©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Human Tryptophan Hydroxylase Gene Promoter
TRANSCRIPTIONAL REGULATION BY cAMP REQUIRES A NEW MOTIF DISTINCT FROM THE cAMP-RESPONSIVE ELEMENT (*)

(Received for publication, October 6, 1994)

Sylviane Boularand(§)(¶) Michèle C. Darmon Philippe Ravassard (¶) Jacques Mallet

From the Laboratoire de Génétique Moléculaire, de la Neurotransmission, et des Processus Neurodégénératifs, C.N.R.S., F91198 Gif-sur-Yvette Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We isolated and sequenced 2,117 nucleotides of the promoter region of the human tryptophan hydroxylase (TPH) gene. Transient transfection in pinealocyte cultures and PC12 cells was used to investigate the human TPH (hTPH) gene promoter activity and its regulation by the cAMP signaling pathway. A region of 2,117 base pairs upstream of the transcription initiation site of the hTPH gene efficiently directed the transcription of a luciferase reporter gene but not in a cell-specific manner. The hTPH promoter activity was significantly enhanced by a cyclic AMP analog in the two cell types. Deletion analysis showed that the promoter region from -73 to +2 is sufficient to direct cAMP-dependent transcription, although it does not contain a motif exhibiting a significant identity to the cAMP-responsive element (CRE) or AP-2 binding site. Following site-directed mutagenesis of the region between -73 and -51, an inverted CCAAT box motif was identified as essential for cAMP inducibility of the hTPH promoter. This sequence between -73 and -51 alone allowed cAMP enhancement of transcription when fused to a heterologous promoter. Additionally, electrophoretic mobility shift assays showed that a specific protein-DNA complex is formed between an oligonucleotide corresponding to the inverted CCAAT box motif and nuclear proteins from pinealocytes treated or not treated with cAMP. Thus cAMP responsiveness of hTPH gene expression is mediated by a cis-acting element, which shares strong identitiy with an inverted CCAAT box and which binds to a constitutively produced nuclear factor.


INTRODUCTION

Tryptophan hydroxylase (TPH) (^1)is the key enzyme in the biosynthetic pathway of the neurotransmitter serotonin. In the central nervous system, TPH is mainly expressed in the brainstem raphe nuclei, where the corresponding neurons project onto almost every region of the brain in a highly divergent manner(1) . Serotonin has been shown to modulate numerous basal brain functions including sleep, thirst, hunger, reproduction, arousal, and awakeness (2, 3) . The synthesis and activity of the TPH enzyme, therefore, has to be tuned to very different physiological situations. It has been shown that glucocorticoids and Ca/calmodulin kinase are able to increase TPH activity (4, 5) and that cAMP treatment increases TPH mRNA levels in primary cultures of raphe neurons(6) , but little is known about the regulation of TPH expression in the central nervous system.

The pineal gland also contains large amounts of TPH. Serotonin is the metabolic precursor of melatonin(1) , which is the main neurohormone produced and secreted in a circadian manner by the pineal gland(7, 8, 9) . This daily fluctuation depends upon the activity of the suprachiasmatic nucleus, from which regulatory signals are transmitted to the pineal gland by the sympathetic fibers of the cervical superior ganglion(10) . Released noradrenaline activates beta-adrenergic receptors, which stimulate adenylate cyclase(11) , thereby activating cAMP-dependent gene transcription. Although TPH is not believed to be the rate-limiting component of melatonin synthesis in the pineal gland, its activity and mRNA level are also subject to a circadian rhythm (12, 13, 14) . Elevated concentrations of intracellular cAMP have also been reported to increase TPH synthesis in the pineal gland(13, 15) . Therefore, cAMP-dependent signaling pathways appear to be a major intracellular relay for environmental stimuli, able to modulate TPH expression at transcriptional or post-transcriptional levels.

There is as yet no direct evidence to show that cAMP or other compounds modulate transcription of the TPH gene. To study in more detail the factors affecting serotonin biosynthesis in the brain, we used a molecular approach to assess the regulation of human TPH. We have studied the genomic organization of the hTPH gene and shown that a single transcriptional initiation site produces a large diversity of TPH mRNAs. This diversity is restricted to the 5`-leader sequence and results from a complex combination of exon and intron splicing in this region (Boularand et al., preceding article; (50) ). These different 5`-ends of the human TPH mRNA might be differentially degraded and translated and thereby involved in the regulation of TPH expression.

In the present study, we show that the 2-kb region upstream of the single human TPH mRNA cap site exhibits the characteristic features of a promoter and is able to drive the expression of a luciferase reporter gene in primary cultures of rat pinealocytes and in PC12 cells. We then demonstrate that the transcriptional activity of the hTPH promoter is modulated by cAMP treatment in the two cell types, and we identify by site-directed mutagenesis a cAMP regulatory motif very similar to an inverted CCAAT box and which confers cAMP responsiveness to a heterologous promoter.


MATERIALS AND METHODS

Plasmid Constructions

KSLuc, a promoterless luciferase vector with a transcription terminator upstream of the single cloning site (HindIII) was constructed by Faust and Catherin (^2)and was used to test the various sequences of the hTPH gene for promoter activity. The SalI-AvaI fragment of the hTPH promoter was isolated from the hTPH genomic clone 12 (preceding article, (50) ). This fragment contained the sequence from position -2117 to +29 relative to the RNA initiation site. Four restriction fragments of the promoter were subcloned into KSLuc upstream of the luciferase reporter gene as follows. PL was obtained by ligating the blunt-ended SalI(-2117)-AvaI(+29) fragment into the HindIII site (made blunt-ended) of KSLuc. Three additional contructions (PL, PL, and PL), truncated at the 5`-end of the hTPH promoter fragment, were produced using ScaI, SspI, and PvuII restriction sites, respectively. ScaI(-1402)-AvaI(+29), SspI(-724)-AvaI(+29), and PvuII(-51)-AvaI(+29) fragments were each subcloned into KSLuc. The constructs PL, PL, PL, and PLDelta3` corresponding to deletions at the 5`- or 3`-end of the 2146-bp hTPH promoter fragment were obtained by PCR amplification using pairs of hTPH-specific primers. PLDelta3` differs from PL by the elimination of 29 nucleotides downstream of the TPH promoter cap site. The PCR products were isolated from low melting point agarose gels (1.5-3%) and inserted into KSLuc. The sequences of the upstream oligonucleotides in constructs PL, PL, PL, and PLDelta3` were, respectively, O (5`-TTGGTTTGGAGAGAATGTCCA-3`), O (5`-TTGTGTGGTTAAGGACGGCC-3`), and O (5`-CTTCTCATTGGCCGCTGC-3`), and the sequence of the downstream primer was either O (5`-CGGGCGCCAGTAGGTGC-3`) (for PL, PL and PL) or O: 5`-GGCCGGCGGCCCCGCG-3` (for PLDelta3`).

The plasmid DeltaTK-Luc (a gift from Dr Hughes de Thé; (16) ) contains a thymidine kinase promoter fragment (-109 to +51) fused to the firefly luciferase reporter gene and was used to subclone wild type or mutated inverted CCAAT box oligonucleotides (Wt and M). The plasmids Wtx3 and Mx3 contain, respectively, three direct tandemly repeated wild type or mutated inverted CCAAT box oligonucleotides. The oligonucleotide sequences used were as follows: Wt, 5`-CTTCTCATTGGCCGCTGCCCAG-3`; M, 5`-CTTCTCTTTAGCCGCTGCCCAG-3`.

Site-directed Mutagenesis

Mutations were introduced into hTPH promoter fragments by site-directed mutagenesis using PCR as described by Higuchi(17) , with the upstream (O) and downstream (O) primers described above as the outside primers and appropriate nested oligonucleotides carrying the desired mutation. The AP-4 binding site (CAGCTG) of PL was mutated to 5`-CTACTT-3` with the oligonucleotide 5`-GGCCGCTGCCCTACTTCTCCGACGC-3` and its reverse complement to give PL. The same procedure was used to produce mutations in the CCAAT box and the RE2 element separately and together. Sequences were as follows: PL (mutated CCAAT box), 5`-CTTCTCTTTAGCCGCTGCCCAGCTG-3`; PL (mutated RE2), 5`-CTTCTCATTGGCCTTTACCCAGCTG-3`; PL (mutated RE2 and CCAAT box), 5`-CTTCTCTTTAGCCTTTACCCAGCTG-3`. All mutations were confirmed by sequencing with the Sequenase kit (U. S. Biochemical Corp).

Cell Culture and DNA Transfection

Pinealocyte Culture

Pineal cells were obtained by the method of Buda and Klein(18) . Briefly, pineal glands from newborn Wistar rats (2 days old), were removed, placed immediately in Joklick medium, and then incubated for 30 min at 37 °C in the same medium containing 0.25% trypsin. The pinealocytes were rinsed with RPMI supplemented with 20% fetal calf serum, dissociated with a Pasteur pipette, and collected by centrifugation. Then, the pinealocytes were resuspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and were plated at a density of 0.25 times 10^6 in polyornithine-coated dishes (diameter, 1.6 cm). After 20 h of culture, pineal cells were transfected with the DNA-lipopolyamine complex according to the method of Loeffler et al.(19) . Briefly, a DNA/lipopolyamine transfection solution containing 0.5 µg of DNA (0.375 µg of the DNA of the plasmid to be tested and 0.125 µg of RSV-CAT) and 5 µg of lipopolyamine (Transfectam) was added to the cells in serum-free Dulbecco's modified Eagle's medium. The cells were incubated for 2 h and then washed and cultured in the maintenance medium.

PC12 Cell Culture

PC12 cells were grown in RPMI 1640 supplemented with 10% horse serum and 5% fetal calf serum. For transient assay experiments, 1 times 10^6 PC12 cells were resuspended in 0.15 ml of serum-free RPMI 1640 and transfected by electroporation using a Bio-Rad gene pulser with 5 µg of each DNA of plasmid to be tested, 3 µg of carrier DNA, and 2 µg of RSV-CAT plasmid to assess transfection efficiency. The cells received a single electrical pulse at 200 V delivered from a total capacitance of 960 microfarads and were placed in serum-containing medium.

Treatment with 8-Bromo-cyclic AMP (8-Br-cAMP)

To study the effect of an increased intracellular cAMP level on hTPH promoter activity, pinealocytes and PC12 cells were treated with 1 mM 8-Br-cAMP 24 h after transfection with hTPH promoter-luciferase fusion constructs and were harvested 20 h later for the luciferase assay.

Luciferase and Chloramphenicol Acetyltransferase Assays

Pinealocytes and PC12 cells were harvested in 1 and 5 ml of phosphate-buffered saline, respectively, centrifuged, and resuspended in 200 µl of lysate buffer (25 mM Tris-phosphate, pH 7.8, 8 mM MgCl(2), 1 mM dithiothreitol, 1 mM EDTA, 1% Triton, 15% glycerol, 1% bovine serum albumin). Cell debris was removed by centrifugation. Luciferase assays were carried out using a Lumat LB9501 (Berthold) luminometer in 150 µl of reaction mixture (0.08 mM luciferin, 0.1 mM ATP, 25 mM Tris-phosphate, pH 7.8, 8 mM MgCl(2), 1 mM dithiothreitol, 1 mM EDTA, 1% Triton, 15% glycerol). Preliminary studies indicated that the luciferase activity in the cell lysate of one transfection was in the linear range of the assay. The luciferase activity resulting from each hTPH promoter construct was normalized to the CAT activity resulting from the co-transfected RSV-CAT vector. CAT assays were performed using the liquid scintillation counting method. The amount of the cell lysate used was always in the linear range of the CAT assay, as assessed using a reference CAT preparation.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay

Nuclear extracts from pinealocytes and PC12 cells were prepared according to the method of Schreiber et al.(20) . The concentration of protein was determined by the Bradford method and was between 1 and 3 mg/ml in nuclear extract preparations. The double-stranded TPH-CCAAT oligonucleotide (Wt) was labeled at the 5`-end by incubation with [-P]ATP and T4 polynucleotide kinase. The binding reactions was performed at 4 °C for 20 min with 0.2 ng of labeled TPH-CCAAT probe, 0.2 µg of poly(dI-dC), and 1 µg of nuclear extract, in the following buffer: 14 mM Hepes, pH 7.8, 80 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 35 µg/ml bovine serum albumin, 10% glycerol. Unlabeled double-stranded oligonucleotides (TPH-CCAAT (Wt), mutated TPH-CCAAT (M), CRE-TH, and POU-IgH) at 250-fold molar excess were used for competition experiments. DNA-protein complexes were separated by electrophoresis on a 5% polyacrylamide gel in 0.25 times Tris borate/EDTA at 10 V/cm. The oligonucleotide sequences used were as follows: Wt, 5`-CTTCTCATTGGCCGCTGCCCAG-3`; M, 5`-CTTCTCTTTAGCCGCTGCCCAG-3`; CRE-TH, 5`-GAGGGGCTTTGACGTCAGCCTGGCCT-3`; POU-IgH, 5`-CTGGGTAATTTGCATTTCTAAA-3`.


RESULTS

Functional Characterization of the Human Tryptophan Hydroxylase Promoter in Cell Cultures

A hTPH genomic DNA clone (12), containing the entire 5`-noncoding region and a 2-kb fragment upstream of the TPH RNA start site was isolated by screening a human genomic library (preceding article, (50) ). The nucleotide sequence of the 5`-flanking region of the hTPH gene was determined (Fig. 1) and revealed the presence of several canonical cis-regulatory elements including a ``TATA box''(21, 22) , a ``CCAAT box'' in an inverted orientation (ATTGGCC; (22) ) and a CCACCC element(23) , which are located at -29 nucleotides, -62 nucleotides and -100 nucleotides, respectively, relative to the transcription start site. Upstream and close to the hTPH promoter cap site, there are other putative binding sites for transcription factors, some of which are required to promote constitutive or induced transcription from many viral or cellular eukaryotic promoters (three potential GC boxes, C/EBP, AP2 and AP4 binding sites, and two 9-bp imperfect direct repeats, RE1 and RE2).


Figure 1: Nucleotide sequence of the 5`-flanking region of the human tryptophan hydroxylase gene. The nucleotide sequence from position -2117 to +32 is numbered relative to the transcription initiation site labeled +1. Putative cis-regulatory elements (TATA box, inverted CCAAT box, CCACC box, GC rich boxes, C/EBP, AP-4 and AP-2 binding sites, and two 9-bp imperfect direct repeats) are underlined.



We first verified that the SalI/AvaI fragment carrying the 2,117 nucleotides upstream of the hTPH RNA initiation site had a promoter activity. Since no mammalian serotonergic neuronal cell line or immortalized pineal gland cell line expressing the TPH gene was available, the hybrid construct (PL) was used to transfect primary cultures of newborn rat pinealocytes. The promoterless luciferase vector (KSLuc) was similarly introduced into these cells as negative control of transfection. The resulting luciferase activity was normalized with respect to the CAT activity expressed from a co-transfected plasmid carrying an RSV promoter-CAT gene fusion (RSV-CAT; (24) ). The 2,117-bp TPH promoter-luciferase construct, in the sense orientation (PL), efficiently directed luciferase expression, whereas the same fragment, in the reverse orientation (PL), did not (Fig. 2). Thus, the 2,117-bp genomic fragment functioned as a promoter in an in vitro assay. To further characterize the regulatory domains of the hTPH promoter that are involved in the basal transcriptional activity of this gene, several deletions were generated, either by enzymatic restriction (PL and PL) or by PCR amplification (PL, PL, and PL) and inserted upstream of the luciferase reporter gene. The luciferase activity expressed by constructions PL, PL PL, PL and PL, which contained 1,402, 724, 252, 204, and 73 bp of the hTPH 5`-flanking region, respectively, showed that deletions of fragments in the region between positions -2,117 bp (PL) and -252 bp (PL) had no significant effect on hTPH promoter activity (Fig. 2). Longer deletions led to a large decrease of the transcriptional activity. Thus, the proximal 252-bp region upstream of the hTPH mRNA initiation site contains information required for promoter function. The transcriptional activity of the shortest construct containing the -73 to +29 bp fragment (PL) was 85% lower than that of PL but 5-8-fold higher than the background level obtained with the promoterless vector (KSLuc) or the construct PL.


Figure 2: Deletional analysis of cis-active DNA elements in the human TPH gene 5`-flanking region. Top, a series of constructions containing various lengths of the 5`-region of the hTPH promoter plus 29 bp of exon 1 were fused to the structural gene for firefly luciferase. The hTPH promoter-luciferase constructs were introduced into primary cultures of rat pinealocytes and PC12 cells for transient expression assays. Luciferase activity is expressed as relative light units (RLU) normalized to CAT activity in the same cell extract (RSV-CAT) expressed in milliunits. The deletion constructs of the hTPH promoter are indicated on the abscissa. PL corresponds to the hTPH promoter fragment of 2,117 bp inserted into the promoterless luciferase vector (KSLuc) in the reverse orientation. Data are mean ± S.E. (bars) values from at least three independent experiments done in triplicate. Bottom, schematic representations of the fragments of the hTPH promoter.



To determine whether the hTPH promoter sequence contained the elements necessary for tissue-specific expression of hTPH gene, the TPH promoter-luciferase fusion constructs were also used to transfect the rat pheochromocytoma cell line (PC12) and human HeLa cells, which do not exhibit TPH activity. Transfected PC12 cells (Fig. 2) and HeLa cells (data not shown) each expressed high levels of luciferase activity. Thus, the transfected sequences did not contain information sufficient for cell type-specific expression. However, PC12 cells transfected with the construct PL containing the hTPH promoter fragment of 252 bp expressed twice the luciferase activity of constructs possessing longer hTPH promoter fragments (PL and PL). This suggests the presence of a weak tissue-specific repressor between positions -724 and -252 on the hTPH gene promoter (Fig. 2).

Transcriptional Activity of the Human TPH Gene Is Regulated by Cyclic AMP

Intracellular cAMP is a major second messenger in pineal gland and is thought to be a key regulator of TPH gene activity. We therefore investigated whether the hTPH promoter can be regulated by a cAMP-dependent mechanism. Pinealocytes were transfected either with the PL plasmid or control plasmid (KSLuc) and treated with 1 mM 8-Br-cAMP for 20 h. The luciferase activity of each construct in the presence and absence of 8-Br-cAMP was compared. Plasmid PL showed a 5-fold increase in luciferase activity after 8-Br-cAMP treatment, whereas no modification of luciferase activity was obtained with the control plasmid (Fig. 3A). Usually, cAMP-regulated genes contain a consensus cis-acting element close upstream of the RNA start site, which mediates the induction of transcriptional activity by cAMP. The hTPH promoter does not contain the canonical CRE (5`-TGACGTCA-3`), but a putative AP-2 binding site (5`-GCCCAGCC-3`) was found at position -100, differing from the consensus sequence, (5`-CCCCAGGC-3`) by two mismatches. To delineate the 5`-end of the TPH cAMP-regulatory region and to determine whether the AP-2 binding site is involved in this response, several 5`-deletions of the hTPH promoter (PL, PL, PL, PL, and PL) were analyzed. The hTPH promoter deletion that removes the AP-2 binding site corresponds to the construct PL. Pineal cells were transfected with each of these constructs and were treated or not treated with 8-Br-cAMP. For all constructions tested, including PL (-73 to +29), 8-Br-cAMP caused a reproducible 4-7-fold induction of luciferase activity as compared with the corresponding untreated transfected cells (Fig. 3A). Thus the AP-2 element is not involved in the hTPH 8-Br-cAMP responsiveness, and the motif confering this inducibility is presumably located either within the 73 bases upstream of the hTPH RNA start site or in the first 29 bases of the 5`-noncoding region of the hTPH.


Figure 3: Luciferase activity of human TPH promoter-luciferase fusion genes in pinealocyte cultures. A and B, pinealocytes were transfected with the hTPH promoter-luciferase plasmids defined in Fig. 2and Fig. 4exposed or not exposed to 1 mM 8-Br-cAMP. Luciferase activity is expressed as relative light units (RLU) normalized to CAT activity in the same cell extract (RSV-CAT) expressed in milliunits. The abscissa indicates the names of the deletion constructs of the hTPH promoter. Data are mean ± S.E. (bars) values from at least three independent experiments done in triplicate. Bottom, the two tables give the values of the basal level of the reporter genes expression (RLU/mUCAT) and the -fold induction of the reporter genes activity after 8-Br-cAMP treatment (*, p < 10; t test).




Figure 4: Nucleotide sequence of the human TPH deletion constructs and position of the three site-directed mutations in the hTPH promoter sequence. The name of each deletion construct is indicated on the left of the corresponding nucleotide sequence. Open boxes indicate the AP-4 binding site. Shaded boxes indicate the inverted CCAAT box and the TATA box. The two arrows indicate the two 9-bp imperfect direct repeats (RE2 and RE1). The transcriptional start site is indicated by the bent arrow. Black boxes indicate the positions of site-directed mutations in the PL, PL, and PL constructs (corresponding to the AP-4 binding site, RE2 element and CCAAT box, respectively). PL73BC contains mutations in two elements: the CCAAT box and RE2.



Two additional plasmids corresponding to 5`- and 3`-deletions of the hTPH promoter were generated by PCR amplification (PLDelta and PL; Fig. 4). PLDelta differed from PL by the elimination of the +2 to +29 bp region. PL plasmid contained only 51 bases upstream of the cap site and lacked both the inverted CCAAT box and the AP-4 binding site. Transfection experiments in pinealocytes with PLDelta gave the same basal luciferase activity and the same induction by 8-Br-cAMP as did PL, indicating that there were no cis-elements in the DNA sequence between +2 and +29 bp required for hTPH promoter activity or cAMP regulation (Fig. 3B). However, there was no increase of the luciferase activity after 8-Br-cAMP treatment and a decrease of the basal level of luciferase activity in pinealocytes transfected with PL, although the transcriptional activity was 2-3-fold higher than the KSLuc background. Therefore, these results suggested that the cAMP-regulatory motif is located between -73 and -51 of the human TPH 5`-flanking region.

The above observations were extended by transfection of PC12 cells with the same TPH promoter-luciferase fusion constructs. The results were entirely consistent with those described above except that the 8-Br-cAMP induction of the hTPH promoter was smaller (a 2-2.5-fold increase; data not shown).

An Inverted CCAAT Box Identified by Site-directed Mutagenesis Is Required for the 8-Br-cAMP Responsiveness of the Human TPH Gene

Three putative motifs entirely cover the 22 nucleotides (between nucleotides -73 and -51) required for the cAMP responsiveness: an inverted CCAAT box, an AP-4 binding site, and a 9-base imperfect direct repeat (RE2). We investigated the role of each of these three elements on the induction of hTPH gene expression by 8-Br-cAMP in pinealocytes and PC12 cells by site-directed mutagenesis (Fig. 4). Synergy between the AP-4 and AP-1 binding sites has been reported to contribute to the cAMP responsiveness of the human proenkephalin promoter(25) . To assess whether the AP-4 binding site is involved in the transcriptional regulation of the hTPH promoter by cAMP, the 5`-CAGCTG-3` AP-4 binding site sequence was mutated to 5`-CTACTT-3`. The resulting construct (PL) was used to transfect pinealocytes. The luciferase activity of PL was almost identical to that of PL in control conditions and after 8-Br-cAMP treatment (Fig. 5A). Thus, the AP-4 binding site did not appear to have any role in the induction of the hTPH promoter activity by cAMP. Mutations of the 9-base imperfect direct element (RE2; 5`-CCGCTGCCC-3`) to 5`-CCTTTACCC-3` and of the inverted CCAAT box (5`-ATTGG-3`) to 5`-TTTAG-3` were similarly tested both independently and together. The mutated construct affecting both the inverted CCAAT box and the RE2 motif in the hTPH promoter sequence (mutant PL) completely inhibited the induction of luciferase activity after 8-Br-cAMP treatment, showing that one or both of the two elements are involved in this response (Fig. 5A). The luciferase activity of mutant PL where only the RE2 element is modified was similar to that of PL, suggesting that the RE2 motif is not involved in the response to cAMP. In contrast, the mutation disrupting the inverted CCAAT box (mutant PL) completely abolished the cAMP responsiveness. Analogous experiments with the four mutated constructs were performed in PC12 cells and confirmed these findings (Fig. 5B). Thus, these results identified the inverted CCAAT box as being involved in the cAMP inducibility of the hTPH promoter.


Figure 5: Mutational analysis of cAMP inducibility of the human TPH promoter in pinealocyte and PC12 cells. Pinealocytes and PC12 cells were transfected with mutated and control hTPH promoter constructs defined in Fig. 4. The relative luciferase activity is an arbitrary unit defined as RLU/mUCAT, set as 100 for the hTPH promoter construct PL in the absence of 8-Br-cAMP. Data are mean ± S.E. (bars) values from at least three independent experiments done in triplicate. A, TPH promoter-luciferase fusion plasmids were introduced into rat pinealocyte cultures and exposed or not exposed to 1 mM 8-Br-cAMP. B, TPH promoter-luciferase fusion plasmids were introduced into 8-Br-cAMP treated and untreated PC12 cells. Bottom, the tables give the values of the relative luciferase activity in the absence of 8-Br-cAMP and the -fold induction of the luciferase activity after 8-Br-cAMP treatment (*, p < 10; t test).



The Inverted CCAAT Box Interacts with DNA-binding Proteins

To further analyze the interaction of the inverted CCAAT box of the hTPH gene with cAMP-dependent factors, we performed electrophoretic mobility shift assays (EMSAs) using nuclear extracts prepared from PC12 cells and pinealocyte primary cultures. The double-stranded P-labeled TPH-CCAAT oligonucleotide (Wt) was incubated with nuclear extracts from 8-Br-cAMP-treated or untreated pineal cells, and the resulting protein-DNA complexes were analyzed by nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography (Fig. 6). EMSA demonstrated that 1 µg of protein extract from pinealocyte nuclei contained a factor able to bind to probe Wt, producing a single retarded band (named complex 1; Fig. 6, lane 2). The same DNA shift pattern was obtained with nuclear extracts from 8-Br-cAMP-treated pineal cells (lane 3), suggesting that the transcription factor(s) bind to this DNA sequence with or without cell stimulation by cAMP. The specificity of this complex was tested by adding a 250-fold molar excess of either unlabeled TPH-CCAAT oligonucleotide (Wt) or mutated TPH-CCAAT oligonucleotide (M) to the binding reaction (Fig. 6, lanes 4 and 5). The competitive displacement of the retarded probe was observed only with the Wt unlabeled TPH-CCAAT oligonucleotide. Then, these results indicated that complex 1 is formed by sequence-specific binding and that the mutated bases within the inverted CCAAT box are necessary for protein-DNA interaction. Finally, we tested whether the nuclear factors composing this complex could also bind a functional CRE sequence; we added an excess of unlabeled oligonucleotide containing the CRE consensus sequence of the rat tyrosine hydroxylase gene(26) , to compete for the binding of pinealocyte nuclear proteins to the labeled TPH-CCAAT probe (Wt). The addition of a 250-fold molar excess of unlabeled CRE-TH did not displace the binding of factors from the probe, demonstrating that transcriptional factors that interacted with the inverted CCAAT box did not belong to the CRE binding protein family (Fig. 6, lane 6). Gel retardation assays using nuclear extracts prepared from PC12 cells gave a pattern of specific bands similar to that obtained with the pinealocytes. EMSA with untreated (data not shown) and 8-Br-cAMP-treated PC12 nuclear extracts showed one major shifted band (named 1) and two minor bands (2 and 3) (Fig. 7, lane 2). A 250-fold molar excess of unlabeled TPH-CCAAT oligonucleotide (Wt), mutated TPH-CCAAT oligonucleotide (M) or an unrelated oligonucleotide (the consensus POU IgH sequence; (27) ) completely displaced complexes 2 and 3 (Fig. 7, lanes 3, 4, and 6), suggesting that the formation of these two complexes was not caused by a sequence-specific binding. In contrast, the lowest mobility complex (complex 1) was displaced only in the presence of 250-fold molar excess of the unlabeled TPH-CCAAT oligonucleotide Wt (Fig. 7, lane 3), indicating that only complex 1 is caused by sequence-specific binding. In addition, as seen with the pinealocyte extracts, no competition with the factor(s) binding the probe Wt was observed by adding of a molar excess of the CRE-TH oligonucleotide. Thus, the effects of 8-Br-cAMP on TPH gene transcription do not appear to be mediated by increasing the amount of cellular factors that bind to this regulatory sequence.


Figure 6: Electrophoretic mobility shift assay (EMSA) with the TPH-CCAAT oligonucleotide in the presence of pineal cell nuclear extracts. Nuclear extracts were prepared from pinealocytes, which were treated or not treated with 8-Br-cAMP. The labeled double-stranded TPH-CCAAT oligonucleotide (Wt) was incubated with pinealocyte nuclear extracts in the presence or absence of a 250-fold excess of unlabeled oligonucleotide competitor. The competitors used were the wild-type (Wt) or mutated TPH-CCAAT oligonucleotide (M) or the consensus CRE-TH of the rat tyrosine hydroxylase gene as indicated above each lane. The arrow denotes the complex (1) obtained with the TPH-CCAAT oligonucleotide (lane 2).




Figure 7: EMSA with the TPH-CCAAT oligonucleotide in the presence of PC12 cell nuclear extracts treated with 8-Br-cAMP. EMSAs were performed with a nuclear extract from PC12 cells and a labeled, double-stranded TPH-CCAAT oligonucleotide (Wt) in the presence or absence of a 250-fold excess of unlabeled oligonucleotide competitor. The competitors used were the wild type (Wt) or mutated TPH-CCAAT oligonucleotide (M), the consensus CRE-TH of the rat tyrosine hydroxylase gene, and POU IgH of the immunoglobulin heavy chain as indicated above each lane. The arrows denote the three complexes (1, 2, and 3) obtained with the TPH-CCAAT oligonucleotide (lane 2).



The Inverted CCAAT Box Motif of the TPH Gene Regulates a Heterologous Promoter in cAMP-treated Pinealocytes

To test whether the inverted CCAAT box module can confer cAMP responsiveness to a heterologous promoter, a 3-fold tandem repeat TPH-CCAAT oligonucleotide containing the wild type inverted CCAAT box motif (Wt) was inserted upstream of the minimal thymidine kinase promoter fused to the luciferase gene (DeltaTK-LUC). The resulting construct was called Wtx3. In addition, we also inserted three copies of the mutated TPH-CCAAT oligonucleotide (M) in the inverted CCAAT box sequence as a putative negative control (Mx3). The sequence of the two oligonucleotides was identical to those of oligonucleotides used for the EMSA (see above). Pinealocytes were transiently transfected with plasmids Wtx3 and Mx3 and treated or not treated with 8-Br-cAMP. The basal activity of these two plasmids was about 3-fold higher than the parental plasmid DeltaTK-Luc (data not shown). After treatment with 8-Br-cAMP, the level of luciferase activity was 2-fold higher in pinealocytes transfected with the wild type (Wtx3) than with the mutated (Mx3) (Fig. 8). Thus, the 22-base sequence located between nucleotides -73 and -51 of the TPH promoter can function in a heterologous context. The cAMP induction of luciferase activity in pineal cells transfected with the construct Wtx3 was weaker than that of a construct that contained the TPH promoter fused to the luciferase gene. This suggests that the activity of the inverted CCAAT box may be affected by the flanking sequences.


Figure 8: Effect of 8-Br-cAMP on activity of the TK-Luc fusion genes. Pinealocytes were exposed or not exposed to 1 mM 8-Br-cAMP after transfection with the plasmids Wtx3 or M3. Wtx3 and M3 were derived from the plasmid DeltaTK-Luc (see ``Materials and Methods'') where, respectively, three tandem repeat sequences of the wild type inverted TPH-CCAAT box oligonucleotide (Wt) and the mutated inverted TPH-CCAAT box oligonucleotide (M) had been inserted upstream of an enhancerless thymidine kinase promoter fused to the luciferase gene. Luciferase activity is expressed as relative light units (RLU) normalized to CAT activity in the same cell extract (RSV-CAT) expressed in milliunits. Bottom, the table gives the values of the basal level of the reporter genes expression (RLU/mUCAT) and the -fold induction of the reporter genes activity after 8-Br-cAMP treatment.




DISCUSSION

We report the identification, sequence, and characterization of the promoter region of the human tryptophan hydroxylase gene. The 2,117-bp genomic region upstream of the hTPH mRNA cap site efficiently promoted the transcription of a luciferase reporter gene in cell cultures. This promoter belongs to the category of genes that possess a TATA box(23) .

Analysis of the basal transcription by progressive deletions delineated a region of about 150 nucleotides from -204 to -51, which appears critical for optimal promoter activity in pinealocytes and PC12 cells. The shortening of the hTPH promoter from 2,117 to 204 nucleotides did not significantly modify the transcriptional activity of the corresponding constructs. In contrast, further deletions of the hTPH promoter up to the nucleotide -73 strongly reduced transcription efficiency. Several putative regulatory elements were found between positions -204 and -73: two G/C rich regions, which could be DNA binding sites for the Sp1 protein; a CCACCC element; an AP-2 binding site; and a C/EBP binding site. All of these sequences are involved in the regulation of transcription of numerous viral and eukaryotic genes (28, 29, 30, 31) . In the case of the hTPH gene, the severance of the CCACCC sequence from the inverted CCAAT box in the construct PL could account for the reduction of TPH promoter activity. Indeed, the CCACCC element of the porphobilinogen deaminase gene interacts with the CCAAT box to modulate basal transcription(32) . A CCACCC element is also required for the basal transcription of the tryptophan oxidase gene (33) and of the beta-globin gene, in combination with the GATA binding site in the latter case(34) . The other cis-regulatory elements of the hTPH gene were not further characterized.

The isolated regulatory region of the hTPH gene does not contain sufficient information to mediate cell-type-specific expression in our experimental conditions, with the exception of a weak tissue-specific repressor located between -724 and -252 bp. The promoter was active both in primary cultures of rat pinealocytes, cells that synthesize large amounts of TPH enzyme, and also in PC12 and HeLa cells, which do not contain detectable levels of TPH mRNA. The regulatory sequences conferring tissue-specific expression could be either nonfunctional in these cultured cells or localized elsewhere on the gene.

The murine TPH promoter has been isolated by Stoll and Goldman (35) and provided us the opportunity to compare its organization with that of the hTPH promoter. No significant identity was found in the two proximal regions of the TPH promoters although they both contain a TATA box, several identical upstream promoter elements including the inverted CCAAT box sequence, the CCACCC motif, and GC boxes. Surprisingly, 70% identity was found in a 450-nucleotide region located, respectively, 1,550 and 520 bases upstream of the transcription initiation site of human and mouse TPH promoters. However, there is no evidence that this conserved region is important either for the control of basal transcription or for the tissue-specific expression of the hTPH mRNA (see below).

cAMP-dependent intracellular signaling plays a major role in the regulation of TPH activity. Indeed, tryptophan hydroxylase activity as well as that of the arylalkylamine N-acetyl transferase, the limiting enzyme in the biosynthesis of melatonin, are modulated by cAMP during the circadian rhythm in the rat pineal gland(13, 15) . Additionally, Foguet et al.(6) have found that TPH mRNA levels are increased by cAMP in rat serotonergic neuronal cultures from raphe nuclei. Therefore, cAMP may act at various different steps of the regulation of TPH expression, both to activate the TPH enzyme via phosphorylation by a cAMP-dependent protein kinase (36, 37) and to induce the transcription of the TPH gene.

We demonstrate that an increase of intracellular cAMP levels stimulated human TPH gene transcription. Transient transfection experiments showed that incubation with a cAMP analog (1 mM 8-Br-cAMP) increased the transcriptional activity of the hTPH promoter in pinealocytes (6-fold) and in PC12 cells (2-fold). Three main cis-acting elements have been identified in a large number of genes that are transcriptionally regulated by cAMP: the consensus CRE sequence 5`-TGACGTCA-3`, which is found in the majority of cAMP-responsive genes (38) , the AP-2 binding site 5`-(C/G)CCCAGGC-3`(39) , and the AP-1 binding sequence 5`-TGAGTCA-3`(40) . None of these elements are implicated in the cAMP-dependent regulation of hTHP gene transcription. Unexpectedly, we found that the inverted CCAAT box was involved in the cAMP induction of the hTPH gene. These arguments are based on the following criteria. First, progressive deletions of the hTPH promoter sequence showed that a 22-bp region between positions -73 and -51 and containing an inverted CCAAT box, an AP-4 site, and an imperfect direct repeat (called RE2), was required to mediate the response of the hTPH promoter to increased cAMP concentrations. Second, site-directed mutagenesis of the inverted CCAAT box completely suppressed the induction of hTPH promoter by cAMP when mutations in the AP-4 and in the imperfect direct repeat RE2 had no effect. Third, the 22-bp region is able to confer cAMP responsiveness to a heterologous (thymidine kinase minimal promoter-luciferase) promoter that is not otherwise regulated by cAMP. These results also excluded a role for the AP-2 binding site (at position -100) as a putative cAMP-responsive site. The mouse TPH promoter also contains an inverted CCAAT box, 55 nucleotides upstream of the transcriptional initiation site. It would be interesting to determine whether or not the cAMP responsiveness of the inverted CCAAT box of the TPH gene is peculiar to the human species. The mouse TPH gene promoter exhibits two additional CRE-like elements (-19 and -157 nucleotides), differing from the canonical CRE by two substitutions. Determination of the role of these sites awaits experimental testing.

A small number of cAMP-inducible genes, including the mouse renin gene (ren1^d, ren2^d), the human myelin basic protein gene, and some of the steroid hydroxylase genes, bear cAMP-inducible sequences dissimilar to the CRE, AP-2, and AP-1 binding sites(41, 42, 43, 44) . For example, a GA box element, which binds SP1-like proteins, confers cAMP responsiveness to the bovine P-450 gene(45) . A consensus GC box is also present in the region required for the transcriptional induction of the human steroid 21-hydroxylase gene (P-450) by cAMP, but this motif has not been definitively shown to be the cAMP-responsive element (43) . The diversity of cAMP-responsive sites has also been extended by the identification in the human myelin basic protein gene promoter of a sequence (5`-CACTTGATC-3`) sharing no identity with any known cis-acting element(42) . Finally, the CCAAT box motif has been implicated, either directly or indirectly, in the cAMP inducibility of several genes. Muro et al.(46) reported that the cAMP responsiveness of the human fibronectin gene required cooperative interaction between a CRE and a CCAAT box. In addition, it has been demonstrated that the CCAAT box sequence alone mediates the regulation of G-protein alpha subunit gene transcription by cAMP (47) . Indeed, this motif inserted upstream of a heterologous promoter is able to confer cAMP inducibility. Therefore, cis-acting elements, which were initially believed to contribute only to basal transcription (CCAAT box, AP-2 binding site, GC box), have now been implicated in the modulation of gene transcription by intracellular signals.

All of these genes exhibiting unconventional cAMP-responsive sites share a common characteristic in that they are belatedly up-regulated after an increase in the intracellular cAMP concentration. Indeed, the cAMP-regulated genes fall into two categories. The major group consists of genes that are rapidly regulated by cAMP and are cycloheximide-insensitive. For example, the cAMP induction of somatostatin and acetyl-CoA carboxylase gene transcription mediated by CRE or AP-2 elements, respectively, occurs within minutes(38, 48) . The corresponding AP-2 and CREB/ATF family of DNA binding proteins are constitutively synthesized and are phosphorylated to transcriptionally active forms. In contrast, the other group of genes is characterized by a delayed transcriptional response to cAMP, which is inhibited by cycloheximide treatment. The increase of gene transcription begins only after a delay of several hours, suggesting that this class of cAMP-regulated genes requires protein synthesis. The kinetic response of human TPH gene transcription to cAMP treatment has not been determined, because no human cell lines expressing the TPH gene are available. However, electrophoretic mobility shift assays showed that the oligonucleotide containing the inverted CCAAT box forms a specific DNA-protein complex giving bands of similar intensity with nuclear extracts from untreated and cAMP-treated rat pinealocytes. Thus, the inverted CCAAT box binding activity is constitutive and does not appear affected by cAMP stimulation. This suggests that the inverted CCAAT box-binding protein is permanently present in the cells. It remains to be determined whether the factor that binds to the inverted CCAAT box needs to be phosphorylated, like the members of the AP-2 and CREB/ATF binding factor family. The consensus CRE sequence does not inhibit the formation of the DNA-protein complex between the wild-type TPH-CCAAT oligonucleotide (Wt) and nuclear extracts from pinealocytes. The transcription factors implicated in the regulation of the TPH gene by cAMP are, therefore, likely to be different from those interacting with the CRE sequence. cAMP induction of the G-protein alpha subunit gene (Galpha) also required a CCAAT box motif but in the sense orientation(47) . It has been demonstrated that the transcription of the Galpha gene is induced only after several hours of cAMP treatment (6 h) and that the CCAAT box binding activity was both constitutive and increased by cAMP treatment, in contrast to what was found for the inverted CCAAT box of the hTPH gene. Therefore, the mechanisms by which cAMP regulates TPH and the Galpha gene transcription seem to be different. The purification and identification of the trans-acting factor(s) that bind to this regulatory element would give further insight into the cAMP-dependent mechanisms regulating hTPH gene transcription.

Finally, Stehle et al.(49) have recently reported that the pineal gland does not contain the CRE binding protein (CREB) but does contain large amounts of ICER, a transcription repressor factor belonging to the cAMP-responsive element modulator family (CREM). ICER levels in the pineal gland exhibit a large circadian fluctuation. It will therefore be of great interest to study whether this factor and others, which remain to be isolated, contribute to the stringent regulation of the TPH gene expression in the pineal gland.


FOOTNOTES

*
This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Medicale, the Association pour la Recherche contre le Cancer, the Institut de Recherche sur la Moëlle Epiniere and Rhône-Poulenc-Rorer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X83212[GenBank].

§
To whom correspondence should be addressed.

Supported by fellowships from the Institut de Recherche sur la Moëlle Epiniere.

(^1)
The abbreviations used are: TPH, tryptophan hydroxylase; CRE, cAMP-responsive element; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; 8-Br-cAMP, 8-bromo-cAMP; EMSA, electrophoretic mobility shift assay; Wt, wild type; M, mutant; TH, tyrosine hydroxylase; IgH, immunoglobulin heavy chain; bp, base pair(s); kb, kilobase(s).

(^2)
W. Faust and A. M. Catherin, unpublished results.


ACKNOWLEDGEMENTS

We thank E. Jean-Gilles, I. Brunet, and D. Samolyk for technical assistance and A. Lamouroux and N. Faucon Biguet for critical comments. A special thanks to P. Vernier for many helpful suggestions and critical reading of this manuscript.


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