Functional Cooperation between Multiple Regulatory Elements in the Untranslated Exon 1 Stimulates the Basal Transcription of the Human GnRH-II Gene

Chi Keung Cheng, Ruby L. C. Hoo, Billy K. C. Chow and Peter C. K. Leung

Department of Obstetrics and Gynecology, University of British Columbia (C.K.C., P.C.K.L.), Vancouver, British Columbia, Canada V6H 3V5; and Department of Zoology, University of Hong Kong (R.L.C.H., B.K.C.C.), Hong Kong, China

Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H30-4490 Oak Street, British Columbia Women’s Hospital, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The wide distribution of GnRH-II and conservation of its structure over all vertebrate classes suggest that the neuropeptide possesses vital biological functions. Although recent studies have shown that the expression of the human GnRH-II gene is regulated by cAMP and estrogen, the molecular mechanisms governing its basal transcription remain poorly understood. Using the neuronal TE-671 and placental JEG-3 cells, we showed that the minimal human GnRH-II promoter was located between nucleotide -1124 and -750 (relative to the translation start codon) and that the untranslated exon 1 was important to produce full promoter activity. Two putative E-box binding sites and one Ets-like element were identified within the first exon, and mutational analysis demonstrated that these cis-acting elements functioned cooperatively to stimulate the human GnRH-II gene transcription. EMSAs, UV cross-linking, and Southwestern blot analyses indicated that the basic helix-loop-helix transcription factor AP-4 bound specifically to the two E-box binding sites, whereas an unidentified protein bound to the Ets-like element. The functional importance of AP-4 in controlling human GnRH-II gene transcription was demonstrated by overexpression of sense and antisense full-length AP-4 cDNAs. Taken together, our present data demonstrate a novel mechanism in stimulating basal human GnRH-II gene transcription mediated by cooperative actions of multiple regulatory elements within the untranslated first exon of the gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
UNTIL RECENTLY, GNRH-I was thought to be the sole hypothalamic regulator controlling the mammalian reproductive processes. However, the identification of a second form of GnRH from chicken hypothalamus (termed GnRH-II) reveals that GnRH-II is the most widely expressed form of GnRH and that its structure is conserved among vertebrates from primitive fish to humans (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). This second GnRH form differs from the mammalian GnRH-I by three amino acid residues at positions 5, 7, and 8 (His5, Trp7, Tyr8-GnRH-I), and the genes encoding GnRH-II have been cloned from monkey and human brains (12, 13). In contrast to GnRH-I, human GnRH-II mRNA is expressed at significantly higher levels outside the brain, particularly in the kidney, bone marrow, and prostate (13). The evolutionary conservation of GnRH-II and its wide distribution in tissues suggest that the neuropeptide has vital biological functions. For instance, it has been reported to suppress the proliferation of some reproductive tissue-derived tumors (14, 15, 16), regulate human chorionic gonadotropin release from the placenta (17), and inhibit ovarian steroidogenesis (18). In addition, GnRH-II has been shown to preferentially stimulate FSH release (19), supporting the notion for the existence of a specific FSH-releasing factor (20, 21). More recently, Chen et al. (22) demonstrated that GnRH-II could stimulate the expression of a 67-kDa laminin receptor in normal and cancer T cells, and subsequent T cell adhesion to laminin, in vitro chemotaxis, and in vivo homing to specific organs.

Several studies have demonstrated that the expressions of the human GnRH-I and GnRH-II genes are regulated differentially. In neuronal medulloblastoma TE-671 cells, the transcription of the GnRH-II gene is strongly up-regulated by cAMP, which only produces a modest stimulation of the GnRH-I promoter activity. This cAMP-stimulated GnRH-II expression is mediated via a putative cAMP-response element located between nucleotide (nt) -860 and -853 (relative to the translation start codon), which is also crucial for the basal transcription of the gene (23). Consistent with this finding, Kang et al. (18) demonstrated that the mRNA expression of GnRH-II in human granulosa-luteal cells was up-regulated by gonadotropins. In addition to cAMP, the expression of these neuropeptides has also been shown to be regulated differentially by 17ß-estradiol in such a way that the estrogen increases the transcription and the steady-state mRNA level of the GnRH-II gene but decreases those of the GnRH-I gene (24). Together with their discrete tissue expression patterns, these observations strongly indicate that the two forms of GnRH play distinct biological roles in humans.

Although the cAMP/protein kinase A signaling pathway has been implicated to mediate full expression of the basal GnRH-II promoter activity (23), the involvement of other molecular mechanisms in controlling the human GnRH-II gene transcription remains obscure. To address this issue, two human GnRH-II-expressing cell lines, neuronal TE-671 and placental JEG-3, were used to identify and characterize additional transcriptional machinery important for the GnRH-II gene expression. In the present study, we demonstrated that two putative E-box binding sites (EBSs) and one Ets-like element (ELE) within the untranslated first exon functioned cooperatively to stimulate the basal transcription of the GnRH-II gene and revealed for the first time that the ubiquitously expressed basic helix-loop-helix (bHLH) transcription factor AP-4 is an enhancer protein for the GnRH-II promoter.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of the Transcription Start Sites for the Human GnRH-II Gene
A primer extension analysis was performed to identify the transcription start sites for the human GnRH-II gene (Fig. 1Go). By using a primer (PE-GII) located at exon 2 of the GnRH-II gene, six extension products were generated when poly A+ RNA from TE-671 and JEG-3 cells was used, and the deduced positions of the transcription initiation sites were located at nt -956, -912, -899, -882, -861, and -858. No extension product was obtained from the control yeast tRNA. In addition, a 5'-rapid amplification of cDNA ends (5'-RACE) reaction was also performed to examine any possible usage of upstream transcription start sites by the GnRH-II gene. Nucleotide sequencing revealed that all of the transcripts had their 5'-ends located approximately from nt -950 to -796, with no evidence for the utilization of upstream transcription start sites by either the neuronal or placental cells (data not shown).



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Figure 1. Identification of the Transcription Start Sites for the Human GnRH-II gene in TE-671 and JEG-3 Cells by Primer Extension Analysis

Five micrograms of poly A+ RNA from TE-671 and JEG-3 cells were hybridized and extended with the primer PE-GII. Six extension products were obtained (marked with arrows) from both cell lines, and their sizes were determined by comparing with a sequencing reaction (T, G, C, A) generated from M13 mp18 DNA. The deduced transcription start sites from these extension products were located at nucleotide positions -858, -861, -882, -899, -912, and -956, respectively. No extension product was detected from the control yeast tRNA.

 
Transcriptional Activities of Various Regions of the Human GnRH-II Gene
To identify putative regions that are important in regulating human GnRH-II gene transcription, a panel of promoter constructs containing various regions of the human GnRH-II gene was generated and examined in human neuronal TE-671 and placental JEG-3 cells (Fig. 2AGo). Transient transfection studies revealed similar promoter activity profiles in these cell lines. The promoter activities were found to be completely abolished in both cell lines when intron 1 was present in constructs p(-2103/+1)-Luc, p(-793/+1)-Luc, and p(-749/+1)-Luc, indicating the presence of very strong negative regulatory elements within this region. Deletion of intron 1 increased the promoter activities of p(-2103/-750)-Luc drastically in TE-671 (210-fold vs. pGL2-Basic) and JEG-3 cells (198-fold vs. pGL2-Basic). In contrast, removal of the untranslated exon 1 from p(-2103/-750)-Luc [i.e. the construct p(-2103/-794)-Luc] reduced the promoter activities by 4.3-fold and 4.5-fold in the neuronal and placental cells, respectively, whereas lower levels of reduction (2.2- to 3.3-fold) were also observed in OVCAR-3, MCF-7, and COS-7 cells (Fig. 2BGo), suggesting that the noncoding exon plays an important role in mediating full expression of the basal GnRH-II promoter activity and that the transcription factor(s) interacting with this region may be widely expressed.



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Figure 2. Analysis of Transcriptional Activities of Various Regions of the Human GnRH-II Gene in Neuronal TE-671 and Placental JEG-3 Cells

A, Upper panel, A diagrammatic representation of the human GnRH-II gene structure adopted from the published sequence (GenBank accession no. AF036329). The locations of the 5'-flanking region, exon 1 (E1), intron 1, and exon 2 (E2) are indicated. The nucleotide position is relative to the translation start codon ATG, which is designated as +1. Lower panel, A series of deletion constructs containing various regions of the GnRH-II gene was transiently transfected into TE-671 and JEG-3 cells by LIPOFECTAMINE Reagent. The RSV-lacZ vector was cotransfected to normalize the transfection efficiency. The relative promoter activity was represented as the fold induction when compared with the promoterless pGL2-Basic vector. B, The constructs p(-2103/-794)-Luc and p(-2103/-750)-Luc were further analyzed in OVCAR-3, MCF-7, and COS-7 cells to examine the enhancer activity of the untranslated exon 1 in other cell types. The relative promoter activity was represented as the percentage of the construct p(-2103/-794)-Luc whose activity was set as 100% after being normalized by ß-galactosidase activity. Values in all panels represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. pGL2-Basic; b, P < 0.05 vs. p(-2103/-794)-Luc.

 
To locate the core active promoter region, a series of 5'-deletion mutants between nt -2103 and -750 was constructed and analyzed in TE-671 and JEG-3 cells (Fig. 3AGoGo). Similar promoter activity profiles were observed in these cell lines. Deletion of the GnRH-II 5'-flanking sequence from nt -2103 to -1124 had no significant effect on the promoter activity. However, removal of the sequence to nt -924 decreased the activities by 1.5-fold and 2.6-fold in the neuronal and placental cells, respectively. Further 5'-deletion from nt -864 to -793 completely abolished the promoter activities, indicating that the untranslated first exon itself has no transcriptional activity. Taken together, these data suggest that the core promoter region lies between nt -1124 and -750 and that exon 1 is likely to serve as an enhancer element to stimulate human GnRH-II gene transcription. The functional relevance of this minimal promoter in directing GnRH-II gene transcription was confirmed by analyzing the construct p(-1124/-750)-Luc in non-GnRH-II-expressing SH-SY5Y (25) and human dermal fibroblast (HDF) cells, which were found to exhibit much lower promoter activities in transient transfection assays when compared with GnRH-II-expressing TE-671, JEG-3, and SK-OV-3 (14) cells (Fig. 3BGoGo).



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Figure 3. Fine Mapping of the Minimal Human GnRH-II Promoter in TE-671 and JEG-3 Cells

A, Progressive 5'-deletion analysis was performed between nt -2103 and -750 by direct PCR amplification of the corresponding regions, followed by subsequent cloning of the amplified fragments into the promoterless pGL2-Basic vector. The GnRH-II promoter-luciferase constructs were cotransfected with the RSV-lacZ vector into TE-671 and JEG-3 cells. B, Comparison of the transcriptional activities of the core promoter [i.e. the construct p(-1124/-750)-Luc in GnRH-II (TE-671, JEG-3 and SK-OV-3) and non-GnRH-II expressing (SH-SY5Y and HDF) cells]. The relative promoter activity was represented as the fold induction when compared with the pGL2-Basic vector in the respective cell lines. Values represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. pGL2-Basic. C, The untranslated exon 1 of the human GnRH-II gene was cloned either upstream or downstream of a heterologous SV40 promoter in the pGL2-Promoter vector in either sense (UsExon 1-pGL2-Promoter and DsExon 1-pGL2-Promoter) or reverse (UrExon 1-pGL2-Promoter and DrExon 1-pGL2-Promoter) orientation. The viral promoter is represented by a white arrow, whereas the untranslated exon is represented by a shaded arrow. The heterologous constructs were transiently cotransfected with the RSV-lacZ vector into TE-671 and JEG-3 cells. The relative promoter activity was represented as the percentage of the pGL2-Promoter vector, the activity of which was set as 100% after being normalized by ß-galactosidase activity. b, P < 0.001 vs. pGL2-Promoter.

 


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Figure 3A. Continued.

 
To ascertain whether exon 1 can function as a stand-alone enhancer, four heterologous constructs, in which the exon was cloned in different positions (upstream or downstream) or orientations (sense or reverse) relative to the simian virus (SV) 40 promoter, were prepared. As shown in Fig. 3CGoGo, no significant change of promoter activity was observed when the exon was cloned upstream of the viral promoter (UsExon 1-pGL2-Promoter and UrExon 1-pGL2-Promoter). However, a 2.9- and 3.5-fold induction of promoter activities were observed in TE-671 and JEG-3 cells, respectively, when exon 1 was situated in a sense orientation and downstream of the viral promoter (DsExon 1-pGL2-Promoter) (i.e. in its native position and orientation within the human GnRH-II gene). In contrast, a strong reduction of promoter activities (69% in TE-671 cells and 56% in JEG-3 cells) was detected when the exon was cloned in the opposite direction (DrExon 1-pGL2-Promoter). Taken together, these findings indicate that exon 1 of the human GnRH-II gene can work independently as a stand-alone enhancer, but its enhancer activity is strictly dependent on its position and orientation relative to the target promoter.

Two Putative EBSs and One ELE within the Untranslated Exon 1 Function Cooperatively to Stimulate Basal GnRH-II Gene Transcription
Two putative EBSs, designated as distal E-box (dE-box, 5'-CAGCTG-3', nt -790 to -785) and proximal E-box (pE-box, 5'-CAGCTC-3', nt -762 to -757), as well as one ELE (5'-AGGA-3', nt -779 to -776) were identified in sense orientations within the untranslated exon 1 of the human GnRH-II gene (Fig. 4Go). The dE-box motif is identical to the consensus AP-4 binding sequence (5'-CAGCTG-3'), whereas the pE-box motif differs from the consensus sequence by one nucleotide and has been identified as a functional E-box element in the Rpe65 gene (26). Site-directed mutagenesis showed that mutation of either one of these motifs could only partially abolish the enhancer activity of the untranslated exon (Fig. 5Go). In TE-671 cells, single mutation at the dE-box, pE-box, or ELE motif resulted in a 33, 38, or 20% decrease in promoter activity, respectively, whereas a 56, 49, or 38% reduction was observed in JEG-3 cells. To examine whether there is any functional cooperation among these cis-acting elements, constructs containing double or triple mutations were prepared and analyzed. As depicted in Fig. 5Go, almost 60 and 80% removal of promoter activities was observed in the neuronal and placental cells when either two of the elements were mutated concurrently. Complete abolishment of the enhancer activity was only achieved when all the three motifs were altered simultaneously, indicating that these regulatory motifs work cooperatively to stimulate basal GnRH-II gene transcription.



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Figure 4. Nucleotide Sequence of the Minimal Human GnRH-II Promoter

Numbers beside the sequence refer to the positions relative to the translation initiation codon, which is designated as +1. Lowercase and uppercase letters represent nucleotides of the 5'-flanking region and exon 1, respectively. The six transcription start sites determined by primer extension analysis are marked by black inverted triangles. Two putative EBSs, namely dE-box and pE-box, as well as one ELE within the untranslated exon 1, are underlined. The cAMP-response element (CRE) that was previously shown to be important for both the basal and cAMP-induced transcription of the human GnRH-II gene is boxed.

 


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Figure 5. Mutational Analysis of the Putative EBSs and the ELE Motif on the Enhancer Activity of the Untranslated Exon 1

A diagrammatic representation of the mutated promoter constructs is shown on the left side of the figure. The EBSs and ELE are shown as shaded rectangles and a gray oval, respectively. Each EBS was mutated by introducing a HpaI restriction site into the core binding site, whereas the sequence of the ELE motif was mutated from 5'-AGGA-3' to 5'-GTTT-3'. Mutations are marked with black crosses. Wild-type [p(-1124/-750)-Luc and p(-1124/-794)-Luc] or mutated GnRH-II promoter-luciferase construct was transiently cotransfected with the RSV-lacZ vector into TE-671 and JEG-3 cells. The relative promoter activity was represented as the percentage of the construct p(-1124/-750)-Luc, the activity of which was set as 100% after being normalized by ß-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. p(-1124/-750)-Luc; b, P < 0.05 vs. p(-1124/-750)-Luc.

 
The bHLH Transcription Factor AP-4 Binds to the Two Putative EBSs, Whereas an Unknown Protein Binds to the ELE
EMSAs showed that DNA-protein complexes of almost identical mobility were formed with oligonucleotides containing the dE-box and pE-box motifs when nuclear extracts from TE-671 and JEG-3 cells were used (data not shown). Formation of these complexes was dose-dependently inhibited by the addition of cold competitors (100- to 500-fold excess) but not by other nonspecific sequences (500-fold excess) such as the consensus nuclear factor-{kappa}B (NF-{kappa}B) or transcription factor IID (TFIID) binding motif (Fig. 6AGo), suggesting specific interactions between the putative EBSs and nuclear proteins. In addition, the formation of these complexes was abolished in the presence of an increasing amount of a consensus AP-4 binding sequence (100- to 500-fold excess) (Fig. 6BGo). In contrast, competitor oligonucleotides carrying HpaI mutations in the EBSs failed to inhibit complex formation (Fig. 6CGo). Antibody supershift assays showed that the bHLH transcription factors E2A (E12 and E47), dHAND, eHAND, upstream stimulatory factor (USF)-1, and USF-2 were not present in these complexes (Fig. 6DGo). By UV cross-linking (Fig. 7AGo) and Southwestern blot analysis (Fig. 7BGo), nuclear factors with molecular mass of 48 kDa [a size that is consistent with that of the bHLH transcription factor AP-4 (27)] from both TE-671 and JEG-3 cells were found to interact with both EBSs specifically. However, since antiserum against AP-4 is not yet commercially available to confirm its presence in the complexes, we performed EMSA reactions using in vitro translated human AP-4 proteins. As shown in Fig. 8AGo, the in vitro translated products bound to the EBSs as single DNA-protein complexes that share electrophoretic mobility similar to that formed with TE-671 nuclear extracts. The specificity of the complexes formed by the in vitro translated products was examined by the addition of an increasing amount of a consensus AP-4 oligonucleotide (100- to 500-fold excess), which could inhibit complex formation in a dose-dependent manner (Fig. 8BGo). Thus, these results indicate that the bHLH transcription factor AP-4 is the nuclear factor binding to the EBSs.



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Figure 6. EMSAs to Characterize the Two Functional EBSs Using Nuclear Extracts Derived from TE-671 and JEG-3 Cells

Synthetic oligonucleotides containing the dE-box and pE-box motifs were annealed to form double-strand DNA, and the probes were end-labeled with 32P and incubated with nuclear extracts (NE) from TE-671 and JEG-3 cells in the absence or presence of competitor oligonucleotides (or antibodies). A, Nuclear extracts, 5 µg, from TE-671 cells were incubated with the radiolabeled EBS probes in the presence of an increasing amount of a cold competitor (100- to 500-fold excess), 500-fold excess of a consensus NF-{kappa}B (cNF-{kappa}B) or TFIID (cTFIID) oligonucleotide. B, Nuclear extracts, 5µg, from TE-671 cells were incubated with the radiolabeled EBS probes in the presence of an increasing amount of a consensus AP-4 binding sequence (cAP-4) (100- to 500-fold excess). C, Nuclear extracts, 5 µg, from TE-671 cells were incubated with the radiolabeled EBS probes in the presence of a different amount of competitor oligonucleotides containing the corresponding mutated EBS sequences (mEBS) (250- and 500-fold excess). D, Nuclear extracts, 5 µg, from TE-671 cells were preincubated with anti-dHAND, anti-eHAND, anti-E2A, anti-USF-1, anti-USF2, or anti-GATA-4 antibody for 30 min at room temperature before addition of the radiolabeled probes. Specific DNA-protein complexes are indicated with black arrows. Similar DNA binding results were also observed when nuclear extracts from JEG-3 cells were used (data not shown).

 


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Figure 7. Identification of a 48-kDa Nuclear Factor Binding to the EBSs by UV Cross-Linking and Southwestern Blot Analyses

A, Nuclear extracts (NE), 20 µg, from TE-671 and JEG-3 cells were incubated with 100 fmol of the radiolabeled EBS probes in the absence or presence of a 500-fold excess of cold competitors. The binding reactions were then exposed to UV for 30 min at 4 C before analyzed by 10% SDS-PAGE. NS, Nonspecific signals. B, Nuclear extracts, 100 µg, from TE-671 cells were separated by a 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. The membrane was then hybridized with the corresponding radiolabeled probes used in the EMSAs.

 


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Figure 8. In Vitro Translated Human AP-4 Proteins Bind Specifically to Both EBSs

A, EMSAs were performed by incubating the radiolabeled EBS probes with 5 µg of TE-671 nuclear extracts (NE), 2 µl of reticulocyte lysate, or 2 µl of in vitro translated (IVT) AP-4 proteins. Two independent preparations of the in vitro translated products [IVT AP-4 (1 ) and IVT AP-4 (2 )] were used. The complexes formed with in vitro translated proteins (indicated with white arrows) share similar electrophoretic mobility with those formed with TE-671 nuclear extracts (indicated with black arrows). B, The radiolabeled EBS probes were incubated with 2 µl of in vitro translated AP-4 proteins in the presence of an increasing amount of a cAP-4 oligonucleotide (100- to 500-fold excess).

 
EMSAs indicated that a nuclear protein commonly expressed in TE-671 and JEG-3 cells bound to the ELE (data not shown), and that the formation of this complex could be inhibited by a cold competitor in a dose-dependent manner (Fig. 9AGo). Mutation of the ELE from 5'-AGGA-3' to 5'-GTTT-3' abolished DNA binding activity of the nuclear factor (Fig. 9AGo). Although the complex formation could be prevented in a dose-dependent manner by a consensus Ets transcription factor binding sequence (Fig. 9BGo), the identity of the ELE-binding factor remained to be determined because the use of five different antibodies targeted against Ets-1, Ets-2, Elk-1, Erg-1, and Erg-2 did not interfere with the complex formation in the supershift assays (data not shown).



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Figure 9. EMSAs to Characterize the ELE Using Nuclear Extracts Derived from TE-671 and JEG-3 Cells

Synthetic oligonucleotides containing the ELE motif were annealed to form double-strand DNA, and the probe was end-labeled with 32P and incubated with nuclear extracts (NE) from TE-671 and JEG-3 cells in the absence or presence of competitor oligonucleotides. A, Nuclear extracts, 5 µg, from TE-671 cells were incubated with the radiolabeled ELE probe in the presence of an increasing amount of a cold competitor (100- to 500-fold excess), cNF-{kappa}B or cTFIID oligonucleotide (500-fold excess), or a different amount of a mutated ELE oligonucleotide (mELE) (250- and 500-fold excess). B, Nuclear extracts, 5 µg, from TE-671 cells were incubated with the radiolabeled ELE probe in the presence of an increasing amount of a consensus Ets transcription factor binding sequence (cEts) (100- to 500-fold excess). Specific DNA-protein complexes are indicated with black arrows. Similar DNA binding results were also observed when nuclear extracts from JEG-3 cells were used (data not shown).

 
Effects of Overexpression of Sense and Antisense AP-4 cDNAs on Human GnRH-II Promoter Activity
To ascertain the functional significance of AP-4 in regulating GnRH-II gene transcription, we constructed expression plasmids encoding sense and antisense AP-4 mRNAs and cotransfected them with the GnRH-II promoter-luciferase construct p(-1124/-750)-Luc. As shown in Fig. 10AGo, overexpression of sense AP-4 mRNA resulted in an up-regulation of the GnRH-II promoter activities in JEG-3 cells, with a maximum of 2-fold induction when 2 µg of expression plasmid were cotransfected. In contrast, forced expression of antisense AP-4 mRNA suppressed the promoter activities in the placental cells. These changes in the GnRH-II promoter activities correlate with the endogenous levels of AP-4 transcripts, as revealed by RT-PCR and Southern blot analysis (Fig. 10BGo). Similar degrees of stimulation and reduction in promoter activities were also observed when equal amounts (1 µg) of expression plasmids were cotransfected into TE-671 cells (data not shown). The down-regulatory effect of antisense AP-4 mRNA expression on the GnRH-II promoter activity was attenuated when the EBSs within the untranslated exon 1 were either deleted [p(-1124/-794)-Luc] or mutated [(dE-box+pE-box)-mut]. In addition, weak but significant stimulation, rather than inhibition of activities, was observed in promoters (those of the human GnRH receptor and human secretin receptor genes) lacking AP-4 binding sites when antisense AP-4 mRNA was introduced into the cells (Fig. 10CGo). The in vivo significance of AP-4 in controlling GnRH-II gene transcription was further investigated by examining the mRNA level of the transcription factor in the non-GnRH-II expressing SH-SY5Y cells. As shown in Fig. 10DGo, the transcript level was found to be much lower in SH-SY5Y cells than in JEG-3 cells. In addition, we found that AP-4 overexpression could induce the activity of p(-1124/-750)-Luc in SH-SY5Y cells by 2.8-fold, a degree that is greater than that observed in JEG-3 cells (1.9-fold) when an equal amount of expression plasmid was cotransfected. Forced expression of antisense AP-4 mRNA consistently caused a weaker repression of the GnRH-II promoter activity in SH-SY5Y cells, and this phenomenon may be due to their scanty endogenous level of AP-4 expression. Taken together, these findings clearly indicate that the bHLH protein AP-4 plays a specific positive role in regulating human GnRH-II gene transcription via the EBSs within the untranslated exon 1 of the gene.



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Figure 10. In Vivo Expression Level of AP-4 Regulates the Promoter Activity of the Human GnRH-II Gene

A, The construct p(-1124/-750)-Luc was cotransfected with an increasing amount (0.25–2 µg) of pcDNA3.1, pcDNA3.1-AP-4 (sense), or pcDNA3.1-AP-4 (antisense) into JEG-3 cells. B, RT-PCR and Southern blot analysis of in vivo levels of AP-4 and GAPDH mRNA transcripts in JEG-3 cells transfected with 1 µg of pcDNA3.1, pcDNA3.1-AP-4 (sense), or pcDNA3.1-AP-4 (antisense). The AP-4 transcripts were hybridized with a [{alpha}-32P]-labeled full-length AP-4 cDNA probe. C, One microgram of p(-1124/-750)-Luc, p(-1124/-794)-Luc, (dE-box+pE-box)-mut, the human GnRH receptor promoter-luciferase construct [hGnRHR p(-1300/-1018)-Luc], and the human secretin receptor promoter-luciferase construct [hSR p(-223/-158)-Luc] was cotransfected with 1 µg of pcDNA3.1 or pcDNA3.1-AP-4 (antisense) into JEG-3 cells. D, Upper panel: Endogenous expression levels of AP-4 mRNAs in JEG-3 and SH-SY5Y cells. Amplification of GAPDH mRNA was performed as a positive control. Lower panel: p(-1124/-750)-Luc, 1 µg, was cotransfected with 1 µg of pcDNA3.1, pcDNA3.1-AP-4 (sense), or pcDNA3.1-AP-4 (antisense) into JEG-3 and SH-SY5Y cells. The RSV-lacZ vector was cotransfected to normalize the transfection efficiency. The relative promoter activity in all panels was represented as the percentage of the control (cotransfection with pcDNA3.1), the activity of which was set as 100% after being normalized by ß-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.001 vs. control; b, P < 0.01 vs. control; c, P < 0.05 vs. control.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In an attempt to identify additional regulatory sequences and transcription factors involved in the transcriptional regulation of the human GnRH-II gene, we have performed a detailed deletion analysis of the human GnRH-II 5'-flanking region in two GnRH-II expressing cell lines, neuronal TE-671 and placental JEG-3. Our present data demonstrated that these cells (and also the ovarian carcinoma OVCAR-3 cells, data not shown) displayed similar promoter activity profiles and that a minimal promoter region (nt -1124 to -750) was sufficient to direct GnRH-II gene transcription in both the neuronal and reproductive cells. These observations are in sharp contrast to previous findings from others and our laboratory which showed that tissue-specific expressions of the human GnRH-I and GnRH receptor genes were mediated, at least partly, by alternative promoter usage such that downstream and upstream promoters (and transcription initiation sites) were selectively used by neuronal and reproductive cell types, respectively (28, 29, 30, 31, 32). These differential regulatory mechanisms in controlling the expressions of the two forms of human GnRH genes may help explain their distinct tissue distribution patterns (13). Sequence analysis revealed that neither TATA box nor initiator element was present upstream of, or overlapping, the six transcription start sites of the human GnRH-II gene. The absence of both TATA box and initiator element is not unique to the GnRH-II promoter as it has also been reported in the mouse thymidylate synthase gene (33), mouse protoporphyrinogen oxidase gene (34), and human Sp1 gene (35). In addition, we did not find any MED-1 element, which is conserved in many TATA- and initiator-less promoters and has been proposed to be important for promoter function (36) downstream of the initiation window of the GnRH-II gene. Further investigations are needed to elucidate the mechanisms that direct transcription initiation of the human GnRH-II gene.

Our deletion analysis indicated that strong repressive element(s) existed in the first intron of the human GnRH-II gene (Fig. 2AGo). To date, a considerable number of genes containing intronic silencers have been identified (37, 38, 39, 40, 41), and it has been suggested that this type of silencers can repress transcription in a number of ways including physical blockage of transcriptional elongation, intronic splicing, and basal transcription apparatus assembly (42). Although the first intron of the GnRH-II gene appeared to work constitutively to suppress promoter functions in various cell types [TE-671 and JEG-3 (Fig. 2AGo) and OVCAR-3 (data not shown)], its silencing activity may be tightly controlled under different physiological or developmental conditions in vivo to allow timely expression of the GnRH-II gene. The functional significance of this intron in repressing GnRH-II gene transcription is currently under investigation, and surprisingly, our preliminary data revealed that its activity could be modulated by certain signal transduction pathways (Hoo, R. L. C., unpublished data), which may have important implications in silencing human GnRH-II gene expression.

The role of untranslated exons in regulating gene expression has been reported in a number of studies including the human zinc-finger ZNF177 gene (43), human serotonin-4 receptor gene (44), human hematopoietic prostaglandin D synthase gene (45), and rat carnitine palmitoyltransferase-Iß gene (46). Unlike many classical enhancers, our present data indicated that the enhancer activity of the untranslated exon 1 of the human GnRH-II gene is strictly dependent on its position and orientation. Notably, a silencing activity of the exon was even detected when it was cloned in an aberrant orientation (DrExon 1-pGL2-Promoter, Fig. 3CGoGo). These observations suggest that binding of transcription factors to the exon may not be symmetrical and that efficient transcriptional activation requires a specific spatial and directional arrangement of protein factors along the sequence. In addition, it should be mentioned in this context that although the primary structure of GnRH-II is conserved over all vertebrate classes, the sizes and the nucleotide sequences of their untranslated first exons vary dramatically, suggesting that the untranslated exon may not possess similar regulatory roles in controlling GnRH-II gene transcriptions in other species as in humans.

Results from EMSAs, UV cross-linking, and Southwestern blot analyses provide solid evidence that the bHLH transcription factor AP-4 is the candidate nuclear protein binding to both EBSs. The wide expression pattern of AP-4 may contribute to the ubiquitous enhancer activity of the untranslated first exon. The transcription factor was originally identified as a cellular protein that bound to the SV40 enhancer and cooperated synergistically with activating protein-1 to stimulate both SV40 late and the human metallothionein-IIA gene transcription in vitro (27). In addition, the bHLH protein has been shown to be involved in cAMP-inducible proenkephalin transcription (47), Tax-mediated transactivation of bovine leukemia virus long terminal repeat (48), and IGF binding protein-2 gene transcription (49). However, the transcription factor has also been demonstrated to repress gene expression in some cases. For instance, binding of AP-4 to the HIV type 1 TATA element was found to compete with the TATA box protein binding to this element, and to inhibit in vitro transcription from the virus long terminal repeat (50). Moreover, cotransfection of an AP-4 expression plasmid could down-regulate the activity of the human angiotensinogen promoter (51). Similar to other members of the family, AP-4 contains a HLH motif and an adjacent basic domain that are necessary and sufficient for site-specific DNA binding. However, unlike other bHLH proteins, AP-4 possesses two additional protein dimerization motifs consisting of leucine repeat elements that were shown to prevent heterodimer formation (52). This property of AP-4 is consistent with our present data which indicated that other bHLH proteins including the ubiquitously expressed immunoglobulin enhancer binding protein E12 were not present in the complexes. Interestingly, previous deletion analysis of the chromatin structure of SV40 late promoter has uncovered a novel function of AP-4 in conferring significant levels of nuclease sensitivity, thus implicating it in the process of chromatin remodeling (53). Additionally, based on its considerable homology with other transcription factors such as Myc and Max, it has also been proposed that AP-4 may bind to its target sequence even when it is incorporated into nucleosomes, thereby allowing other factors to bind in a cooperative fashion (53). As noted below, the results of our present study are in agreement with the function of AP-4 in that it may serve as a nucleating protein to facilitate interactions with other transcription factors.

Given that the two EBSs are juxtaposed with the ELE motif and the presence of multiple protein-dimerization domains in AP-4, it is postulated that the cooperative action between the EBSs and ELE may involve direct protein-protein interactions, which may lead to synergistic transactivation of the target gene. Evidence supporting this view comes from a previous finding which demonstrated that USF-1 could interact with the Ets domain of Ets-1 via its HLH domain to cooperatively activate HIV-1 expression (54). In addition, the leucine zipper repeats of AP-4 may also participate in the formation of higher-order complexes because it has been reported that the Ets domain of PU.1 can interact with the basic leucine zipper region of NF-IL6ß, leading to a synergistic transcriptional activation of an artificial promoter containing both Ets and NF-IL6ß consensus binding sequences (55). Interestingly, a similar arrangement of two EBSs and one ELE was also found in the immunoglobulin µ heavy-chain gene enhancer, and transcriptional synergy between the bHLH proteins E47 and TFE-3 requires the Ets domain protein Ets-1, which may function to bridge the transactivation domains of the flanking bHLH proteins (56). Based on this phenomenon, it is logical to speculate that the weak transactivation response (~2-fold) observed (when only AP-4 cDNA is overexpressed, Fig. 10AGo) may be due to the concomitant requirement of an intermediate Ets protein, which is essential to mediate functional cooperation between the bHLH transcription factors. However, unlike the so-called "protein-tethered transactivation" (57), our present EMSA results indicated that both AP-4 and the unidentified Ets-related protein participated in DNA binding, suggesting that the cooperative effect may be the result of an overall enhanced binding affinity to the target sequences. Nevertheless, whether the ELE-binding protein belongs to an existing or an unknown member of the Ets family of transcription factors remains to be elucidated. Such investigation should greatly facilitate our understanding of the mechanistic action of the exonic enhancer in stimulating GnRH-II gene transcription and the identification of potential heterodimerization partners for the bHLH protein AP-4, which is so far unknown.

Although in the present study we demonstrated that exon 1 constitutively stimulated the basal transcription of the human GnRH-II gene, we cannot rule out the possibility that its enhancer activity is modulated by certain extracellular stimuli. It has been well documented that the transcriptional activity of bHLH proteins is tightly regulated by phosphorylation both positively and negatively. Thus, the serine/threonine kinases 3pK and MK2 were shown to interact with and phosphorylate E47 protein and repress its transcriptional activity (58), whereas overexpression of casein kinase II increased the transcriptional activities of MRF4 and MyoD in vivo through a mechanism involving phosphorylation of E47 (59). Furthermore, Bain et al. (60) demonstrated a direct connection between the Ras-ERK MAPK signaling cascade and HLH proteins in a common pathway involved in thymocyte positive-selection. Likewise, many Ets-domain transcription factors are known to represent nuclear targets of some signaling pathways (61), and in particular, all members of the ternary complex factor subfamily of the Ets-domain proteins have been shown to be ERK targets, and also to respond differentially to the related c-Jun N-terminal kinase and p38 MAPK pathways (62). Because MAPKs are known to respond to an extraordinarily diverse arrays of extracellular stimuli, it is tempting to speculate that under different physiological conditions, the transcriptional activities of AP-4 and the ELE-binding protein may be modulated accordingly, which in turn regulate the rate of basal transcription of the human GnRH-II gene.

In conclusion, we have identified multiple regulatory elements within the untranslated exon 1 of the human GnRH-II gene, which function cooperatively to stimulate the basal transcription of the gene, and demonstrated that the bHLH transcription factor AP-4 is an enhancer protein for the GnRH-II promoter.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Cell Culture
Human neuronal medulloblastoma TE-671, human choriocarcinoma JEG-3, human ovarian adenocarcinomas OVCAR-3 and SK-OV-3, human breast adenocarcinoma MCF-7, and African green monkey kidney COS-7 cells were obtained from American Type Culture Collection (Manassas, VA). The HDF and SH-SY5Y cells were kindly provided by Dr. N. Auersperg (Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada) and L. T. O. Lee (Department of Zoology, University of Hong Kong, Hong Kong, China). The cells were maintained in DMEM (Invitrogen, Inc., Burlington, Canada) supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT). Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2 in air. Cells were passaged when they reached about 80% confluence using a trypsin/EDTA solution (0.05% trypsin and 0.53 mM EDTA).

Plasmid Construction and Site-Directed Mutagenesis
Human GnRH-II promoter-luciferase construct p(-2103/+1)-Luc (all numbering is relative to the translation start codon ATG) was prepared by PCR of the corresponding region of the GnRH-II gene (i.e. nt -2103 to +1) using human genomic DNA (CLONTECH Laboratories, Inc., Palo Alto, CA) as the template and subsequent cloning of the amplified fragment into the promoterless pGL2-Basic vector (Promega Corp., Nepean, Canada). The authenticity of the DNA fragment was confirmed by nucleotide sequencing. Deletion constructs except p(-1524/-750)-Luc and p(-1962/-750)-Luc were generated by amplification of the corresponding regions using the p(-2103/+1)-Luc as the template. The constructs p(-1524/-750)-Luc and p(-1962/-750)-Luc were prepared by digesting p(-2103/-750)-Luc with a combination of NheI/KpnI and SpeI/KpnI, respectively, followed by self-ligation. Heterologous SV40 promoter constructs were prepared by PCR amplification of exon 1 of the human GnRH-II gene and subsequent cloning of the fragment into the SmaI (upstream) or HindIII (downstream) site of the pGL2-Promoter vector (Promega Corp.). Site-directed mutants were generated by PCR using a forward primer initiating from nt -1124 and reverse primers containing the desired mutations, with the plasmid p(-1124/-750)-Luc as the template. An HpaI restriction site (5'-GTTAAC-3') was introduced into each of the putative EBSs, whereas the ELE (5'-AGGA-3') was mutated to 5'-GTTT-3'. The PCR for wild-type and mutant GnRH-II promoter-luciferase constructs was carried out for 30 cycles with denaturation for 30 sec at 94 C, annealing for 1 min at 50 C, extension for 1 min at 72 C, and a final extension for 15 min at 72 C. Human AP-4 expression plasmid (pCMV-AP-4) was kindly provided by Dr. R. Tjian (Howard Hughes Medical Institute, University of California, Berkeley, CA). The full-length AP-4 cDNA was released by restriction digestion and blunt ended, followed by cloning into the pcDNA3.1 vector (Invitrogen) in both orientations. The human GnRH receptor promoter-luciferase construct p(-1300/-1018)-Luc has been described previously (32), whereas the human secretin receptor promoter-luciferase construct p(-223/-158)-Luc was kindly provided by Y. Y. Kwok (Department of Zoology, University of Hong Kong, Hong Kong, China). Plasmid DNA for transient transfection was prepared using the QIAGEN Plasmid Midi Kits (QIAGEN, Chatsworth, CA) following the manufacturer’s suggested procedures. The concentration and quality of DNA were determined by measuring absorbance at 260 nm and by agarose gel electrophoresis, respectively.

Transient Transfection and Reporter Gene Assays
Transient transfections were carried out using LIPOFECTAMINE Reagent (Invitrogen) following the manufacturer’s suggested procedures. To correct for different transfection efficiencies of various luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was cotransfected into the cells with the GnRH-II promoter-luciferase construct. Briefly, 4 x 105 of cells were seeded into six-well tissue culture plates before the day of transfection. One microgram of GnRH-II promoter-luciferase construct, 0.5 µg of RSV-lacZ plasmid, and an indicated amount of AP-4 expression plasmid were cotransfected into the cells under serum-free conditions. After 5 h of transfection, 1 ml of medium containing 20% fetal bovine serum was added and the cells were further incubated overnight (18 h). After incubation, the old medium was removed and the cells were cultured for another 24 h with normal fresh medium containing 10% fetal bovine serum. Cellular lysates were collected with 150 µl reporter lysis buffer (Promega Corp.) and assayed for luciferase activity with the Luciferase Assay System (Promega Corp.). Luminescence was measured using a Lumat LB 9507 luminometer (EG&G, Berthold, Germany). ß-Galactosidase activity was measured using the ß-Galactosidase Enzyme Assay System (Promega Corp.) and used to normalize for varying transfection efficiencies. Promoter activity was calculated as luciferase activity/ß-galactosidase activity.

RNA Isolation and Primer Extension Analysis
Poly A+ RNA was isolated from 1 x 108 cells using the PolyATtract System 1000 (Promega Corp.) following the manufacturer’s suggested procedures. Primer extension analysis was performed essentially the same as previously described (32) using oligonucleotide PE-GII, which is located between nt +55 and +32 of the human GnRH-II gene. Briefly, the primer was end-labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase (Invitrogen) and hybridized with 5 µg poly A+ RNA for 42 C overnight. The RNA was then reverse transcribed at 42 C for 2 h with 20 U Superscript RNaseH-reverse transcriptase (Invitrogen), and the reaction was stopped by the addition of RNaseA (20 µg/ml). The extended products were purified by phenol-chloroform extraction and analyzed on a 6% polyacrylamide/7.0 M urea gel. A sequencing ladder (T, G, C, A) was generated from M13 mp18 DNA using the universal primer provided in the T7 Sequencing Kit (Amersham Pharmacia Biotech, Morgan, Canada) and was used as a size standard.

5'-RACE
5'-RACE was performed using the SMART RACE cDNA Amplification Kit (CLONTECH Laboratories, Inc.) following the manufacturer’s suggested procedures. Briefly, first-strand cDNA was constructed from 2 µg poly A+ RNA. An antisense human GnRH-II exon 4-specific primer and an antisense exon 3-specific primer were used as the outer and nested primers, respectively. The PCR product was cloned into pUC18 and sequenced.

EMSA
Oligonucleotides containing the putative EBSs (dE-box: 5'-GTCCTGCAGCTGCCTGAA-3'; pE-box: 5'-CATCCACAGCTCTTCCTT-3'), ELE (5'-GCCTGAAGGAGCCATCTC-3'), consensus AP-4 binding sequence (5'-CACCCGGTCAGCTGGCCTACACC-3'), and mutated sequences (dE-box-mut: 5'-GTCCTGGTTAACCCTGAA-3'; pE-box-mut: 5'-CATCCAGTTAACTTCCTT-3'; ELE-mut: 5'-GCCTGAGTTTGCCATCTC-3') were synthesized by the Oligonucleotide Synthesis Laboratory (University of British Columbia, Vancouver, Canada) and annealed to form double-strand DNA. Underlined sequences represent mutated nucleotides. The oligonucleotides for the two EBSs and the ELE motif were designed such that they contained only their respective core binding sequences. Consensus Ets transcription factor, NF-{kappa}B, and TFIID oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Probes for EMSAs were end-labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase and separated from unincorporated radionucleotides by Microspin G-25 columns (Amersham Pharmacia Biotech). Nuclear extracts were prepared from TE-671 and JEG-3 cells as described previously (32). Protein concentrations were determined by a modified Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). In vitro translated human AP-4 proteins were generated by the TNT Coupled Reticulocyte Lysate System (Promega Corp.). EMSA was carried out in a 20-µl reaction containing 20 mM HEPES (pH 7.5), 50 mM NaCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 1 µg poly (dI:dC), 5 µg nuclear proteins (or 2 µl in vitro translated AP-4 proteins), and 50 fmol radiolabeled probe (30,000 cpm). For the competitive assays, competitor oligonucleotides were added simultaneously with the radiolabeled probes. For the supershift assays, nuclear extracts were preincubated with 3 µg anti-dHAND, anti-eHAND, anti-E2A, anti-USF-1, anti-USF-2, or ant-GATA-4 antibody (Santa Cruz Biotechnology, Inc.) for 30 min at room temperature before the addition of the radiolabeled probes. The binding reaction was incubated at room temperature for 15 min and then separated by a 6% polyacrylamide gel containing 0.5 x 0.09 M Tris-borate and 2 mM EDTA, pH 8.0, at constant 200 V and at 4 C. Before loading of samples, the gel was prerun for 90 min at 100 V. After electrophoresis, the gel was then dried and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) at -70 C.

UV Cross-Linking and Southwestern Blot Analysis
UV cross-linking was performed as previously described (63). Briefly, end-radiolabeled oligonucleotides used in the EMSAs were incubated with 20 µg of nuclear extracts for 15 min at room temperature in a volume of 40 µl. After incubation, the binding reaction mixtures were exposed to UV (254 nm) for 30 min at a distance of 2 cm from the light source. The cross-linked reaction was stopped by the addition of 8 µl of 6x Laemmli gel loading buffer and then boiled for 5 min before being separated by 10% SDS-PAGE. After electrophoresis, the gel was dried and the products were detected by autoradiography.

Southwestern blot analysis was performed as described previously (64). Nuclear extracts (100 µg) from TE-671 cells were separated by 10% SDS-PAGE and then transferred onto a nitrocellulose membrane (Hybond-C, Amersham Pharmacia Biotech). Transferred proteins were allowed to renature in TNED buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 0.1 mM EDTA (pH 7.5), and 1 mM dithiothreitol] containing 5% milk overnight at room temperature. Afterward, the membranes were rinsed three times in the TNED buffer and incubated overnight at room temperature with 11 ml of the same buffer containing 75 pmol of the radiolabeled probes used in the EMSAs. After hybridization, the membrane was washed three times (10 min each) with the TNED buffer, dried, and subjected to autoradiography.

RT-PCR and Southern Blot Analysis
Total RNA was extracted from JEG-3 and SH-SY5Y cells by TRIZOL Reagent (Invitrogen) and reverse transcribed using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech) following the manufacturer’s suggested protocols. Primers specific for human AP-4 (GenBank accession no. BC010576) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GenBank accession no. M33197) were designed based on the published sequences. The PCR was carried out for 32 cycles for AP-4 and 22 cycles for GAPDH, with denaturation for 30 sec at 94 C, annealing for 1 min at 60 C, extension for 1 min at 72 C, and a final extension for 15 min at 72 C. The authenticities of the PCR products were confirmed by Southern blot analysis. For the Southern blot analysis, the PCR products were separated by agarose gel electrophoresis and then transferred onto a nylon membrane (Hybond-N, Amersham Pharmacia Biotech), followed by hybridization with the corresponding [{alpha}-32P]-labeled AP-4 or GAPDH cDNA probe at 65 C overnight. Radioactivities of the hybridized products were visualized by autoradiography at -70 C with Kodak X-OMAT AR films (Eastman Kodak Co.).

Data Analysis
For transfection assays, data were shown as the mean ± SEMof triplicate assays in three independent experiments. For the primer extension, EMSAs, UV cross-linking, and Southwestern blot analyses, all studies were performed at least twice, and consistent results were obtained between experiments. Data were analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison test using the computer software PRISM (GraphPad Software, Inc., San Diego, CA). Data were considered significantly different from each other at P < 0.05.


    ACKNOWLEDGMENTS
 
We thank Dr. R. Tjian, L. T. O. Lee, and Y. Y. Kwok for providing the pCMV-AP-4, the SH-SY5Y cells, and the human secretin receptor promoter-luciferase construct. Also, we would like to express special thanks to Dr. C. M. Yeung for his assistance during the course of this study.


    FOOTNOTES
 
This work was supported by grants from the Canadian Institutes of Health Research. C.K.C. is a recipient of the British Columbia Research Institute of Children’s and Women’s Health Studentship Award. P.C.K.L. is a Distinguished Scholar of the Michael Smith Foundation for Health Research.

Abbreviations: bHLH, Basic helix-loop-helix; EBS, E-box binding site; ELE, Ets-like element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDF, human dermal fibroblast; NF-{kappa}B, nuclear factor-{kappa}B; nt, nucleotide; RACE, rapid amplification of cDNA ends; RSV, Rous sarcoma virus; SV40, simian virus 40; TFIID, transcription factor IID; USF, upstream stimulatory factor.

Received for publication December 13, 2002. Accepted for publication March 20, 2003.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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