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Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (LINE), Dorothy Hodgkin Building, Whitson Street, University of Bristol, Bristol BS1 3NY, UK
(Requests for offprints should be addressed to A-M O’Carroll; Email: A.M.OCarroll{at}bristol.ac.uk)
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, glucocorticoid receptor and CCAAT enhancer binding protein (C/EBP)
transcription factors. Site-directed mutagenesis identified an individual Sp1 motif that plays a major role in activation of the APJR promoter and also demonstrated constitutive transcriptional regulation of the promoter by estrogen and glucocorticoid receptors. Promoter regulation by the cAMP-dependent signal cascade was also shown.
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Introduction |
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The recent cloning of the human apelin receptor (APJ receptor (APJR)) gene (O’Dowd et al. 1993) and mouse and rat cDNAs (Devic et al. 1999, O’Carroll et al. 2000) indicated this receptor as a membrane glycoprotein with seven membrane-spanning hydrophobic domains characteristic of the G-protein-coupled receptor superfamily. The pattern of expression of the rat receptor implies that APJR has tissue-specific functions in adult animals. High levels of receptor mRNA expression are found in the brain, the parenchyma of the lung, a subpopulation of glomeruli in the kidney, the heart, the corpora lutea of the ovary, and isolated cells of the anterior lobe of the pituitary (O’Carroll et al. 2000). APJR expression is restricted in the CNS, primarily to the medial parvocellular regions of the hypothalamic PVN (pPVN) and scattered magnocellular neurons of the magnocellular PVN and SON, sites that control hypothalamic-pituitary–adrenal (HPA) axis and hypothalamo-neurohypophysial system (mHNS) activity (Yang et al. 1977). We and others have found that APJR mRNA and apelin immunoreactivity is expressed within a proportion of magnocellular PVN and SON vasopressinergic neurons (Reaux et al. 2001, O’Carroll et al. 2003), where the two peptides are segregated within distinct subcellular compartments within these cells (Reaux-Le Goazigo et al. 2004). Extrahypothalamic structures – such as the piriform cortex, the dentate gyrus, the nucleus of the lateral olfactory tract and the dorsal raphe nucleus – also express APJR mRNA (De Mota et al. 2000).
The molecular basis for the regulation of APJR gene transcription is not known. There is evidence however, to indicate that the steady-state levels of the gene transcripts for this receptor are altered by osmotic stimulation, glucocorticoids, stress, and during development (O’Carroll & Lolait 2003, O’Carroll et al. 2003). Additionally, a study on water deprivation shows a significant increase in both the number and labeling intensity of magnocellular apelin-immunoreactive cells in the rat SON and PVN (Reaux-Le Goazigo et al. 2004). This evidence suggests that the receptor plays an important role in regulating both hypothalamic mHNS and HPA axis activity. As a first step towards understanding the molecular mechanisms underlying how APJR gene expression is regulated, and thus ultimately influences the maintenance of homeostasis and the regulation of stress and cardiovascular responses, we have isolated and characterized the rat APJR gene and its 5'-flanking region and identified functional sequences controlling APJR gene expression.
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Materials and methods |
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A rat cosmid library in pWE15 (BD Biosciences Clontech, Palo Alto, CA, USA) was screened using a [-32P]dATP tailed coding region oligonucleotide probe, B78A (5'-CTGGCCATTGTCAGGCCAGTGGCTAA CGCTCGACTAAGGCTGCGAGTC-3'), specific for the APJR. Filters were hybridized overnight in 3 x SSC at 60 °C and were washed in 1 x SSC, 0.1% SDS at the same temperature. Digestion of the positive cosmid clone with NotI endonuclease yielded a fragment of > 20 kb which was subcloned into pGEM-4Z (Promega) and sequenced using BigDye terminators (Applied Biosystems, Foster City, CA, USA).
Rapid amplification of 5'-cDNA ends (5'-RACE)
5'-RACE was performed using the SMART RACE cDNA amplification kit (BD Biosciences Clontech) with 1 µg rat heart poly A+ RNA (Ambion, Huntingdon, UK) as the starting material. The initial PCR amplification (1 cycle at 94 °C for 1 min, 35 cycles at 94 °C for 5 s, 58 °C for 30 s and 72 °C for 2.5 min and a final extension cycle of 72 °C for 5 min) was performed using the adaptor-specific primer provided in the kit together with the APJR-specific primer RACE-1 (5'-AGATGTCAG CTGAGCGTCTCTTTTCTC-3') (Fig. 1
) directed towards the 5' end of the APJR genomic clone. The primary PCR product was purified and a nested amplification was performed using the anchor primer and an APJR- specific primer, RACE-2 (5'-AGGCCAT TGCCTGTGGTGCCTAGAAGG-3'). The amplified PCR fragments were subcloned into pCR2.1 by TA cloning (TA cloning system, Invitrogen) and sequenced using BigDye terminators (Applied Biosystems).
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A 500 bp probe, spanning the putative transcription start points was generated by PCR with two specific oligonucleotide primers: forward primer RP1, 5'-AGAG AAGGTGGGACCAGGAGG-3'; reverse primer RP2, 5'-CCCGTAGTAGTTGTAACCAT-3' (Fig. 1
). The PCR product was gel purified and subcloned into pGEM-4Z (Promega). The construct was linearized with EcoR1 and a [32P]UTP antisense riboprobe was synthesized with the MAXIscript in vitro transcription kit (Ambion, Austin, TX, USA). An RNase protection assay was performed using the RNase Protection Kit (Roche). In brief, total heart RNA samples (100 µg) were resuspended in hybridization buffer (40 mM Pipes, 400 mM NaCl, 1 mM EDTA, 80% deionized formamide (v/v), pH 6.4) containing 3 x 105 c.p.m. of the radioactively labelled antisense riboprobe. Samples were denatured at 95 °C, hybridized overnight at 45 °C and non-hybridized RNA was digested with RNase A (3.5 µg) and RNase T1 (25 U) for 30 min at 37 °C. Hybrids were then treated with Proteinase K (50 µg) in SDS (20%) for 15 min at 37 °C, extracted with phenol/chloroform and precipitated in ethanol. The final product was resuspended in loading buffer (80% formamide (v/v), 0.1% xylene cyanol, 0.1% bromo-phenol blue, 2 mM EDTA) and analyzed on a 6% polyacrylamide/7 M urea gel. The gel was exposed to Kodak XAR film at –80 °C with intensifying screens overnight and the protected band was sized relative to RNA size markers (Century Plus markers, Ambion).
Primer extension analysis
The oligonucleotide primer PE1 (5'-GCTGCACCTC TACCTTCCTGG-3') (Fig. 1) was used for primer extension analysis, performed using the primer extension system–AMV reverse transcriptase kit (Promega). The primer was end-labelled with [-32P]ATP (Perkin Elmer Life Sciences, Buckinghamshire, UK) and annealed to 1 µg rat heart poly A+ RNA (Ambion) at 55 °C for 20 min. The annealed product was reverse transcribed at 42 °C for 30 min and then subjected to analysis on an 8% denaturing polyacrylamide gel and visualized by autoradiography.
DNA constructs
To test promoter activity, a series of constructs was generated by PCR from the 5' end of the APJR genomic clone. The primers contained appropriate restriction sites to facilitate ligation into the multiple cloning sites of the pGL3-Basic luciferase reporter vector (Promega) in sense and antisense orientations. In this way, a series of constructs (3, 1.6, 1.35, 1.2, 1 and 0.15 kb) was generated consisting of deletions of the APJR putative promoter region. For deletion constructs, forward primers were as follows: for the 3102 bp construct (called 3 kb), 5'-CTATCCTAGCATCCTCCCATA-3'; for the 1667 bp construct (called 1.6 kb), 5'-AGTTCTGGCC CATTTCATTCT-3'; for the 1390 bp construct (called 1.35 kb) 5'-TCTTCCTGCGGAGCCATGGGGA-3'; for the 959-bp construct (called 1 kb), 5'-TCTTTCCT TTTGTGACTCTGT-3'; for the 157 bp construct (called 0.15 kb), 5'-CTCCCAGAGAGGCTAGTTCTG-3'. The antisense primer was located at bp –7 relative to the initiating ATG. Two additional constructs were generated by PCR: a 1232 bp (called 1.2 kb) DNA fragment (bp –1397 to –165) and an 0.8 kb DNA fragment (bp –966 to –165). These are identical to the 1.35 and 1 kb fragments respectively, but lack the first 158 nucleotides. These constructs were subcloned into the SacI site of pGL3-Basic in sense and antisense orientations.
Cell culture, transfections and reporter gene assay
Chinese hamster ovary (CHO-K1) (modified alpha essential medium, 10% fetal bovine serum (FBS), 2 mM glutamine, penicillin (1000 U/ml)/streptomycin (1000 µg/ml) (P/S)), COS-7 (Dulbecco’s modified essential medium, 10% FBS, 2 mM glutamine, P/S) and SH-SY5Y (Ham’s F12: Eagle’s minimum essential medium (1:1), 15% FBS, 1% non-essential amino acids, 2 mM glutamine, P/S) cells were maintained at 37 °C under 95% air/5% CO2 conditions. SH-SY5Y cells were used at passages 15–18. Twenty-four hours before transfection, cells were plated into 24 well plates at 90% confluency in medium without antibiotics. APJR promoter–luciferase construct DNA was transfected into SH-SY5Y, COS-7 and CHO-K1 cells at concentrations of 0.5 and 1.0 µg/well using Lipofectamine 2000 (Invitrogen). Renilla luciferase (4 ng/well; Promega) was co-transfected with the construct DNA for correction of transfection efficiency. Background levels of luciferase activity were determined by transfection of the empty pGL3-Basic vector. Five hours after transfection, SH-SY5Y cells were incubated in Optimem (Invitrogen) for 2 h and then maintained in standard SH-SY5Y medium containing antibiotics for 24 h. CHO-K1 and COS-7 cells were maintained in transfection medium for 24 h. Cells were then harvested in passive lysis buffer (Promega) and luciferase and Renilla activities were measured by the dual-luciferase assay (Promega) using a Berthold Technologies Lumat LB 9507 luminometer. Experiments were performed at least three times in triplicate.
Hormonal treatment included ß-estradiol (E2), forskolin and dexamethasone (all at 1 nM–5 µM) (Sigma-Aldrich). Cells were incubated in the presence or absence of hormones for an additional 36 h.
DNase I footprinting
DNase I footprint analysis was performed to define the approximate region of protein–DNA interaction. Overlapping DNA templates which covered the promoter region from bp –189 to –650 were PCR amplified from rat genomic DNA, gel purified, quantified and labelled at the 5' end with T4 polynucleotide kinase and [-32P]ATP. DNase 1 footprinting was performed using rat heart and rat lung nuclear extract obtained from Active Motif (Carlsbad, CA, USA). DNA templates were incubated with nuclear extract (25 µg) for 10 min in a final volume of 50 µl containing: 200 mM NaCl 25 mM Tris–HCl (pH 8), 50 mM KCl, 6.25 mM MgCl2, 0.5 mM EDTA, 10% glycerol and 0.5 mM dithiothreitol (DTT). After addition of 50 µl of a solution of 5 mM CaCl2 and 10 mM MgCl2, DNA was digested at room temperature with DNase 1 (3 µl of 0.05 U/µl) for 75 s. The reaction was stopped with 90 µl of buffer containing: 200 mM NaCl, 30 mM EDTA, 1% SDS and 100 µg/ml yeast RNA. The reaction products were purified by phenol–chloroform extraction and ethanol precipitation, and were analysed on a 6% polyacrylamide/7 M urea gel together with a 
X174 Hinf I DNA ladder and manual sequencing reactions (Sequenase version 2.0; USB, Cleveland, OH, USA). The gel was exposed to Kodak XAR film at –80 °C with intensifying screens and the positions of the footprints determined.
Electrophoretic mobility shift assay (EMSA)
Synthetic sense and antisense oligonucleotides (Table 1) were used as probes in gel-shift and super-shift assays. The upper and lower strands of each probe were labelled at the 5' end with T4 polynucleotide kinase and [-32P]ATP and purified on NucAway spin columns (Ambion). The end-labelled sense and antisense probes were mixed, denatured at 65 °C for 3 min and allowed to anneal by cooling to room temperature. Both rat heart and rat lung nuclear extracts (5 µg, Active Motif) in 10 µl final volume EMSA binding buffer (50 mM Tris–HCl (pH 7.5), 250 mM NaCl, 2.5 mM DTT, 2.5 mM EDTA, 5 mM MgCl2, 20% glycerol and 0.25 mg/ml poly(dI-dC)) were incubated at room temperature for 20 min, after which probes (2–3 x 104 c.p.m.) were added and the reactions allowed to incubate at room temperature for a further 30 min. Competition binding assays were performed by the addition of 100-fold molar excess of non-radioactive competitor oligonucleotide and incubation at room temperature for 20 min before addition of labelled probe. For antibody super-shift assays, 2 µg of anti-Sp1, CCAAT enhancer binding protein-(C/EBP) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), –glucocorticoid receptor or estrogen receptor-(ER) (Abcam Ltd, Cambridge, UK) antibodies were added to the mixture and incubated at 4 °C for 30 min before addition of labelled probe. The incubation was then continued at room temperature for a further 30 min. The DNA–protein complexes were resolved by electrophoresis on a 5% non-denaturing polyacrylamide gel in 0.5 x TBE (44.5 mM Tris, 44.5 mM Boric acid, 1 mM EDTA, pH 8.3) buffer and visualized by autoradiography by exposure to Kodak XAR film at –80 °C with intensifying screens.
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The rat APJR 5'-flanking region 1 kb construct, bp –966 to –7 subcloned into the pGL3-Basic luciferase vector, was used as a template for the mutagenesis reactions. Four Sp1 sites, and individual estrogen response element (ERE), glucocorticoid response element (GR) and C/EBP sites in the APJR promoter were deleted using the QuikChange II site-directed mutagenesis kit (Stratagene). The 2Sp1, 3Sp1, 4Sp1 and 5Sp1 sites were deleted from the luciferase construct using primers mut2Sp1a/mut2Sp1b, mut3Sp1a/mut3Sp1b, mut4Sp1a/mut4Sp1b and mut5Sp1a/mut5Sp1b respectively (Table 1). The ERE, GR and C/EBP sites were deleted using primers mutEREa/mutEREb, mut6Sp1a/mut6Sp1b and mutCEBPa/mutCEBPb respectively. Eighteen cycles of PCR were run and, after digestion with DpnI for 2 h, products were transformed into XL1-Blue super-competent cells. Site mutations were confirmed by sequence analysis.
Statistical analysis
Results are expressed as mean ± S.E.M. values. All data were analysed using the Statistical Package for Social Sciences, version 9.0 (SPSS Inc., Chicago, IL, USA), ANOVA followed by Scheffé’s F test. P < 0.05 was considered statistically significant.
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Results |
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Six genomic clones were isolated after screening a rat cosmid library in pWE15 using an oligonucleotide probe specific for the rat APJR. All six clones were found to be identical following Southern blot analysis. Digestion of the clone with NotI endonuclease yielded a fragment of ~20 kb which hybridized to the APJR cDNA probe. The genomic fragment was subcloned into pGEM-4Z and was shown by sequence analysis to contain the APJR gene. The genomic clone was found to extend ~8 kb 5' of the initiation codon. Fragments of the 5'-flanking region of the genomic clone were sequenced (these sequence data have been submitted to the GenBank database under accession number AY942137 [GenBank] ) and comparison with the rat cDNA clone (GenBank accession number AF184883 [GenBank] ) revealed that the receptor coding region and the 5'-flanking region over the first 227 bases is intronless. The 5'-flanking region shares 78% homology with the human APJR gene sequence over the first 79 bases (GenBank accession number U03642 [GenBank] ) and 86% identity with the mouse APJR gene sequence over the first 248 bases (GenBank accession number NM 011784).
Analysis of the 5'-flanking sequence
The nucleotide sequence of 4 kb of the 5'-flanking region of APJR was determined on both DNA strands and the sequence of the proximal 1.8 kb is shown in Fig. 2. The sequence contains two putative TATA boxes at bp –2295 and –1881 that may be too distant from the transcription initiation site to be functionally relevant, and a putative CAAT box at position –1257. Several potential transcription factor binding sites were also identified in the proximal promoter region, using the TESS-String-based Search (//www.cbil.upenn.edu/tess/) and AliBaba2/Transfac 6.0 (//wwwiti.cs.uni-magdeburg.de/~grabe/alibaba2/) computer programs, including a cluster of putative Sp1 recognition sequences, consensus sites for AP1 and AP2, putative EREs (bp –237 and –1049), an ERE half-site (bp –404) and a number of putative GRs. In addition, other putative elements present are sites for Pit-1a, GATA-1–2–3, MAZ, Oct-1, NF-
, USF, C/EBP and GCRE.
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5'-RACE was performed on heart poly A+ RNA in order to locate the transcription start site of the APJR mRNA transcript. Sequence analysis of the two products observed, 383 and 346 bp (Fig. 3A), mapped two main transcriptional start sites at positions bp –247 and –210 relative to the initiating ATG. The positions of the transcriptional start sites were confirmed by two different experimental approaches: RNase protection and primer extension analyses. For RNase protection, total RNA from rat heart was hybridized to a [32P]UTP antisense riboprobe derived from the genomic clone. Two protection products were obtained with the 500 bp probe – a major product of ~276 bp and a minor product of ~239 bp (Fig. 3B) – which mapped the transcriptional start points to positions consistent with the results obtained by 5'-RACE. Primer extension analysis using a [-32P]ATP-labelled primer directed against the sequence upstream of the initiating methionine revealed an extension product of 51 nucleotides (Fig. 3C), which is consistent with the major product obtained by RNase protection. A predicted second extension 14 bp product was not resolved in our gels. Taken together, these experimental approaches identify two transcriptional start sites at bp –247 and –210.
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To localize the promoter region regulating APJR expression and confirm promoter activity, a series of constructs was generated by PCR from the 5'-flanking region of the APJR gene. A series of seven 5'-truncated fusion constructs were generated containing varying lengths of 5' DNA located upstream of the translation initiation site ligated to the coding region of the luciferase gene in the pGL3-Basic reporter vector in both sense and antisense orientations. Reporter constructs were transfected into SH-SY5Y cells, a neuroblastoma cell line that expresses the native APJR gene under culture conditions. To test the cell-type specificity of the promoter activity, the deletion constructs were also transfected into CHO-K1 and COS-7 cells which do not endogenously express APJR (O’Carroll, unpublished data). Transient transfection of the series of constructs (Fig. 4) showed that, in SH-SY5Y cells, the three largest constructs (3.0, 1.6 and 1.35 kb) did not exhibit promoter activity significantly above that of the empty vector. Transfection with the shorter 1.2, 1.0 and 0.8 kb constructs, however, showed clear promoter activity with 5- to 6-fold greater activity than the empty vector. The shortest promoter construct, 0.15 kb, showed no promoter activity. Transfection of the same promoter constructs into CHO-K1 and COS-7 cells showed similar patterns of luciferase activity, however transfection into COS-7 cells resulted in higher levels of luciferase activity (up to 13-fold greater than the empty vector) and promoter activity was seen with the 1.35 kb construct in this cell line. The promoter constructs, subcloned in antisense orientations used as controls, did not increase luciferase activity (data not shown).
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To determine the approximate location of the cis-elements that bind to nuclear extracts, DNase I footprint analysis of the core promoter region was performed using rat heart and rat lung nuclear extracts. The data for rat lung nuclear extracts showed that as many as seven footprints were identified within the gene’s proximal promoter region. Using a probe against the bottom DNA strand labelled at its 5' end at bp –189, five prominent regions of nuclease protection were observed in rat lung extracts at bp –206 to –218, –227 to –242, –259 to –284, –298 to –326 and –331 to –343 (Fig. 5A, FP1–5 respectively) that correspond to putative Sp1, Sp1/ERE, Sp1, Sp1/MAZ and C/EBP binding sites respectively (Fig. 6). Using a probe labelled at its 5' end at bp –650, two regions of nuclease protection were observed at bp –564 to –525 and at –606 to –580 (Fig. 5B, FP6 and FP7) that correspond to putative GR/PR/Sp1 binding sites (Fig. 6). Similar results were obtained using rat heart nuclear extracts (data not shown).
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To identify the trans-acting factors that bind to the putative binding sites, EMSAs were performed using a series of nine oligonucleotides corresponding to the sequences of the putative binding sites (Table 1) and rat lung nuclear extracts. As shown in Fig. 7, one major band (indicated by arrows) and several minor DNA–protein complexes were observed with seven individual probes. No DNA–protein complexes were observed using probes 1Sp1 or 7Sp1 (data not shown). A 100-fold molar excess of unlabelled consensus Sp1, ERE, GR or C/EBP oligonucleotides (self-competitor, Fig. 7A–G, lanes 3) completely inhibited the complex formations, while in the presence of unlabelled non-specific competitor (AP2 competitor, Fig. 7A–G, lanes 4), the specific bands remained. These competition assays demonstrate DNA–protein binding specificity.
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, super-shift bands were observed using anti-Sp1 antibody with DNA–protein complexes generated with the 2Sp1, 3Sp1, 4Sp1 and 5Sp1 probes (Fig. 7A, C, D and E respectively, lanes 5). No super-shift was observed with DNA–protein complex 6Sp1 using anti-Sp1 antibody (Fig. 7 G, lane 5). However, as a putative GR motif was also predicted within this region, anti-glucocorticoid receptor antibody was used in an antibody super-shift assay with radiolabelled 6Sp1 probe. Incubation with anti-glucocorticoid receptor antibody resulted in a super-shift (Fig. 7 G, lane 6) indicating functionality of the GR motif in the 6Sp1 oligonucleotide region. Super-shift bands were observed using anti-ER antibody with DNA–protein complex ERE (Fig. 7B, lane 5) and anti-C/EBP antibody with DNA–protein complex C/EBP (Fig. 7F, lane 5). These results strongly indicate that Sp1, ERE, GR and C/EBP motifs in the promoter region of the APJR gene may be responsible for APJR gene basal promoter activity through interaction with Sp1, ER, glucocorticoid receptor and C/EBP respectively. Similar DNA–protein interactions were obtained using rat heart nuclear extracts (data not shown). Mutational analysis of putative Sp1, ERE, GR and C/EBP motifs within the upstream promoter
To confirm that Sp1-, ERE-, GR- and C/EBP-motifs in the promoter region are responsible for APJR gene basal promoter activity through interaction with Sp1, ER, glucocorticoid receptor and C/EBP, and to determine the relative contribution of each site to basal promoter activity, the four Sp1 motifs, the ERE, GR and C/EBP motifs in the APJR 5'-flanking region 1 kb construct, bp –966 to –7, were deleted by site-directed mutagenesis. These mutants were transiently transfected into SH-SY5Y cells. In comparison with the wild-type vector (APJR 5'-flanking region 1 kb construct, bp –966 to –7, subcloned into the pGL3-Basic luciferase vector), basal transcriptional activity of the APJR promoter was decreased by deletions of all individual motifs except the 4Sp1 motif, indicating their involvement in constitutive transcriptional activity of the promoter. Deletion of the 2Sp1, 5Sp1, ERE and C/EBP motifs caused significant reductions of promoter activity (37%, 74%, 51% and 42% respectively) whereas deletion of 3Sp1 and 6Sp1(containing putative GR motif) motifs decreased transcriptional activity only marginally (23% and 24% respectively) (Fig. 8). Interestingly, deletion of the 5Sp1 motif resulted in a reduction in basal transcriptional activity (74%) to background levels found with the promoterless vector pGL3-Basic (not shown).
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To investigate the functional significance of potential promoter elements in the APJR 5'-flanking region identified by consensus sequence, the responses to various hormones on 1 kb of APJR promoter were measured. As shown in Fig. 9, treatment with 17ß-estradiol and dexamethasone (at concentrations of 1 nM–5 µM) did not significantly influence activity of the rat APJR promoter in SH-SY5Y cells. In contrast, treatment with forskolin at concentrations of 1 and 5 µM significantly down-regulated promoter activity.
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Discussion |
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To understand the molecular mechanisms underlying regulation of the rat APJR gene we characterized the genomic structure of an isolated rat APJR genomic clone and analyzed the transcriptional regulation. The 5' end of the APJR genomic clone was sequenced to allow definition of the 5' end of the transcript and potential promoter sequences. The sequence obtained confirmed the published DNA sequence of rat APJR and extends it at the 5' end. The clone spans > 20 kb, including more than 8 kb upstream of the reported cDNA sequence and the coding region. Comparison of the genomic and cDNA clones revealed that the receptor coding region is intronless. Using 5'-RACE and rat heart poly A+ RNA, two main putative transcription start sites were located at 247 and 210 nucleotides upstream from the ATG initiating codon. This result was confirmed by RNase protection and primer extension analyses.
Although the 5'-flanking region of the rat APJR gene near the transcription start site lacks a TATA box, there is one potential CAAT box and several AP-1, AP-2 and Sp1 motifs present. Associated until recently with constitutively expressed housekeeping genes, numerous highly regulated genes including developmentally regulated and tissue-specific genes have been shown to have TATA-less promoters (Minowa et al. 1992a,b, Collins et al. 1993, Gao & Kunos 1993). In some genes that have promoters lacking TATA boxes, binding of Sp1 has been shown to play a critical role in transcription initiation (Pugh & Tjian 1990, 1991). The 5'-flanking APJR sequence has potential Sp1 elements near the transcriptional start sites and interestingly, also has two MAZ sites that may be sites of regulation in TATA-less promoters (Parks & Shenk 1996). It is interesting to note that the promoter region (2 kb upstream of the initiating methionine) of the human APJR gene (GenBank accession number U03642 [GenBank] ) is also TATA-less but does contain a number of Sp1 and AP-1 motifs. Putative binding sites for transcription factors that may be involved in tissue specificity were also identified on the rat APJR promoter. The transcription factor Pit-1 is specifically expressed in the pituitary (Ingraham et al. 1988), while the octamer-binding protein Oct-1 – which can activate eukaryotic promoters that lack a TATA box (Elsholtz et al. 1990) – is expressed in cells of the anterior pituitary.
To assess whether the cloned 5'-flanking sequence of the APJR gene functions as a cell-type- specific promoter, transient expression assays were performed with seven DNA fragments in an APJR-expressing neuroblastoma cell line (SH-SY5Y) as well as in two cell lines which do not express the APJR gene under culture conditions (CHO-K1 and COS 7). While transfection efficiency was similar in all three cell lines, levels of luciferase activity after transfection varied between the cell lines, with CHO-K1 cells showing the lowest levels of activity, followed by SH-SY5Y cells (increase of about 2-fold) and COS-7 cells (increase of about 3- to 4-fold). This difference in luciferase activity between cell lines may be due to species-specific or cell-specific differences in the control of transcription. The presence of promoter activity in cell lines that do not express the APJR suggests however that the cis elements required for tissue specificity are not present in the promoter fragments used in the transfection studies but may be located upstream of bp –3000.
In all three cell lines, transient expression of the two largest constructs, 3 and 1.6 kb, did not exhibit promoter activity significantly over background levels. In SH-SY5Y and CHO-K1 cells, the 1.35 kb fragment also exhibited no significant increase in promoter activity over the empty vector; however, this fragment showed activity in COS-7 cells. Analysis of the 1.2 kb fragment, which is identical to the 1.35 kb fragment but lacks the proximal 158 bp (bp –165 to –7) of the 5'-flanking region, revealed an appreciable increase in promoter activity over the 1.35 kb fragment in all three cell lines (4- to 6-fold in SH-SY5Y and CHO-K1 cells), suggesting that the region between bp –165 and –7 contains negative regulators of APJR expression in these cells. Removal of region bp –1397 to –966 to generate the 1 kb fragment also resulted in an increase of promoter activity suggesting that there are also potential negative regulatory elements in this region. Transient transfection of the 0.8 kb fragment, which lacks both of these regions (bp –1397 to –966, and –165 to –7), resulted in the greatest levels of luciferase activity in all three cell lines tested. The shortest fragment (0.15 kb) had minimal promoter activity. Thus the putative region promoting transcription is located between bp –966 and –165.
The 0.8 kb fragment having the highest transcriptional activity in SH-SY5Y cells was found to contain consensus sequences for transcription factors such as GATA-1, -2, -3, ER, GR, Oct-1, C/EBP, AP-1, -2 and contained numerous Sp1 sites. DNase I footprint analysis identified protected sequences in this promoter region that include putative GR, ERE, C/EBP and Sp1 motifs. To determine which nuclear proteins recognize these putative regulatory sites, these regions were used as probes in EMSA and super-shift assays. In this way, we identified four major DNA–protein complexes capable of binding the Sp1 transcription factor, and three additional complexes capable of binding ER, glucocorticoid receptor and the C/EBP transcription factor respectively.
To determine whether these sites are involved in the functional regulation of the gene, mutation constructs were transiently transfected into SH-SY5Y cells (which express ER, ERß (Lee et al. 2003) and glucocorticoid receptor (Gold et al. 1999)). These site-directed mutation analyses demonstrated a dependence of basal transcriptional activity of the APJR promoter-reporter gene construct on three of the four Sp1 sites, and on the ERE, GR and C/EBP sites. As the transcriptional activity was not completely lost upon individual mutations of the 2Sp1, 3p1, 6Sp1 (containing overlapping functional GR motif), ERE and C/EBP sites, binding of other transcription factors to multiple sites may be necessary for full promoter activity. However, deletion of the 5Sp1 site completely abolished promoter activity within these cells, suggesting that the 5Sp1 motif plays a major role in activation of the APJR promoter. Sp1 is expressed in most tissue types and is the prototype of the Sp family of transcription factors binding to GC-box motifs in promoters, regulating transcriptional activation of promoters lacking TATA consensus sequences either by recruitment of transcription factors or by prevention of promoter methylation.
Interestingly, disruption of the ERE and GR motifs led to a loss of promoter activity, thus suggesting a hormone-independent promoter induction with ER and glucocorticoid receptor, and indicating an important role for these sites in constitutive transcriptional activity of this promoter and thus a role in regulation of basal levels of APJR. Although, conventionally, ligand-bound ERs homo- or heterodimerize and bind to consensus EREs, unliganded ER induction of the human corticotropin-releasing hormone-binding promoter, that is dependent on ERE half-sites, has also been described (van de Stolpe et al. 2004). That study suggests that unliganded ERs may form transcriptionally active heterodimers with transcription factor components that are known to contribute to constitutive promoter activity, e.g. AP1 or cAMP response element-binding protein (CREB), or, alternatively, interact with basal transcriptional components on the promoter (e.g. transcription factor IIB) to augment basal transcriptional activity.
The presence of putative GR motifs within the 5'-flanking region is consistent with our hypothesis that APJR expression is dependent upon circulating corticosteroids – recent studies in our laboratory demonstrate that APJR mRNA transcripts can be regulated by osmotic stimulation and by changes in serum corticosteroid concentrations (O’Carroll & Lolait 2003, O’Carroll et al. 2003), changes in APJR mRNA levels that may be a consequence of changes in APJR gene transcriptional activity. As is the case for the vasopressin gene promoter, osmotic regulation (e.g. dehydration) may be manifested via cAMP, levels of which increase in the SON after the onset of an osmotic stimulus (Carter & Murphy 1989), indirectly acting upon C/EBP response elements in the APJR gene. To determine if the regulatory elements identified in the 5'-flanking region of the APJR gene may mediate changes in APJR expression, the responses to various hormones on 1 kb of APJR promoter were measured. Surprisingly, in our cell transfection studies we did not see any significant changes in promoter activity by treatment with 17ß-estradiol or dexamethasone using the 1 kb construct in SH-SY5Y cells. Thus it seems that additional physiologically important transcription factor binding sites for these hormones may be located 5' of the analyzed promoter region or they may act through an indirect mechanism or at a post-transcriptional level. Gel-shift analysis indicated that the ERE and GR sites have the potential to be functional binding sites, however it may be that other unidentified factors in addition to ER are necessary for generation of a response from the APJR promoter to estrogen, as has been shown for the chicken riboflavin carrier protein gene (Bahadur et al. 2005). Induction and/or repression of promoter activity may also be a balance of integrative activities of several functional promoter elements as has been shown for the regulation of osteocalcin gene transcription by glucocorticoids (Aslam et al. 1995). The mechanism of ER- and glucocorticoid receptor-mediated transcriptional regulation of the APJR promoter remains unclear, but it is clear that these receptors may have a role to play in regulation of basal levels of APJR.
Interestingly, we did observe a significant inhibition of APJR promoter activity by treatment with forskolin. Forskolin is thought to transduce its effects by control of the cAMP signal cascade, therefore cAMP responsive elements may be located in the region between bp –1200 and the start site. Although no typical cAMP responsive elements have as yet been identified in the 5'-flanking region, other regulatory sequences present in this region such as AP-2, Pit-1 or Sp1 may also mediate cAMP-dependent gene regulation (Imagawa et al. 1987, Roesler et al. 1998, Chen et al. 1999, Gronning et al. 1999). For example, it has been suggested that cAMP may influence the interaction of Sp1 with cofactors shared with other transcription factors involved in gene regulation (Ahlgren et al. 1999). Further analyses are ongoing to elucidate the molecular basis of the regulation of the APJR promoter by forskolin.
There are a number of repressors that may bind to the putative repressor region bp –1397 to –966. This region contains consensus sequences for two GATA-1 motifs and numerous Sp1 sites. GATA-like sites have been shown to bind the DNA-binding proteins Ash1p and TRPS1 that actively repress gene transcription (Malik et al. 2001, Maxon & Herskowitz 2001) while members of the Sp-multigene family can act both as transcriptional repressors and activators. Further studies are required to identify the proteins binding to these sequences, while mutational analysis of these sites will reveal whether they have any functional role in transcriptional activity of the APJR promoter.
In conclusion, the results described here are a first step towards understanding the molecular mechanisms underlying APJR tissue-specific expression and its regulation of hypothalamic mHNS and HPA axis activity. We have determined the structure of the gene and provide evidence that the promoter is under complex regulation by Sp1, C/EBP, estrogen and glucocorticoid protein complexes. Sp1 is implicated as a major transcriptional regulator of APJR promoter activity. We have also shown regulation of the promoter in SH-SY5Y cells by the cAMP-dependent signal cascade. This study provides the groundwork for further studies on APJR gene regulation and function, and provides important insights into the complex regulation of APJR expression.
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Received 11 October 2005
Accepted 2 November 2005
Made available online as an Accepted Preprint 21 November 2005
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