Serotonin (5-hydroxytryptamine, 5-HT) ()is a neurotransmitter used by one of the most extensive signaling systems found in the brain(1) . Neurons that release serotonin have axons that project widely throughout the brain, and a family of related receptors has been identified that mediates its biological effects. These receptors were originally classified based on differential affinities for agonists and antagonists, and subsequent sequence analysis of cloned cDNAs has shown that most of the serotonin receptors belong to the family of membrane-spanning, G-protein-coupled receptors(2, 3) .

Serotonin receptor expression is highly tissue-specific, and in cases that have been studied, each serotonin receptor family member seems to have a characteristic pattern of regional expression within the brain (2, 3) . This restricted pattern of expression is probably a critical point of control for mediating specific biological responses to serotonin that are believed to affect brain development, arousal, learning and memory, sensory perception, and higher cognitive functions (1, 4, 5, 6) .

The serotonin 1a (5-HT1a) receptor has several features that make it an interesting candidate for study of gene regulation. It is expressed in a restricted pattern in the brain. 5-HT1a receptor mRNA has been detected by in situ hybridzation in the hippocampus, midbrain raphe nuclei, and cerebral cortex(7, 8, 9) . 5-HT1a receptor expression is regulated during development; it is present transiently in the rat cerebellum(10, 11, 12, 13) . The levels of 5-HT1a receptor and its mRNA apparently are regulated by hormones(14) , and the 5-HT1a receptor itself may be an important regulator of gene expression through its coupling to a G-protein that negatively regulates adenylate cyclase(15, 16) . All of these characteristics probably contribute to the roles thought to be played in the brain by the 5-HT1a receptor, influencing mood, behavior, and regulation of neuroendocrine function (17, 18, 19, 20, 21, 22, 23, 24, 25, 26) .

Here we report that the 5`-flanking sequences of the human and mouse 5-HT1a genes contain complex TATA-less promoters that specify numerous RNA 5` ends extending through a 700-base pair region. In addition, we have characterized a G/C-rich DNA sequence motif in the promoter that participates in transcriptional regulation. We cloned a cDNA encoding a protein that interacts with this sequence element, and found that it was the MAZ (Pur-1, Zif87) DNA-binding protein(27, 28, 29) . In previous studies, MAZ was cloned based on its interactions with sequences in the c-myc and insulin gene promoters. Both of these genes contain TATA box elements. Our data indicate that MAZ may also play an important role in regulating expression from TATA-less promoters like the 5-HT1a receptor gene promoter, and in some cases, the G/C-rich binding site may serve as a transcription initiator sequence.

Analysis of G/C-rich transcriptional control elements is complicated by the relatively large number of different DNA-binding proteins that are capable of interaction with GC-rich DNA sequences(30) . Consistent with this fact, we have found that the MAZ binding sites located in the 5-HT1a receptor 5`-flanking region also interact with transcription factor Sp1(31) . Transient expression experiments revealed that both MAZ and Sp1 can stimulate expression from the 5-HT1a receptor 5`-flanking region, but MAZ was the more potent activator.

METHODS

Cell Lines and Transfections

The Q5B and Y8 cell lines were generated by targeted tumorigenesis (32) . Transgenic mice carried the SV40 virus tumor antigen (SV40 T-Ag) coding region under the control of the 5`-flanking region of the human 5-HT1a receptor gene. Transgenic mice were generated by pronuclear injection of fertilized eggs from C57Bl/6J DBA F mice using standard procedures(33) . Cell lines were derived from brain tissue from 6-8-week-old transgenic mice. Brain tissue was dissociated from transgenic animals using modifications of published procedures (34, 35) and were prepared from the dorsal raphe nucleus and surrounding tissue(36) . The dissected tissue was minced with a razor blade and then incubated 30 min at 37 °C in 1 ml of phosphate-buffered saline that contained 1% glucose and 0.025% trypsin. The treated tissue fragments were collected by centrifugation and resuspended in 1 ml of phosphate-buffered saline supplemented with 1% glucose, 0.1 mM EDTA, and 0.2% bovine serum albumin. Cells were dissociated by tituration with a fire-polished Pastuer pipette. Debris was allowed to settle out of solution for about 5 min, and the cell supernatant was transferred to a new tube and mixed with an equal volume of DMEM containing 10% fetal bovine serum. The cells were then collected by centrifugation and plated on a poly-L-lysine-coated 35-mm tissue culture dish in DMEM containing 10% fetal bovine serum and nonessential amino acids. After incubation for 12 h, nonadherent cells and debris were washed away, and the cells were fed with fresh medium. The medium was changed every 2-3 days until colonies were visible. Individual cell colonies were cloned to establish cell lines. The Q5B and Y8 cell lines were derived from different transgenic mouse lines.

For transient expression assays, cells were transfected by the calcium phosphate precipitation method (37, 38) when approximately 50% confluent. Each 10-cm plate received a total of 40 µg of DNA that included the appropriate plasmid DNAs and sheared salmon sperm DNA in the amounts specified in the figure legends. Cultures were incubated with the calcium phosphate precipitate for 10-14 h (6-8 h for U-87 MG cells) before the cells were washed and fed with fresh medium. For analysis of CAT and luciferase activity, cells were harvested 72 h after transfection and lysed by three freeze-thaw cycles in 100 mM potassium phosphate buffer (pH 7.8) containing 1 mM 2-mercaptoethanol. CAT assays were performed as described previously(39) , and relative CAT activity was quantified after thin layer chromatography using a PhosphorImager. Luciferase activity was quantified using reagents from Analytical Luminesence Laboratory (San Diego, CA). RNA was prepared from transfected cells, as described below, by lysis in guanidinium isothiocyanate. Protein expression in transfected cells was monitored by Western blotting (37) using antibody specific for the flu epitope tag (antibody 12CA5; Boehringer Mannhiem) and antibody against transcription factor Sp1 (anti-PEP2 antibody, Santa Cruz Biotechnology Inc., Santa Cruz, CA).

Promoter and cDNA Cloning

The MAZ cDNA was cloned by screening a HeLa cell cDNA phage expression library (Clontech, Palo Alto, CA) for DNA binding activity that recognized oligonucleotide -21G/C (nucleotides -6 to -35 in the human sequence) shown in Fig. 1and Fig. 8. Both DNA strands were designed with 4-base overhanging complementary ends. The probe was prepared by labeling 2 µg of each oligonucleotide in a reaction containing 1 mCi of [-P]ATP and polynucleotide kinase followed by a chase period including 2 mM unlabeled ATP to ensure phosphorylation of the oligonucleotides. After labeling, the oligonucleotides were precipitated with ethanol, and then the DNA strands were resuspended and combined in 50 mM Tris-HCl (pH 7.4), 10 mM MgCl, 1 mM spermidine, heated to 90 °C, and cooled slowly to room temperature to anneal the oligonucleotides. ATP and dithiothreitol (DTT) was added to 2 mM and 10 mM, respectively, 100 units of ligase were added, and the ligation reaction was incubated at 15 °C overnight. Plaque screening was performed with the oligomerized probe oligonucleotide as described previously(37, 44) . 6 10 plaques were screened, and one phage was isolated that expressed the appropriate DNA binding activity. The cDNA insert from the positive phage was subcloned and sequenced. The resulting MAZ cDNA sequence was about 200 bp longer at the 5` end than the human sequences reported previously.

Figure 1: 5-HT1a receptor gene promoter DNA sequences and RNA 5` ends. The human 5`-flanking sequence is presented on the top line and the mouse sequence is the lower line. The sequences were aligned with the GCG Gap program. The boxed nucleotides represent positions were 5` termini were mapped using nuclease S1. An arrowhead below a nucleotide in the mouse sequence indicates that this was a 5` termini identified from mouse brain RNA using the RACE technique. Asterisks over two regions in the human DNA sequence identify the sequence of two oligonucleotides, -21G/C (nucleotides -6 to -35) and -628G/C, which contain G/C-rich sequence motifs and were used in experiments presented below.

Figure 8: Summary of MAZ binding sites and RNA 5` ends in the minimal promoter. The human promoter sequence from -207 to +33 is shown labeled with the boundaries of the footprints (sites I-IV) generated by MAZ. Sites I-IV are indicated by shaded boxes. Sp1 footprints are indicated by cross-hatched boxes. RNA 5` ends mapped in Fig. 3are shown above the sequence and marked with an arrow in the direction of transcription. Open arrowheads above the DNA sequence labeled a-e provide estimates of were the 5` termini of CAT (Fig. 9C) and luciferase (Fig. 9D) RNAs mapped in cotransfection experiments. In vitro transcription start sites (Fig. 10) mapped within the site I region are designated by closed arrowheads. The -21G/C oligonucleotide sequence is indicated by a solid bar between the DNA strands.

Figure 3: Characterization of RNA 5` ends originating from the human 5-HT1a receptor gene 5`-flanking region. A, the DNA structure of the transgene present in the Q5B cell line is illustrated by rectangles at the bottom of the panel. Above the DNA diagram are the probe DNAs used to map 5` ends with the S1 nuclease assay. The P-labeled 5` end is identified by a filled circle. B, analysis of mRNA 5` ends originating from the human promoter sequence. The details of this experiment are essentially as described in Fig. 2. Hybridizations were performed with 50 µg of total RNA at 50 °C. RNAs: Q5B cell RNA (Q5B); HeLa cell RNA (H). Bands representing 5` ends are marked by arrows and brackets. The nucleotide position relative to the 5-HT1a receptor translation start site is marked adjacent to the sequencing reactions.

Figure 9: Effect of elevated levels of MAZ and Sp1 on activity of the proximal 5-HT1a receptor promoter region. A, U-87 MG cells were cotransfected with a luciferase reporter plasmid (p5HT1a/-225luc) containing the sequence from -6 to -225 relative to the translation start site from the receptor gene plus an expression vector with no inserted gene (Vector), the vector expressing an epitope-tagged Sp1 protein (Sp1), or the vector expressing an epitope-tagged MAZ protein (MAZ). The luciferase activity is expressed relative to the activity observed when the expression vector was cotransfected with the reporter plasmid. The results from six independent experiments are shown. The values of induction by MAZ or Sp1 are expressed relative to the values obtained when the the vector DNA was cotransfected. B, transfected cells were tested for expression of MAZ or Sp1 by Western blot assay. Left panel, overexpressed proteins were detected using antibody specific for the epitope tag. The extract in lane 1 (Mock) was derived from mock-transfected cells that received only salmon sperm DNA; extract in lane 2 (Vec) was derived from cells cotransfected with the reporter, and the expression vector with no cDNA insert, extracts in lanes 3 and 4, were derived from cells cotransfected with the luciferase reporter and the MAZ or Sp1 expression vector, respectively. Right panel, extracts from mock-transfected and Sp1 expression vector-transfected cultures were tested by Western blot using an Sp1-specific antibody. C, effect of MAZ expression on steady-state levels of CAT reporter mRNA. RNA was examined by nuclease S1 assay from cells that did not receive reporter plasmid (lane 1), cells that received the reporter alone (lane 2), and cells that received the reporter (p1a/-225CAT) plus MAZ expression plasmid (lane 3). The RNA was detected by hybridization at 44 °C with a probe specific for the reporter RNA. Bands representing CAT RNA 5` ends are indicated by arrows. The nucleotide position within the 5`-flanking sequence is marked adjacent to the sequencing reactions. D, effect of Sp1 expression on steady-state levels of luciferase reporter (p1a/-225luc) mRNA. RNA was examined, as described above, by nuclease S1 assay from cells that did not receive reporter plasmid (lane 1), cells that received the reporter alone (lane 2), and cells that received the reporter plus MAZ (lane 3) or Sp1 (lane 4) expression plasmids. Designations are as in C.

Figure 10: Transcription initiator activity associated with site I. A, in vitro transcription was performed with HeLa whole cell extract and a plasmid template containing a cloned copy of the -21G/C oligonucleotide ( Fig. 1and Fig. 8) or the adeno-associated virus p5+1 initiator element(70) . The structure of the template DNAs is indicated at the top of the figure. Lanes 1 and 2 were negative control reactions that lacked extract or template, respectively. RNA synthesis was detected by primer extension. The size limits of primer extension products with 5` ends occurring within the cloned oligonucleotide sequence is indicated by an arrow bounded by brackets. The 5` ends detected in this experiment and examined in more detail in B are designated by a open box at the right side of the autoradiogram. B, transcription start sites were mapped by primer extension and analyzed on a sequencing gel next to sequencing reactions (A, G, C, and T). The promoter region examined is represented above each autoradiogram. The bracketed arrows show the limits of the site I sequence and the open boxes at the sides of the autoradiograms identify the same series of bands in each autoradiogram in A and B. The nucleotide positions along the sequence of the human promoter sequence is identified by arrowheads. The experiment analyzed in lanes 1-6 was performed with RNA isolated from the Q5B cell line (Fig. 3) or the negative control HeLa (H) cell line. In vitro transcription reactions were analyzed in lanes 7-17. Negative control transcription reactions(-) contained no template and (+) indicate addition of template.

Figure 2: Mouse 5-HT1a receptor mRNA characterization. A, representation of the genomic 5-HT1a receptor gene. The probe DNAs that were used for S1 analysis are indicated above the boxes that depict the 5`-flanking sequence, coding region, and 3`-noncoding (NC) domain. The P-labeled 5` ends of probe DNAs are represented by black circles. The primers used for RACE (SR30, 31, and 33) and a Northern blot probe are drawn below the DNA diagram. B, detection of 5-HT1a receptor mRNA in preparations of mouse brain RNA by Northern blot. RNA was subjected to electrophoresis in 1% formaldehyde-agarose gels and transferred to nitrocellulose filters. In the panel labeled Poly A, 10 µg of poly(A) or poly(A) RNA was analyzed from brain (Br) or liver (L). In the panel labeled Total, 25 µg of total RNA were analyzed from three brain regions (midbrain, Mb; cerebral cortex, Cx; cerebellum, Cb), liver (L), and HeLa cells (H). The positions of RNA size markers (in kb) are indicated. C, S1 nuclease analysis of 5-HT1a receptor RNA 5` ends. The 5` end-labeled probes illustrated in A were hybridized to 200-250 µg of the total RNA indicated above each lane. Hybridization was performed for 10 h at 50 °C except for reactions containing probe 2 that were hybridized at 55 °C. After digestion of hybrids with S1 nuclease, the reaction products were subjected to electrophoresis on a 6% DNA sequencing gel. Bands representing 5` ends are marked by arrows and brackets. Chemical sequencing reactions were performed with probe DNA to prepare DNA sequence ladders (GA and AC). The nucleotide position relative to the 5-HT1a receptor translation start site is marked adjacent to the sequencing reactions. Designations for RNA sources are as in B.

For expression in eucaryotic cells, the MAZ cDNA was cloned downstream of the human cytomegalovirus immediate early transcriptional control region in plasmid pCGN(45) . The entire MAZ cDNA was fused in frame with an amino-terminal 9-residue sequence encoding the influenza virus heamagglutinin epitope tag recognized by monoclonal antibody 12CA5 (Boehringer Mannhiem). The cDNA was cloned into pQE-11 (Qiagen Inc., Chatsworth, CA) to allow synthesis in Escherichia coli of MAZ containing 6 amino-terminal histidine residues. The Sp1 cDNA sequence, obtained from plasmid pAct-Sp1(46) , was also inserted into pCGN to generate an epitope-tagged Sp1 variant for expression in eukaryotic cells.

Computer-aided sequence analysis was performed with the GCG Wisconsin Package version 8.0 (Genetics Computer Group, Inc., Madison, WI).

RNA Mapping

cDNA clones representing the 5` termini of the mouse brain 5-HT1a receptor mRNA were cloned by the RACE (rapid amplification of cDNA ends) technique (40, 47) using whole brain RNA that was treated with DNase I. Control reactions performed as described below, but without reverse transcriptase, indicated that the DNase-treated RNA was free of detectable DNA. Primer SR30 (5`-ATGAATCCAGGGACGTTGTGGT-3`), complementary to nucleotides 34 to 50 in the coding region, was used to prime cDNA synthesis with Super Script reverse transcriptase (Life Technologies, Inc.). The cDNA was purified and modified by addition of a polydeoxyadenosine sequence to the 3` end with terminal transferase. The complementary strand of the cDNA was synthesized by one PCR cycle (94 °C, 1 min; 47 °C, 5 min; 72 °C, 10 min) in the presence of the adapter primer SR12 (5`-GCTCTGGATCCAAGTCTAGA(T)-3`), and then the DNA was amplified by 30 PCR cycles (94 °C, 1 min; 60 °C, 1.5 min; 72, 3 min) using gene-specific primer SR32 (5`-GTTGTTGCCCTGGCCAAGACT-3`, complementary to nucleotides 13-32) and the adaptor primer without the poly(T) sequence. The PCR product was cloned, and recombinant plasmids were identified by colony hybridization using oligonucleotide SR33 (5`-ATCCATGCCTGCCTGCACTCC-3`, complementary to nucleotides +6 to -16) as the probe DNA.

Northern blots were performed essentially as described earlier(48) . RNA was subjected to electrophoresis in formaldehyde agarose gels and transferred to nitrocellulose filters. Prehybridization and hybridization was performed at 42 °C in buffer containing 50% formamide, 0.25 M sodium phosphate (pH 7.2), 0.25 M sodium chloride, 1.0 mM EDTA, 1% SDS, 2 Denhardt's solution(49) , 100 µg/ml sheared salmon sperm DNA, and 10% dextran sulfate. High stringency washes were performed at 60 °C in 0.1 SSC(49) , 0.05% SDS.

RNA 5` termini were detected by nuclease S1 mapping(50, 51, 52) . Appropriate DNA probes were P-labeled at the 5` end with polynucleotide kinase (kinase reaction buffer: 50 mM Tris (pH 7.5), 10 mM MgCl, 1 mM spermidine, 10 mM DTT, 10% glcerol, 0.05% Nonidet P-40), and hybridized to RNA (50-250 µg) in a final volume of 40 µl of 40 mM PIPES (pH 6.4), 400 mM NaCl, 1 mM EDTA, 80% formamide. The nucleic acids were denatured at 70 °C for 10 min, then hybridized 10-12 h at temperatures determined empirically (see figure legends). S1 digestion was performed at 37 °C for 2 h by adding 0.4 ml of a solution containing 250 mM NaCl, 1 mM ZnCl, 30 mM sodium acetate (pH 4.6), and 1000-2000 units/ml nuclease S1. The reaction products were precipitated and analyzed on DNA sequencing gels using sequence reactions of probe DNAs as markers (53, 54) .

DNA-Protein Interactions

DNA band-shift assays (57, 58) were performed by a modification of Hendrickson et al.(59) . Probe DNAs were prepared by 3`-end labeling with the appropriate [-P]dNTP and T7 DNA polymerase. 15 µl of binding reactions contained 2 µl of protein diluted to the appropriate concentration, 10 mM Hepes (pH 7.9), 60 mM KCl, 0.1 mM EDTA, 250 µg of bovine serum albumin/ml, 2 µg of poly(dA-dT)poly(dA-dT) (Pharmacia), 0.5 µg of sonicated and denatured salmon sperm DNA, 0.5 mM DTT, 0.05% Nonidet P-40, 5% gylcerol, and P-labeled probe DNA (10-20 fmol). Reactions were incubated 15-30 min at 25 °C and loaded onto a 4% (40:1 ratio of acylamide to bisacrylamide) native polyacrylamide gel run in Tris-EDTA buffer (10 mM Tris-HCl (pH 7.4), 1.0 mM EDTA). Electrophoresis was at 250 V (20-25 mA) for 2-3 h with buffer recirculation at 4 °C.

DNase I footprinting (60) was performed as described previously(51) . Probe DNAs were labeled at the 5` end with polynucleotide kinase and [-P]ATP or labeled at the 3` end with DNA polymerase (Sequenase, U. S. Biochemical Corp.) and the appropriate [-P]dNTP(49) . 60-µl binding reactions contained the amount of recombinant MAZ protein specified in the figure legends, 10 mM Hepes (pH 7.9), 60 mM KCl, 0.1 mM EDTA, 0.1 µg of poly(dA-dT)poly(dA-dT), 0.5 mM DTT, 0.05% Nonidet P-40, 5% gylcerol, 100 µg of bovine serum albumin/ml, 1 mM magnesium acetate, and approximately 10 fmol of labeled probe DNA. Reactions were incubated on ice for 10 min with all components except the probe, then the probe DNA was added, and the reaction was incubated 15-30 min at 25 °C. DNase I (2-10 units; Promega) was added, and the incubation was continued 1 min at room temperature. The reaction was stopped with 250 µl of buffer containing 350 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2% SDS, 7 M urea, and 50 µg/ml yeast RNA. The reaction products were purified by phenol-chloroform extraction and ethanol precipitation and were analyzed on DNA sequencing gels with chemical sequencing reactions of probe DNAs as markers(53, 54) .

In Vitro Transcription

RESULTS

5-HT1a Receptor Promoter Structure

Initially, we screened cell lines derived from neuroblastomas (SK-N-SH, SK-N-MC, and IMR-32), a glioma (C6), and astrocytomas (U-87 MG, U-373 MG, and CCF-STTG1) for the presence of 5-HT1a receptor mRNA using a ribonuclease protection assay that was sensitive enough to detect the mRNA in total mouse and rat brain RNA (data not shown). None of the cell lines expressed detectable amounts of the receptor mRNA. So we mapped murine mRNA 5` ends using mouse brain RNA and analyzed human 5` ends using a cell line produced from the the brain of a transgenic mouse that expressed the SV40 T-Ag gene under the control of the human 5-HT1a receptor 5`-flanking region. These cells expressed nestin RNA (data not shown), suggesting that they may represent a neuronal precursor cell type(63) .

Fig. 2A illustrates the probes used for mouse brain RNA mapping experiments, and Fig. 2B displays a Northern blot analysis of RNA used for 5`-end mapping experiments. The Northern blot analysis showed that the 5-HT1a receptor mRNA was present in the poly(A) fraction of brain RNA and, as expected, was undetectable in liver RNA. The receptor mRNA appeared as a broad band of estimated size between 5 and 8 kilobases. This broad size distribution implies that alternative RNA processing or transcription start site selection occurs during the synthesis of the 5-HT1a receptor mRNA. Differences must be limited to the 5`- and 3`-noncoding regions because DNA sequence analysis has indicated that the receptor coding region is not interrupted by introns(15, 42, 64) .

We next prepared RNA from three brain regions: the cortex, cerebellum and midbrain. In this case (Fig. 2B), we examined total RNA by Northern blot and the corresponding hybridization signal is weaker than observed with brain poly(A) RNA. No 5-HT1a receptor RNA was detected in the negative control mouse liver and HeLa cell RNAs, but the RNA was present in all brain samples, and it was most abundant in the midbrain and cortex (Fig. 2B). Consistent with these results, the 5-HT1a receptor mRNA has been detected in specific structures within these regions by in situ hybridization(7, 8, 9) .

The mRNA 5` ends were mapped by hybridization with 5` end-labeled DNA probes followed by nuclease S1 digestion. The various probe DNAs used for hybridization are shown schematically in Fig. 2A. These overlapping probes allowed us to examine a large portion of the 5`-flanking region for 5` ends. Individual probe DNAs were hybridized to total brain RNA then digested with nuclease S1. The nuclease-resistant reaction products were subjected to electrophoresis on a sequencing gel using a DNA sequence ladder generated from the probe DNA (Fig. 2C). Numerous apparent 5` ends, represented by bands of varying intensities, were scattered over a large area (>700 bp) of the mouse 5-HT1a flanking sequence. The S1 assay detected receptor RNA in all three brain regions tested (Fig. 2C), and the relative abundance of the mRNA in the different regions followed a similar trend as in the Northern assay (Fig. 2B).

Probe 1 allowed clear visualization of start sites nearest the receptor coding region. The region between -15 and -45 gave rise to some of the strongest bands in this domain. Several bands were visible about five bases above the strong signal at -45, but these bands also appeared in the negative control lane (L) and presumably are not specific. Two additional proximal clusters of RNA termini were located between -105 and -125. Numerous weaker bands mapped further 5` of -125, locating 5` ends for less abundant RNAs. A substantial percentage of the probe DNA was retained near the top of the gel, indicating that there was a significant number of 5` ends that mapped to regions distal to the coding region.

Probe 2 provided a closer examination of the region from -190 to about -400. This region contained numerous apparent 5` ends, but they were of reduced abundance compared to those detected by probe 1 that mapped closer to the receptor coding region. Again, a substantial portion of the signal was retained near the top of the gel. Probes 3 and 4 resolved the more intense bands compressed at the top of the previous two gels. More than seven clusters of 5` ends were detected upstream of -620, extending as far as -760.

To confirm that the 5-HT1a receptor gene 5`-flanking sequence generated transcripts with many different 5` ends, we used a second approach to analyze the structure of the 5` ends. Our goal was not to confirm every start site but to show agreement, by a second method, with some of the start sites detected with the S1 nuclease assay. We chose the RACE method (47) because it is based upon a distinctly different approach involving primer extension, PCR amplification, and cloning rather then a nuclease digestion assay. The primers used in the RACE procedure are shown schematically in Fig. 2A. The RNA used for RACE was a different preparation then that used for the nuclease mapping, and this RNA was treated with DNase I to reduce the potential for cloning of genomic DNA. Whole brain RNA was subjected to reverse transcription using primer SR30 and the resulting cDNA was modified by addition of a poly(A) stretch at its 3` end to provide a sequence that would anneal to an adapter primer for PCR. The cDNA pool was amplified by PCR with the adapter primer and nested primer SR32, the products were cloned, and nested primer SR33 was used as a probe for colony hybridization. The 5` ends identified by sequencing RACE clones are marked by arrowheads in Fig. 1. Each of the 5` ends identified by this analysis mapped to a 5` end domain identified in the S1 analysis, providing a strong indication that our mapping results are accurate. The 5` termini identified by RACE were restricted to the proximal grouping of 5` ends mapped by S1 nuclease digestion, and this is not surprising since the primer extension step would not be expected to copy the longest 5` RNA sequences at high efficiency. None of the RACE clones revealed any evidence for splicing within the 5-HT1a receptor gene 5`-untranslated region.

The results presented above indicated that transcription initiates within the mouse 5-HT1a receptor gene at a large number of sites in a region spanning greater than 700 bp. Consistent with this observation, the mouse 5`-flanking sequence does not contain any evident TATA box homologies. If this type of complex transcription initiation arrangement plays an important role in regulating the mouse 5-HT1a receptor gene, we would expect this promoter structure to be evolutionarily conserved. Therefore, we analyzed the 5` ends generated by the human 5`-flanking region using cell lines established from the brains of transgenic mice that contain a copy of the SV40 T-Ag coding region fused to 1.2 kbp of human 5-HT1a receptor upstream flanking sequence (Fig. 3A).

Using probes specific for the transgene (Fig. 3A), we determined the positions of RNA 5` ends produced from within the human flanking sequence in two cell lines derived from different transgenic animals: Q5B cells (Fig. 3B) and Y8 cells (data not shown). Each probe generated numerous nuclease S1-resistant bands indicating that the human promoter also gives rise to a large number of RNAs that differ at their 5` ends, as was the case for mouse brain RNAs. In fact, many of the 5` ends mapped in the human promoter correspond in location to ends mapped in the mouse upstream region (Fig. 1), suggesting that the generation of multiple RNA termini is an evolutionarily conserved feature of this gene.

5-HT1a Receptor Promoter Function

Figure 4: Transient expression analysis of the human 5-HT1a receptor gene promoter. A, representation of the reporter gene constructs used in these experiments. The approximate location of major RNA 5` ends is show above the DNA diagram and restriction enzyme sites used to make deletions are shown below. B, bar graphs that summarize the results of transient expression assays performed in four different cell types. Cells were transfected with 10 µg of appropriate CAT reporter plasmid, and to control for transfection efficiency, cells were cotransfected with a luciferase reporter gene (2 µg) linked to the promoter and enhancer of either the SV40 virus or Moloney murine leukemia virus. Transfections were repeated four to six times to allow calculation of a mean value and standard deviation. The values for CAT activities were calculated after thin layer chromatography and quantification with a PhosphorImager. Values were expressed relative to the activity obtained with the full-length promoter construct (p5HT1a/-1176CAT, shaded bar) that was set at a value of 1. Names of reporter constructs are indicated below the x axis of the bar graph: reporter plasmid lacking a promoter insert (Vec); reporter with -6 to -1176 sequence (1a/-1176); reporter with -6 to -225 (1a/-225); reporter with herpes simplex virus thymidine kinase gene promoter (HSV TK).

The p5HT1a/-1176CAT construct was functional in all of the cell types tested and capable of generating 4-5-fold more CAT activity than the negative control plasmid. In three cell types (HeLa, U-87 MG, and Q5B), the activity of the thymidine kinase promoter was higher than that of p5HT1a/-1176CAT. The difference was highest in Hela cells where the activity of the thymidine kinase promoter was five times higher than the serotonin receptor promoter. In contrast, p5HT1a/-1176CAT and the thymidine kinase promoter generated equal levels of CAT activity in the SK-N-SH neuroblastoma cells.

The more proximal 5`-flanking segment (p5HT1a/-225CAT) was more active than the longer receptor promoter segment in U-87 MG cells and 3-5-fold more active than the thymidine kinase promoter in HeLa, U-87 MG, and SK-N-SH cells. However, the more upstream sequences had little inhibitory effect in the Q5B cells, which were selected for the presence of an active 5-HT1a receptor promoter. In these cells, p5HT1a/1176CAT and p5HT1a/-225CAT exhibited similar activity. This suggests that the upstream domain contains one or more elements that inhibit activity of the 5-HT1a promoter in cells where the gene is not expressed.

MAZ and Sp1 Bind at Multiple Sites within the 5-HT1a Receptor Promoter

Initial in vitro transcription experiments indicated that the -6 to -35 domain, which contains a G/C-rich element (Fig. 1, sequence marked by asterisks) could independently direct transcription (data not shown and Fig. 10), so we asked whether the DNA segment could interact with one or more proteins in a band-shift assay. The G/C-rich oligonucleotide (-21G/C, Fig. 1and Fig. 8) was labeled with P, incubated with a HeLa cell nuclear extract, and DNA-protein complexes were assayed by electrophoresis. Two prominent complexes were observed (Fig. 5A, complexes A and B). These major complexes appeared to represent specific protein-DNA interactions because unlabeled homologous oligonucleotide inhibited their formation (Fig. 5A, compare lanes 2 and 3). Several minor complexes were detected, but they were less susceptible to competition with the homologous oligonucleotide, indicating that they are probably nonspecific.

Figure 5: DNA-protein complexes formed with the -21G/C motif. A, band-shift assay of DNA-protein complexes formed with HeLa cell nuclear extract proteins (1 µg), purified MAZ (2.5 ng), and purified Sp1 (10 ng). The variable components of each binding reaction are listed above the appropriate lane in the gel. Competitor DNA oligonucleotides were included in the binding reaction at a 100-fold molar excess over the probe DNA. The competitor DNAs were: -21G/C, the G/C-rich motif sequence located between -6 and -35 in the human 5-HT1a receptor promoter; -628G/C, the G/C-rich motif found located between -613 and -642 in the human promoter; Myc/MAZ, the MAZ binding site (ME1a1) in the human c-myc promoter; E1b/Sp1, the Sp1 binding site in the adenovirus E1b promoter; P5 YY1, the negative control competitor DNA homologus to the binding site (P5+1) in the adeno-associated virus P5 promoter for the transcription factor YY1. The specific A and B complexes are located by arrows. B, schematic representation of the MAZ DNA-binding protein illustrating some of the more notable features found in the primary amino acid sequence including six zinc finger motifs as well as proline-rich, alanine-rich, and glycine-rich domains.

To identify the proteins present in the A and B complexes, we used the -6/-35 oligonucleotide to screen a phage expression library for the presence of HeLa cell cDNAs encoding proteins able to bind the G/C-rich sequence. We isolated one phage encoding a specific binding protein from 6 10 plaques, and subcloned and sequenced its cDNA insert. A GenBank search revealed that the same cDNA was cloned previously, based on its ability to interact with sequence elements present in the promoters of the c-myc and insulin genes, and termed MAZ, Pur1, or Zif87(27, 28, 29) . MAZ contains six CH-type zinc finger motifs(65) , as well as domains rich in proline, alanine, or glycine (see Fig. 5B).

To test whether MAZ was responsible for one or more of the complexes formed between the -21G/C oligonucleotide (see Fig. 1and Fig. 8) and proteins in the nuclear extract, we compared the mobility of DNA-protein complexes containing recombinant MAZ to the mobility of complexes formed with extract. In addition, we suspected that the slowly migrating complex A contained Sp1, so we included recombinant Sp1 this experiment. The complexes formed with recombinant MAZ and Sp1 proteins comigrated with complexes B and A from nuclear extract, respectively (Fig. 5A, compare lanes 2, 9, and 16). To confirm the identity of the complexes we employed competition experiments with unlabeled oligonucleotides that were included in band-shift reactions at a 100 fold molar excess relative to the labeled probe. The competitor DNAs used included the MAZ binding site from the human c-myc promoter (Myc/MAZ)(27, 29) , the Sp1 binding site from the adenovirus E1b promoter (E1b/Sp1)(66) , the oligonucleotide identical to the probe (-21G/C), another G/C-rich sequence from an upstream region of the 5-HT1a receptor promoter (-628G/C), and, as a negative control, a binding site for transcription factor YY1 from the adeno-associated virus P5 promoter (P5/YY1)(67) . The ability of the competitor DNAs to inhibit MAZ binding to the probe oligonucleotide (E1b/Sp1 > -21G/C, -628G/C > Myc/MAZ) was quite similar to the effect that these competitors have on complex B (Fig. 5A, compare lanes 2-7 with lanes 10-14), arguing that complex B contains MAZ protein. Also, the previously characterized MAZ binding site in the c-myc promoter (the ME1a1 site) effectively competed for the protein that formed complex B (Fig. 5A, lane 4). Recombinant Sp1 binding to the labeled probe DNA was inhibited by the competitors (E1b/Sp1 > MYC/MAZ > -21G/C, -628G/C) to an extent that closely resembled the effect produced on complex A (Fig. 5A, compare lanes 2-7 with lanes 17-21), consistent with the conclusion that complex A contains Sp1. As expected, the negative control YY1 binding site competitor had little effect on the formation of any of the DNA-protein complexes.

In summary, the band-shift analysis indicated that MAZ and Sp1 can both interact with two different G/C-rich motifs from the human serotonin receptor promoter. In addition, the experiment showed that MAZ and Sp1 are capable of interacting with a similar range of G/C-rich sequence elements.

Footprint Analysis of MAZ and Sp1 Binding to the Proximal Promoter Region

Using a probe labeled at its 5` end at position 125, two prominent regions of nuclease protection were observed as the MAZ protein level was increased (Fig. 6A, sites I and II). Between sites I and II, at about -35, there are several unprotected bases that define a boundary between these two binding sites. In site I, MAZ binding provided nearly complete protection of the nucleotides between -5 and -30 (indicated by a solid bar) and weakly protected about 15 bases to the 3` side of the primary region of protection (shaded bar). In site II, MAZ binding strongly protected bases between -53 and -67, protected nucleotides to either side of this domain less completely, and induced a hypersensitive site at -80 (asterisk). The footprint designated site III was weaker then sites I and II, protecting bases between -92 and -117 and inducing hypersensitivity at bases -99 and -100 at the highest MAZ concentrations. Protected and hypersensitive bases were also evident to the 5` side of site III. Site IV comprised the weakest footprint, but it nevertheless contained both protected and hypersensitive bases.

Figure 6: Localization of MAZ binding sites within the proximal 5-HT1a receptor gene promoter region by DNase I protection assay. Variable amounts of purified recombinant MAZ protein were incubated with P- labeled probe DNA followed by limited digestion with DNase I. The reaction products were subjected to electrophoresis in 6% sequencing gels along with DNA sequence ladders (GA and AC). The amount of MAZ protein (ng) is indicated above the appropriate lane. The probe used in the experiment shown in A was labeled at its 5` end at nucleotide position 125 (the template or bottom strand), and the probe used in the reactions shown in B was labeled at its 5` end at position -225 (the non-template or top strand). Regions of DNase I protection are indicated by solid black rectangles (I-IV) and regions with less pronounced indications of protein interactions are marked by lightly shaded rectangles. Hypersensitive bases are indicated by asterisks. The nucleotide position within the 5`-flanking sequence is marked adjacent to the sequencing reactions.

To examine these MAZ binding sites further, the opposite DNA strand was 5`-end-labeled at position -225 (Fig. 6B). Protection within site IV was clearly evident on this strand. The most strongly protected nucleotides in site IV span -160 to -175, but the protection in this region spans 40 bases, possibly because site IV includes two adjacent MAZ binding sites. Sites II and III exhibit weakly protected bases on this strand, and site I is strongly protected on this strand as it was on the other strand.

Next, the footprinting patterns obtained with MAZ were compared to those of Sp1 in the proximal promoter region (Fig. 7). Footprints generated by MAZ are designated by solid bars. The MAZ footprints at sites III and IV are less prominent than observed in Fig. 6, because lower MAZ concentrations were used in this experiment. The Sp1 footprint pattern was different than that produced by MAZ. When the DNA was 5`-end-labeled at position 125, Sp1 protected a sequence within site I that was less extensive than the sequence protected by MAZ (Fig. 7A, cross-hatched bar). This footprint was expected because this region was included in the -21G/C oligonucleotide that bound to Sp1 in the band-shift assays described earlier (Fig. 5). Sp1 also interacted weakly with site II on this DNA strand, while Sp1 binding was not evident at sites III and IV. When the opposite DNA strand was analyzed, the probe was 3`-end-labeled at position 125. Sp1 binding was evident within sites I, II, and III, but not at site IV (Fig. 7B).

Figure 7: Comparison of MAZ and Sp1 interactions with the minimal promoter region. Both DNA strands of the minimal promoter were analyzed by DNase I protection essentially as described in Fig. 6. Probe DNAs were labeled at nucleotide position +125 on the 5` end (A) or 3` end (B). The quantity of protein used in each reaction is indicated above each lane of the autoradiogram. MAZ footprints (Fig. 6, sites I-IV) are represented by a solid black bar on the right side of the figure, and the footprints generated by Sp1 are represented by a cross-hatched bar. Nucleotide positions are indicated next to the sequence ladders (GA and AC).

Finally, the footprint pattern was examined when both proteins were present. In these reactions, Sp1 was maintained at the highest protein mass tested (50 ng), and the amount of MAZ was varied. When the DNA was 5`-end-labeled at position 125 (Fig. 7A), MAZ could compete for occupancy of sites I and II in the presence of Sp1. MAZ binding in the presence of Sp1 was revealed by the larger footprint that occurred as the MAZ concentration was increased. When the complementary strand was assayed by 3`-end labeling at position 125 (Fig. 7B), MAZ again bound at site I in the presence of Sp1, as evidenced by the extended footprint characteristic of MAZ. In contrast, the dominant interaction at sites II and III resembled the Sp1 footprints, indicating that on this strand Sp1 may bind more effectively than MAZ to sites II and III.

The results of the footprint analyses are summarized in Fig. 8. The proximal segment of the 5-HT1a receptor promoter has the potential to interact with both MAZ and Sp1 at multiple sites. Site I can bind to both proteins, but it seemed that both DNA strands favored the interaction with MAZ. Interactions in sites II and III on the nontranscribed strand appeared to favor MAZ, but Sp1 binding was predominant on the template strand under these assay conditions. The DNA strand-specific nature of the DNA-protein interactions observed when both proteins were present suggests that MAZ and Sp1 may interact simultaneously with sites II and III. Only MAZ binds at site IV. Messenger RNA 5` ends were mapped to many locations within this region of the promoter. The most abundant 5` ends reside between -15 and -50, a region that overlaps site I and extends into the region between sites I and II.

Stimulation of the 5-HT1a Proximal Promoter by Overexpression of MAZ or Sp1

When the MAZ expression plasmid was cotransfected with the reporter, the level of gene expression increased 10-20-fold (Fig. 9A) over control transfections including the expression vector. When the Sp1 expression construct was used the levels of luciferase activity were increased 2-10-fold. In six independent assays, overexpression of MAZ always increased luciferase expression to a substantially greater level than that achieved with Sp1.

The amounts of epitope-tagged MAZ and Sp1 were monitored in transfection experiments by Western blotting using antibody directed against the epitope tag. The 57-kDa MAZ protein was clearly present in the appropriate transfected cell extracts (Fig. 9B, lane 3). In cells transfected with the Sp1 expression vector, a substantial quantity of a large protein was produced that migrated as a broad band (Fig. 9B, lane 4). The large size (over 90 kDa) was consistent with this band being epitope-tagged Sp1. The broad band size probably was due to varying degrees of glycosylation that normally occur during Sp1 synthesis(68) . To confirm that the epitope-tagged Sp1 was similar in mobility to endogenous Sp1, we analyzed cell extracts with anti-Sp1 antibody (Fig. 9B, lanes 5 and 6). The large band in the cells transfected with the Sp1 expression plasmid was recognized by anti-Sp1 and the polypeptides of largest relative mobility in this band comigrated with both the endogenous Sp1 detected in the extract from mock transfected cells and the band detected by the anti-epitiope antibody.

The luciferase assays demonstrate the ability of MAZ and Sp1 to enhance expression from the proximal promoter reporter construct. To show that the effect of MAZ and Sp1 was to stimulate RNA accumulation, we examined the steady-state levels of the reporter mRNAs. We used a nuclease S1 assay, similar to those described in Fig. 2and Fig. 3, to quantify RNA levels and locate RNA 5` ends. Fig. 9C shows that the level of CAT reporter mRNA was increased substantially when MAZ was overexpressed. No bands were visible in the negative control reaction, bands were barely detectable when the MAZ expression plasmid was not included in the transfection mix, and multiple bands were detectable when the MAZ expression plasmid was present (Fig. 9C, lanes 1-3). 5` ends evident in this experiment near -40, between -90 and -130 and near -160, also were detected in the assays of the human 5`-flanking region described in Fig. 3. Similar results were obtained when Sp1 was overexpressed (Fig. 9D), although, consistent with the luciferase assays, Sp1 induced luciferase RNA to a lesser extent than that observed for MAZ.

We conclude that MAZ and Sp1 both stimulate the activity of the proximal 5-HT1a receptor promoter, and both activators induce the accumulation of reporter mRNAs with 5` ends that correspond to termini present in cells where the human promoter is constitutively active. Overexpression of MAZ has a greater effect than overexpression of Sp1.

Transcription Initiator Activity in the Site I Region of the Proximal Promoter

To determine if the in vitro transcription reaction was accurately representing start site selection in vivo, we compared the in vitro start sites to those utilized in vivo in the Q5B cell line. Primer extension analysis (Fig. 10B) was performed on RNA from the Q5B cell line or RNA synthesized in whole cell extract from templates containing either the minimal promoter or a template containing only the site I sequence. As earlier S1 nuclease mapping experiments showed ( Fig. 1and Fig. 3), heterogenous 5` ends were detectable by primer extension (Fig. 10, lanes 1-6) in the site I region (Fig. 10B, bracketed arrow) of the promoter controlling T-Ag expression in the Q5B cell line. In vitro RNA synthesis initiated within the boundaries of site I was also detectable from both template DNAs (Fig. 10B, lanes 7-17).

From this comparison it seems reasonable to conclude that start site selection within site I is similar in vivo and in vitro, and that this result suggests that the in vitro transcription reaction accurately reflects the situation in vivo. The selection of initiation sites within site I also appeared to be a property of this sequence; it seemed to function similarly in vitro in the context of surrounding promoter sequences or as an independent sequence element. In addition, the experiments in Fig. 10show that the ability of site I to mediate transcription initiation in vitro was not an artifact of the plasmid backbone because it was tested within the context of two different vectors (Fig. 10, A and B). In summary, it appears that the site I DNA sequence has the properties of a transcription initiator sequence.

DISCUSSION

Several lines of evidence support the conclusion that complex promoters reside within the 5`-flanking domains of the human and mouse 5-HT1a receptor genes. RNA 5` end mapping experiments demonstrated that numerous RNA 5` termini were positioned within a region of more than 700 bp in both the mouse and human 5`-flanking sequence (Fig. 1Fig. 2Fig. 3). Since some of the mouse brain RNA termini were mapped by two different methods, nuclease S1 and RACE ( Fig. 1and Fig. 2C), we are confident that we have localized bona fide 5` ends which are indicative of transcription start sites. In addition, promoter activity was observed in transient expression assays when the human 5`-flanking sequence was linked to a reporter gene ( Fig. 4and Fig. 9), and stable cell lines were isolated that expressed SV40 T-Ag from the human 5`-flanking sequence, demonstrating the ability of the promoter to function when integrated into genomic DNA (Fig. 3).

The reason for the complex arrangement of numerous start sites spread over an extended domain remains obscure, but the fact that this arrangement is conserved in the human and mouse transcriptional control region argues that it likely has some functional significance. The heterogeneous start sites generate mRNAs that differ in their 5`-untranslated region. Perhaps these mRNAs are differentially regulated at the level of translation in different cell types. Alternatively, the transcription initiation sites may be regulated independently, providing a potential mechanism to differentially control transcription of the gene in different cell types and brain regions. An assemblage of active transcription complexes along a large G/C-rich region may provide greater regulatory options then found in typical promoters that are centered around a TATA box. Greater flexibility could be generated by the ability of the G/C-rich sequences to interact with a relatively large number of different DNA binding proteins, like MAZ, Sp1 and a number of other factors (30) that may participate in the assembly of active transcription complexes.

The results of the transient expression assays point to another potential role of the promoter arrangement. When some of the strongest start sites were deleted from the distal region of the human promoter, the remaining proximal promoter fragment was more active (Fig. 4). The proximal promoter fragment (-6 to -225) was as much as 15-fold more active than the 1.2-kbp promoter fragment when transfected into HeLa cells. This result may simply indicate that the upstream region contains cis-acting DNA sequences that repress transcription, but it is also conceivable that active transcription in the distal region of the promoter may interfere with transcription in the proximal promoter region possibly by a mechanism that involves transcription readthrough. Examples of interplay between transcription units that result in promoter occlusion have been described before (71, 72, 73, 74, 75, 76) .

Consistent with the ability of overexpressed MAZ or Sp1 to stimulate activity of the proximal 5-HT1a receptor promoter (Fig. 9), both proteins bind at multiple sites within the promoter (Fig. 5Fig. 6Fig. 7). Both MAZ and Sp1 are expressed in most if not all tissue types, including the brain(27, 77) , and seem reasonable candidates for factors that sponsor constitutive basal expression of the 5-HT1a receptor promoter. Tissue-specific regulatory factors presumably modulate the ability of MAZ, Sp1, and additional, as yet unidentified, factors to activate the promoter. Overexpression of MAZ consistently induced the activity of the 5-HT1a receptor promoter to a greater extent than overexpression of Sp1. However, it is difficult to assess the significance of this observation since it is possible that MAZ but not Sp1 was limiting in the cells used for the assays.

Sp1 binding sites have been identified in promoters that lack TATA sequences (30) as well as adjacent to TATA boxes(31, 66) . MAZ binding sites may also be found in both types of promoter environments. The 5-HT1a gene promoter is an example of a complex TATA-less promoter that interacts with MAZ. MAZ binding sites have also been identified adjacent to TATA boxes in the case of myc and insulin genes(27, 28, 29) . Apparently, then, both MAZ and Sp1 can function in conjunction with a downstream TATA box or participate in transcription from a TATA-less promoter.

It is generally thought that many G/C-rich, TATA-less promoters can bind one or more Sp1 molecules that recruit specific cofactors (such as TATA-binding protein associated factors, TAFs) which in turn bind to TFIID(30, 78, 79, 80) . These interactions enable Sp1 to participate in the assembly of the transcription initiation complex by helping to anchor TFIID at the promoter, allowing TFIID to interact with the other basic transcription factors and polymerase. In the case of the 5-HT1a receptor gene promoter, our data suggest that at least certain G/C-rich motifs may function through an interaction with either Sp1 or MAZ. The possibility that MAZ plays a critical role in establishing active transcription complexes on the 5HT1a receptor gene promoter gains support from our experiments showing that MAZ can activate the proximal promoter region in transient assays (Fig. 9). Further, site I interacts strongly with MAZ and this DNA sequence can function like an initiator element in whole HeLa cell transcription extracts (Fig. 10). We can not be certain that MAZ or Sp1 is responsible for the initiator activity. However, our results are consistent with the interpretation that MAZ and/or Sp1 can serve to attract key elements of the basal transcriptional machinery to a promoter, helping to establish a transcription initiation complex in the absence of a binding motif for TFIID.

A MAZ binding site consensus sequence (Fig. 11) can be derived by alignment of previously identified MAZ binding sites together with those described in our studies. The motif is very similar to the Sp1 binding site consensus(31) . Consistent with this similarity, we found that Sp1 can bind to three of four MAZ binding sites located in the proximal promoter region. Like the G/C-rich motifs in the serotonin receptor promoter, the c-myc promoter G/C-rich motif has been shown to bind both MAZ and Sp1(81) . The ability of MAZ to interact with at least some Sp1 binding sites, and vice versa, suggests that the activity of some G/C-rich motifs potentially results from interactions by multiple transcription factors at the same or overlapping sequences. Indeed, a variety of factors besides Sp1 and MAZ have been shown to interact at G/C-rich motifs (30) . Presumably one or another factor will generally win out and be the predominant DNA-binding moiety residing at any specific motif. The winner could be determined by differences in the affinity of factors for specific G/C elements as well as by the functional concentration of individual factors within the nucleus. However, it is also conceivable that multiple factors might interact with a single G/C-rich motif. Our footprint analysis of the proximal 5-HT1a receptor promoter in the presence of both Sp1 and MAZ is a case in point (Fig. 7). At sites II and III within the proximal promoter, Sp1 appears to preferentially contact the nontranscribed strand, while MAZ interacts with the template strand of DNA. Work is in progress to ascertain whether the two proteins can simultaneously contact the same DNA sequence and, if so, to explore the functional consequence of such a complex interaction.

Figure 11: MAZ binding site comparison. A, MAZ binding sites (I-IV) identified by footprinting and band-shift experiments are aligned. B, MAZ consensus sequence with the Sp1 consensus (31) shown for comparison. C, sites shown to bind recombinant MAZ in other studies (27, 28, 29, 81, 83) .

Finally, some of the conclusions drawn from our analysis of the 5-HT1a receptor gene promoter may be applicable, in a more general sense, to other TATA-less promoters. In particular, we have demonstrated that MAZ, in addition to Sp1, may be an important regulator of TATA-less promoters. Also, we have identified a transcription initiator sequence (site I) that may be utilized by other TATA-less promoters. Initiator consensus sequences are generally rich in pyrimidines with a conserved, central CA dinucleotide, and the majority of transcription initiates at the A residue(82) . The site I sequence is also very rich in pyrimidines (72%), but it does not contain the conserved CA dinucleotide and it directs transcription at multiple sites. These differing characteristics may identify the site I initiator as a new type of initiator.

FOOTNOTES

*

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) U33820[GenBank], mouse serotonin 1a receptor gene promoter; Z11168[GenBank], human serotonin 1a receptor gene promoter; U33819[GenBank], MAZ DNA-binding protein.

§

Associate of the Howard Hughes Medical Institute and a postdoctoral fellow of the National Cancer Institute during this research.

¶

American Cancer Society Professor and an Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed. Tel.: 609-258-5992; Fax: 609-258-1704; :tshenk{at}molbiol.princeton.edu.

()

The abbreviations used are: 5-HT, 5-hydroxytryptamine; DMEM, Dulbecco's modified essential medium; CAT, chloramphenicol acetyltransferase transferase; DTT, dithiothreitol; PIPES, piperizine-N,N`-bis(2-ethanesulfonic acid); PCR, polymerase chain reaction; TF, transcription factor; T-Ag, tumor antigen; RACE, rapid amplification of cDNA ends.

ACKNOWLEDGEMENTS

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