(Received for publication, October 6, 1994)
From the
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.
Tryptophan hydroxylase (TPH) ()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
-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.
The plasmid TK-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`.
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).
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 (PL and
PL
; Fig. 4). PL
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
PL
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).
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).
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).
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 TK-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.
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
-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, ren2
), 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
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 subunit gene
(G
) also required a CCAAT box motif but in the sense
orientation(47) . It has been demonstrated that the
transcription of the G
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
G
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X83212[GenBank].