(Received for publication, November 14, 1996, and in revised form, December 16, 1996)
From the Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892
A 10-100-fold rhythm in the activity of
arylalkylamine N-acetyltransferase (AA-NAT; EC 2.3.1.87)
controls the rhythm in melatonin synthesis in the pineal gland. In some
mammals, including the rat, the high nocturnal level of AA-NAT activity
is preceded by an ~100-fold increase in AA-NAT mRNA. The increase
in AA-NAT mRNA is generated by norepinephrine acting through a cAMP
mechanism. Indirect evidence has suggested that cAMP enhances AA-NAT
gene expression by stimulating phosphorylation of a DNA-binding protein (cAMP-responsive element (CRE)-binding protein) bound to a CRE. The
nature of the sites involved in cAMP activation was investigated in
this report by analyzing the AA-NAT promoter. An ~3700-base pair
fragment of the 5-flanking region of the rat AA-NAT gene was isolated,
and the major transcription start points were mapped. The results of
deletion analysis and site-directed mutagenesis indicate that cAMP
activation requires a CRE·CCAAT complex consisting of a near-perfect
CRE and an inverted CCAAT box located within two helical turns.
The rhythmic nocturnal increase in plasma levels of melatonin (1) in mammals is due to norepinephrine (NE)1 stimulation of melatonin production in the pineal gland. NE release is regulated by the endogenous circadian oscillator in the suprachiasmatic nucleus (2), which is connected to the pineal gland by a multisynaptic pathway. NE controls melatonin production by regulating the activity of the penultimate enzyme in the melatonin biosynthetic pathway, serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AA-NAT), EC 2.3.1.87) (3). The second messengers involved are cAMP and Ca2+i (3-7).
The importance of transcriptional events in the regulation of AA-NAT activity varies remarkably on a species-to-species basis (8). An absolute requirement for de novo transcription is most evident in the rat (8, 9), where an ~100-fold increase in AA-NAT mRNA is required for the ~100-fold increase in AA-NAT activity to occur. The mechanism involved in turning on expression of the AA-NAT gene does not require de novo protein synthesis (9). Rather, it appears to be initiated by cAMP-dependent phosphorylation of cAMP-responsive element (CRE)-binding protein (CREB) (10) or another member of this growing family (11, 12).
The molecular basis of cAMP stimulation of expression of the rat AA-NAT gene has not yet been investigated at the level of the promoter, and it is not known whether cAMP acts directly or indirectly on the AA-NAT gene. Here we describe the isolation of the rat AA-NAT promoter region and report the results of a functional analysis focused on the question of NE- and cAMP-dependent activation. The results of this study indicate that cAMP can effect gene activation from the AA-NAT promoter. Furthermore, it appears that cAMP acts through a CRE·CCAAT complex, which consists of a near-perfect CRE located within two helical turns of an inverted CCAAT box.
AA-NAT promoter sequences were
identified using the Promoter FinderTM DNA Walking kit (CLONTECH, Palo
Alto, CA) in conjunction with the nested AA-NAT primers 652, 581, and
587 (see below) derived from the 5-flanking regions of AA-NAT. The PCR
conditions were recommended by the manufacturer. Genomic fragments were
subcloned into pCR3 (Invitrogen, San Diego, CA) for sequencing by the
dideoxynucleotide chain termination method (13). To generate
promoter/reporter hybrid constructs, different PCR-generated fragments
of the AA-NAT promoter region were introduced into the XbaI
site in pCAT-Basic (Promega, Madison, WI) or into the NheI
site in pGL3-BASIC (Promega). These vectors carry the bacterial
chloramphenicol acetyltransferase (CAT) and firefly luciferase (LUC)
reporter genes, respectively.
For S1
nuclease protection analysis, a 289-nucleotide end-labeled
single-stranded probe was synthesized that was complementary to
positions 207 to +82 in the AA-NAT gene (relative to the start of
transcription); synthesis was by asymmetric PCR driven by primer 581 (see below). The probe was gel-purified and added (50,000 cpm) to 30 µg of total pineal RNA in 20 µl of S1 hybridization buffer (80%
deionized formamide, 40 mM Pipes, pH 6.4, 400 mM NaCl, and 1 mM EDTA, pH 8). The mixture was
then denatured (10 min, 65 °C) and hybridized overnight at 44 °C.
S1 nuclease digestion was carried out by adding 300 µl of S1 nuclease
buffer (0.28 M NaCl, 50 mM sodium acetate, pH
4.5, 4.5 mM ZnSO4, 10 µg of sheared salmon
sperm DNA, and 240 units of S1 nuclease). After a 1-h incubation at
37 °C, 80 µl of stop buffer (4 M ammonium acetate, 20 mM EDTA, pH 8, and 40 µg/ml tRNA) were added. After
ethanol precipitation, samples were boiled and electrophoresed on a 6%
sequencing gel. A dideoxynucleotide sequencing reaction of
267/CAT
primed with primer 581 was run in parallel to locate the transcription
start point.
Dissociated rat pinealocytes (5 × 106 cells) were prepared by trypsinization essentially as described (14) with minor modifications. Briefly, 50 pineal glands (Taconic Farms Inc., Germantown, NY) were separated from residual afferent nerve fibers and the surrounding leptomeninges, partially teased apart, and rinsed in 10 ml of BGJb (Life Technologies, Inc.). Glands were incubated (37 °C, 95% O2 and 5% CO2) in 5 ml of DMEM containing 0.2% trypsin and 40 µg/ml DNase I (Boehringer Mannheim) for 50 min. Trypsinization was ended by the addition of 10 ml of DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (DMEM+). Partially dissociated glands were allowed to sediment and triturated in 3 ml of DMEM+ supplemented with 70 µg of DNase I. Pineal cells were passed through a small funnel-shaped mesh (125-µM opening; Bellco Glass, Inc., Vineland, NJ) to a new tube, spun at 1000 × g for 5 min, and plated in two wells of a six-well plate (Costar Corp., Cambridge, MA) in 2 ml of DMEM+. Twenty-four hours later, cells in suspension were harvested, collected, and resuspended in serum-free Opti-MEM (Life Technologies, Inc.; ~0.5 × 106 cells/ml); and 0.4-ml samples were transferred to individual wells in a 24-well plate (Costar Corp.). Transfections were performed by overlaying the cells with a precipitate of 3 µl of LipofectAmineTM (Life Technologies, Inc.) and 2 µg of DNA for 30 min before adding ~2 × 108 adenovirus shuttle particles to enhance transfection efficiency (15). Eighteen hours later, 0.5 ml of DMEM+ were added. Where indicated, dibutyryl cAMP (Bt2cAMP) was added to the cultures at this point. Cells were harvested 48 h later. CAT assays were performed as described (16). Luciferase activity was measured with the luciferase assay system (Promega) according to the manufacturer's recommendation. Transfection of primary rat pituitary cells or fibroblasts was done using the same procedure as described above. COS-7 and C6 cells (American Type Culture Collection, Rockville, MD) were transfected using LipofectAmine following the manufacturer's recommendations.
OligonucleotidesThe synthetic DNA oligonucleotides and
primers referred to in this study were synthesized using an Applied
Biosystems 381B DNA synthesizer. Nested primers for promoter cloning
were as follows (positions are relative to start of transcription):
primer 652 (positions +177 to +158),
5-CAT GGG TAT CTG GCC ACT GA-3
; primer 581 (positions +82 to
+63), 5
-TCC CCA CCA CAG AGC TGG TCA CAC TGG-3
; and primer 587 (positions
14 to
34), 5
-CCT GAC AGC ATG TGA TGG CTC A-3
. Oligonucleotides used in EMSA and for site-directed mutagenesis were as
follows (only upper strand is shown): natCRE,
5
-GAA AAG CTT AGT ACC ACC GAT GAC GCC AGC CCT CAG CAG TCT AGA GC-3
; natCREmut,
5
-GAA AAG CTT AGT ACC ACC GAT TAA ACC AGC CCT CAG CAG TCT AGA GC-3
; AP-1, 5
-CGC TTG ATG AGT CAG CCG GAA-3
; CCAAT-binding
transcription factor, 5
-CCT TTG GCA TGC TGC CAA TAT-3
;
sstCRE (where sst is somatostatin),
5
-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3
; caCRE, 5
-GAG CCC GTG ACG TTT ACA CTC ATT C-3
; AP-2,
5
-GGA TTA TAG TTA GAC CCC AGG CTT AGC CCA TGC TCT CC-3
; CCAAT,
5
-CAG CCC TCA GCA GGA TTG GGT CAG GGC CTG ACT ACC-3
; CCAATmut1,
5
-CAG CCC TCA GCA GTC TAG AGTCAG GGC CTG ACT ACC-3
; CCAATmut2
5
-CCT CAG CAG GTATGG GTC AGG GCC T-3
; and
CRECCAAT+5, 5
-GAT GACGCC AGC CCT CGA GTA AGC AGG ATTGGG TC-3
.
Pineal glands were quick-frozen on dry ice. To
prepare extracts, a 30-µl sample of ice-cold buffer C (20 mM Hepes, pH 7.9, 1.5 mM MgCl2,
0.42 M NaCl, 0.2 mM EDTA, 1 µg/ml aprotinin,
1 µg/ml leupeptin, 1 mM sodium fluoride, 5 µM sodium orthovanadate, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and
25% glycerol) was added to two glands. The glands were homogenized on
ice with 20 strokes of a tight-fitting 1.5-ml microtube pestle. Lysates
were centrifuged at 12,000 rpm for 10 s, vortexed, and refrozen on
dry ice for 5 min. They were next incubated in ice water for 15 min and
centrifuged at 12,000 rpm for 15 min at 4 °C. This procedure
extracted 40-50 µg of soluble protein/pineal gland. EMSA was
performed using a 32P-radiolabeled double-stranded
oligonucleotide probe containing the natCRE or natCCAAT sequence (see
Fig. 1) and 3 µl of whole pineal extract as described previously
(17). Competition EMSA was performed as described above with a 200-fold
molar excess of unlabeled double-stranded oligonucleotides added before
the probe or 1 µl of one of the following antisera (added 20 min
after the probe): anti-c-Fos-(3-16) (SC-52X), anti-Fra-1-(3-22)
(SC-183X), anti-Fra-2-(285-299) (SC-57X), anti-FosB-(102-117)
(SC-48X), anti-JunD-(329-341) (SC-74X), and anti-CREM-1 (SC-440X)
(Santa Cruz Biotechnology, Inc. Santa Cruz, CA) or rabbit anti-rat
CREB-(1-205) polyclonal antiserum (Upstate Biotechnology, Inc., Lake
Placid, NY).
Site-directed Mutagenesis
In vitro mutagenesis of the CRE and CCAAT cis-acting elements within the AA-NAT promoter was done with the QuickChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's recommendations. Initial screening of putative mutant clones was performed by BsaHI and XbaI restriction endonuclease digests, respectively. Mutations were then confirmed by dideoxynucleotide sequencing. Promoter fragments were subsequently excised with XbaI and inserted into an NheI-linearized pGL3-BASIC reporter vector.
Putative regulatory
regions in the sequence immediately upstream of the AA-NAT coding
sequence were isolated using a ligation-mediated PCR-based technique as
described under "Experimental Procedures." This generated an
~3700-bp genomic fragment that contains the first intron (intron 1;
Fig. 1A) located in the 5-untranslated region after position +114; the intron is 1628 bp
long.2
The 2160-bp putative promoter sequence was analyzed to determine if it
contains known transcription factor-binding sites (Fig. 1A).
This failed to identify a canonical TATA box; however, an A/T-rich
sequence resembling a TATA box (ATTAGAAT) is located 38 bp upstream of
the start of transcription, as defined below. This putative TATA box is
preceded by a GC-rich region (positions 47 to
65), which could
function as an SP1-binding (18) and/or AP-2-binding (19) site. An
inverted CCAAT box (natCCAAT) exists at position
120. In addition, we
detected an alternate purine-pyrimidine repeat (20) between positions
1165 and
1104, an AP-1-like element (21) at position
32, and a
CRE-related sequence at position
139 (natCRE). The natCRE sequence
(TGACG
CA) closely resembles (seven out of eight) the
perfect CRE consensus sequence (TGACG
CA) (22, 23).
The
transcription initiation site was established using S1 nuclease
protection analysis of total RNA isolated from
Bt2cAMP-treated pineal glands. Three transcription start
points were detected (Fig. 1B), the strongest of which
generates the longest leader sequence (173 nucleotides). The leader
sequences generated by the weaker start points are 148 and 136 nucleotides long. The upstream initiation site is located within an
apparent initiator consensus sequence, characterized by an initiating A
with a 3-pyrimidine-rich flanking sequence (Fig. 1C) (24,
25).
Regions
necessary and sufficient for AA-NAT promoter activity were identified
using partial promoter constructs. These contained decreasing portions
of the longest 5-flanking genomic fragment placed in front of the CAT
reporter gene.
Primary pinealocytes were transiently transfected with this series of
constructs and tested for basal and stimulated CAT activity (Fig.
2). CAT activity was low in untreated cells in all
constructs. Bt2cAMP treatment produced comparable levels of
CAT expression from a family of intronless promoter deletion constructs
with 5-boundaries ranging from positions
2160 to
267 and
encompassing sequences up to position +82. Similar results were
obtained following treatment with NE or a selective
-adrenergic
agonist (isoproterenol), but not following selective
-adrenergic
treatment (phenylephrine + propranolol; data not shown). Maximum
Bt2cAMP induction was achieved using a promoter construct
that begins at position
207, spans the entire length of intron 1, and
ends just prior to the first codon (
207/iCAT). However, a similar
construct containing >90% of intron 1 but no 5
-untranslated region
(+371/CAT) failed to drive detectable CAT enzyme activity, indicating
that the key elements promoting Bt2cAMP responsiveness are
not likely to reside in intron 1. Taken together, these results suggest
that the region between positions
267 and +82 is sufficient for
conferring cAMP responsiveness. This region contains the natCRE and
natCCAAT sequences described above.
The 267/CAT construct fragment was also able to drive
cAMP-dependent expression of CAT when tested in non-pineal
cells, including rat glioma C6, COS-7, and primary rat pituitary cell
cultures (Table I). It follows that this fragment does
not confer tissue specificity, nor does it require pineal
gland-specific proteins to support cAMP responsiveness.
|
The capacity of natCRE to recruit specific DNA-binding proteins was
tested using a 30-bp-long double-stranded oligonucleotide centered
around position 139; this probe does not contain the natCCAAT box.
EMSA analysis revealed that this natCRE oligonucleotide formed
complexes with pineal proteins (Fig. 3A). The
patterns of retarded species were not dramatically different if
extracts were prepared from pineal glands harvested during the day or
night (data not shown), indicating that similar or identical binding proteins are present at both times.
To determine if this EMSA pattern reflected specific interactions, excess unlabeled CRE-containing oligonucleotides (somatostatin or c-fos CRE) were added (Fig. 3A); this blocked the appearance of most retarded species. In contrast, excess unlabeled oligonucleotides containing either CCAAT-binding transcription factor- or AP-1-binding sites did not alter this pattern. Accordingly, formation of these complexes is CRE-dependent.
To test for the presence of specific transcription factors among the proteins associated with natCRE, selected antisera were used (Fig. 3A, lanes 6-12). An anti-CREB antiserum modified the EMSA pattern by preventing the formation of two nucleoprotein complexes (asterisks). However, antisera directed against several bZIP-containing transcription factors, belonging to the Fos, Jun, and CREM families, did not. Taken together, these results indicate that natCRE can specifically interact with several pineal proteins, including a CREB-like protein(s).
The function of the natCRE sequence was tested by site-directed
mutagenesis in the context of the minimal promoter construct 267/CAT.
The mutation involved three base changes
(T
A
CCA
T
A
CCA), including a G/T transversion at
position
141, which disrupts CRE function (26). The triple mutation
abolished binding to specific nuclear proteins (data not shown). In
transfection studies using primary pinealocytes, the
CREmut/CAT reporter construct partially reduced the
response to Bt2cAMP (60-80% inhibition) (Fig.
3B) as compared with the response exhibited by the unmutated control. The possibility that this apparent inhibition was due to
uncontrolled reporter gene-dependent artifacts was ruled
out using equivalent firefly luciferase constructs, indicating that the
triple mutation partially reduced effects of cAMP (Fig.
4B, wt/LUC versus
CREmut/LUC).
The above results indicate that natCRE plays a key role in translating
the adrenergic stimulus into an increase in AA-NAT gene transcription,
consistent with predictions regarding the role of CREB phosphorylation
in activation of the rat AA-NAT gene (10). However, a discrepancy is
apparent: the triple natCRE mutation completely abolished binding, but
only partially abolished the effects of Bt2cAMP on promoter
activity. This raised the possibility that cAMP-mediated gene
activation requires another element(s) in the 267 construct. The
basis of this residual responsiveness was examined below.
As indicated above, an inverted CCAAT box is located downstream and two helical turns away from natCRE. The CCAAT box is of special interest because of two observations: a CRE·CCAAT complex in the fibronectin gene promoter is necessary for full cAMP activation of gene expression (27-29), and an inverted CCAAT box mediates cAMP activation of expression of the apparently CRE-less tryptophan hydroxylase promoter in primary pinealocytes (30).
To test whether natCCAAT binds pineal proteins, we analyzed the region containing the inverted natCCAAT sequence by EMSA using whole pineal protein extracts. The double-stranded oligonucleotide used did not contain natCRE. A distinct nucleoprotein complex was generated using either day or night pineal extracts. Proteins in this complex are referred to here as CCAAT-binding proteins (CATBPs) (Fig. 4A). Binding specificity was demonstrated by successful competition with a 200-fold molar excess of the wild-type natCCAAT sequence (Fig. 4A, compare lanes 7 and 8). In contrast, binding was not inhibited by double-stranded oligonucleotides containing either of two mutated natCCAAT oligonucleotides (Fig. 4A, lanes 9 and 10) or by AP-2 or AP-1 sequences (lanes 11 and 12). These studies indicate that natCCAAT specifically binds to pineal CATBPs.
The contribution of the natCCAAT site to gene expression was determined
by site-directed mutagenesis of position 120 within the minimal
promoter (construct
267) using the firefly luciferase reporter
system. Promoter activity of the resulting construct (CCAATmut/LUC) was reduced by 60-80% when compared with
that obtained using wt/LUC and CREmut/LUC (Fig.
4B).
These observations raise the possibility that both natCRE and natCCAAT are important for inducible promoter activity. To determine whether both sites are required to achieve full transactivation, we generated a double mutant without functional natCRE and natCCAAT sites ((CRECCAAT)mut/LUC). Whereas the single mutant constructs CREmut/LUC and CCAATmut/LUC were partially active, the double mutant (CRECCAAT)mut/LUC was essentially refractory to cAMP stimulation (Fig. 4B). This supports the interpretation that both sites participate in cAMP regulation of the AA-NAT gene.
When the natCRE and natCCAAT sites are considered within the context of coiled DNA, their centers are separated by approximately two turns of a helix and are therefore likely to be in phase. To address the question of whether this precise alignment is crucial for cAMP-inducible promoter activity, phasing was disrupted by inserting a 5-bp sequence between the two elements (Fig. 4B, CRECCAAT+5/LUC). This did not affect promoter activity, indicating that precise phasing between bound factors does not appear to be important when the distance between these sites is not significantly affected.
Transcription plays a pivotal role in the nocturnal stimulation of
AA-NAT in the rat. This is evident from the findings that NE and cAMP
protagonists, including Bt2cAMP, increase AA-NAT mRNA levels ~100-fold (9) and that the transcription blocker actinomycin D
blocks the NE cAMP stimulation of AA-NAT mRNA and activity (3,
9). We have suspected that cAMP acts to increase AA-NAT mRNA
through cAMP-dependent phosphorylation of CREB resident at a CRE site. This was based on the evidence that NE acts through a cAMP
mechanism to phosphorylate CREB in the pineal gland and that the
protein kinase A antagonist (Rp)-8-CPT-cAMP-S
inhibits cAMP-dependent induction of AA-NAT activity
(10).
The studies
presented here appear to support this notion because a near-perfect CRE
(natCRE) is found in a 2160-bp fragment of the rat AA-NAT promoter.
Functional dissection by deletion analysis revealed that an
natCRE-containing region from positions 267 to +82 confers
cAMP-dependent responsiveness to either CAT or luciferase
reporter genes in primary pinealocytes. Furthermore, the natCRE core
sequence binds a CREB-like moiety present in whole pineal protein
extracts, and mutagenesis of the natCRE site reduces promoter activity
and binding, indicating that it functions as a bona fide
CRE. Accordingly, it appears very likely that natCRE mediates cAMP
stimulation of expression of the rat AA-NAT gene, presumably in
response to cAMP-dependent phosphorylation of resident CREB
molecules.
In addition to this site, a neighboring sequence appears to be involved
in cAMP responsiveness because disruption of natCRE in the 267/+82
region of the promoter does not completely abolish the response to NE
or Bt2cAMP. The most likely element involved in this
residual cAMP responsiveness is the inverted CCAAT box (natCCAAT) at
position
120. natCCAAT represents a perfect match in reverse
orientation to a known regulatory site referred to as the CCAAT box.
Such sites are found in many promoters and appear to control gene
expression through interaction with members of a growing family of
different CATBPs (31-35). The natCCAAT element appears to exert a
positive effect on transcription because site-directed mutagenesis
abolished binding activity and decreased the level of reporter gene
activity under both basal and stimulated conditions (Fig.
4B). In addition, a double mutant with a disrupted
CRE·CCAAT complex displayed only basal luciferase activity after
Bt2cAMP stimulation. This finding, together with the
partial decrease obtained after mutagenesis of either site alone, is
consistent with the hypothesis that both elements are necessary to
achieve full activation of the AA-NAT promoter by cAMP.
It should be noted that there are reports in the literature of CRE-CCAAT cooperation and of cAMP activation being mediated by a CCAAT box. As indicated above, a CRE·CCAAT complex is found in the fibronectin gene promoter (27-29), in which occupancy of the CCAAT box is facilitated by the CRE. In addition to this example of a CRE-CCAAT interaction, there also is evidence that the CCAAT box can mediate activation of gene expression in the absence of a CRE; this comes from the tryptophan hydroxylase gene (30). This points to the possibility that cAMP may act through CATBPs, perhaps via phosphorylation, as it does through CREB to control gene expression. It is of further interest to note that tryptophan hydroxylase is very strongly expressed in the pineal gland, suggesting that CATBPs may play a common role in this tissue in gene expression.
Turning Off Expression of the AA-NAT GeneThis investigation
focused on the mechanisms through which cAMP activates expression of
the rat AA-NAT gene and thereby generates the rhythmic ~100-fold
increase in AA-NAT mRNA. Mention should be made here of some of the
possible mechanisms that turn off expression of this gene, based on the
structure of the promoter revealed in this study and current thinking
about this issue. One mechanism that will significantly reduce
expression of this gene involves a decrease in cAMP, which would
terminate positive downstream effects of this second messenger. An
additional theoretical mechanism involves cAMP-dependent
coinduction of negative transcription factors such as Fra-2
(os-
elated
ntigen-
)
(17) and the inducible cAMP early repressor (ICER) (36), which could
repress transcription through binding to AP-1 and CRE sites,
respectively.
Levels of mRNA encoding both factors increase in the pineal gland
during the night period of a typical lighting cycle providing 10-12 h
of darkness (17, 36). The increase in fra-2 mRNA drives a >100-fold rhythm in Fra-2 protein that is generally similar to the
increase in AA-NAT activity. We suspect that Fra-2 protein binds to
AP-1 sites in the AA-NAT promoter, negatively affecting transcription.
One such site, identified above at position 32, is located in close
proximity to the major transcription start point. Fra-2 strongly binds
to AP-1 sites, but does not possess a strong transactivation domain
(37). Binding of Fra-2-containing AP-1 complexes at this location could
conceivably disrupt the assembly of the basic transcription machinery,
inhibiting cAMP activation of AA-NAT expression. Inhibition of Fra-2
protein synthesis may explain why the cAMP-induced increase in AA-NAT
mRNA is greater if protein synthesis is blocked (9).
Accordingly, it is possible to hypothesize that Fra-2 could be part of a complex AA-NAT gene regulatory mechanism controlled by cAMP, in which cAMP rapidly turns on expression of the AA-NAT gene through phosphorylation of CREB and activation of the natCRE·CCAAT complex and coincidently induces expression of the fra-2 gene and accumulation of Fra-2 protein, which then progressively turns off expression of the AA-NAT gene.
In contrast to the close association of the rhythm in fra-2 mRNA and Fra-2 protein, the rhythm in ICER mRNA is not associated with a remarkable increase in ICER protein; rather, under typical lighting schedules, ICER protein appears to be relatively constant and stable (38). Accordingly, it seems reasonable to suspect that ICER and CREB proteins are present at all times of the day and that they compete for CRE occupancy. As a result, the relative abundance of ICER and CREB pools at these sites becomes an important factor because it could influence the magnitude of the AA-NAT response. Although the relative amount of ICER and CREB might not change under typical fixed laboratory lighting schedules, their relative abundance appears to change in response to very long and very short nights; for example, ICER levels are highest if animals are maintained in lighting cycles with 20-h dark periods (38). This could gradually increase the ICER/CREB ratio and might influence the magnitude of the AA-NAT rhythm. Such a mechanism could underlie seasonal changes in melatonin production.
Potentially Important Features of the AA-NAT PromoterOur analysis revealed two additional features of the AA-NAT promoter that deserve comment. First, in addition to the data obtained by S1 nuclease protection analysis, two observations are consistent with the identification of the major transcription start point. One is the location, 38 bp upstream, of a TATA-like element. The other is the location of the transcription start point within a putative initiator sequence. This sequence belongs to a family of RNA polymerase II start sites (24, 25) and is present in other dynamically regulated genes, including homeotic genes and genes expressed during immunodifferentiation (for review, see Ref. 39). It seems possible that the dynamic regulation of AA-NAT and these genes may involve interactions of this element with the same or similar nuclear factors (40).
A second point relates to the issue of translational control and the
finding that large changes in sheep AA-NAT activity and protein3 can occur with little or no change
in levels of AA-NAT mRNA. It is reasonable to suspect that this
translational control might be a conserved feature of AA-NAT
regulation. In this regard, some features of the rat AA-NAT 5-flanking
region are relevant. Translation is known to be influenced by long
leaders with extensive secondary structure (41, 42). Such extensive
secondary structure is likely to form in the 173-bp leader sequence
according to computer analysis of this region (43); this predicts the
formation of an elongated stem-loop structure with a free energy of
54.1 kcal/mol (data not shown). It will be of interest to investigate
the possible role of the leader sequence upon translational regulation
of AA-NAT.
The promoter of the AA-NAT gene has been isolated and partially characterized. It appears that a CRE and an adjacent inverted CCAAT box function as a combined target site for the adrenergic signaling cascade that activates expression of this gene. The pattern of expression of the AA-NAT gene is similar to that of an immediate-early gene in that it is rapidly activated in the absence of de novo protein synthesis (9). Thus, rapid induction of AA-NAT gene transcription appears to rely on adrenergically induced post-translational modification and/or recruitment of pre-existing trans-acting factors belonging to the CREB and CATBP families.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U77455[GenBank].
We express our gratitude to Brian O'Connell for the generous gift of the adenovirus shuttle particle stock used in this study for transfection of primary cell cultures.
Following submission of this manuscript, Folkes et al. reported the identification of natCRE (Folkes, N. S., Borjigin, J., Snyder, S., and Sassone-Corsi, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14140-14145). In addition, they found a rhythm in AA-NAT mRNA in the normal mouse and in mice lacking ICER and found that the amplitude of the AA-NAT mRNA rhythm in mice lacking ICER was larger. This confirms the proposal here that ICER does not play a dynamic role in rhythmically turning off AA-NAT gene expression, but does play a role in determining the amplitude of the increase in AA-NAT mRNA.