoma; hSR, human secretin receptor; PANC-1, human pancreatic ductule carcinoma.

Received for publication June 24, 2003. Accepted for publication November 11, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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