©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Rat Adrenergic Receptor Gene Middle Promoter Contains Multiple Binding Sites for Sequence-specific Proteins Including a Novel Ubiquitous Transcription Factor (*)

(Received for publication, October 11, 1994; and in revised form, December 19, 1994)

Bin Gao Mark S. Spector George Kunos (§)

From the Departments of Pharmacology and Toxicology and Medicine, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Transcription of the rat alpha adrenergic receptor (alphaAR) gene in the liver is controlled by three promoters that generate three mRNAs. The middle promoter (P2), located between -432 and -813 base pairs upstream from the translation start codon and lacking a TATA box, is responsible for generating the major, 2.7-kilobase mRNA species expressed in many tissues (Gao, B., and Kunos, G.(1994) J. Biol. Chem. 269, 15762-15767). DNase I footprinting using rat liver nuclear extracts identified three protected regions in P2: footprint I (-432 to -452), footprint II(-490 to -540), and footprint III (-609 to -690). Putative response elements in footprints I and III were not analyzed except the AP2 binding site in footprint III, which could be protected by purified AP2 protein. Footprint II contains four sites corresponding to half of the NF-I consensus sequence, but DNA mobility shift assays indicate that this footprint binds two proteins distinct from NF-I: a ubiquitous CP1-related factor and another novel factor, termed alpha-Adrenergic Receptor Transcription Factor (alphaARTF), which binds to two separate sites in this region. The alphaARTF is widely distributed, with the highest amounts found in brain, followed by liver, kidney, lung, and spleen, but no detectable activity in heart. Deletions of alphaARTF binding sites nearly abolished P2 promoter activity, which suggests that the alphaARTF is essential for the transcription of the alphaAR gene in most tissues.


INTRODUCTION

alpha(1)-Adrenergic receptors (alpha(1)AR) (^1)play an important role in key components of the sympathoadrenal response to stress, such as peripheral vasoconstriction, increased cardiac contractility, and hepatic glycogenolysis. Both pharmacological and molecular cloning studies have indicated the existence of multiple subtypes of alpha(1)AR with unique tissue distributions (see reviews by Harrison et al.(1991) and Ford et al.(1994)). One of these subtypes, the alphaAR, has been identified in a variety of mammalian tissues, with the highest amounts being present in the liver. The expression of the alphaAR gene in the rat is regulated in a complex and tissue-specific manner. For example, hypothyroidism decreases the level of alphaAR mRNA in the liver, but increases it in the heart and the lungs (Lazar-Wesley et al., 1991). The tissue distribution of the alphaAR message in the rat generally parallels that of the alphaAR binding sites, with the highest levels present in the liver, followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen (Lomasney et al., 1991). The rat alphaAR gene is composed of two exons and a single large intron of at least 16 kb in length (Gao and Kunos, 1993), and has three transcripts in the liver of 2.3, 2.7, and 3.3 kb in length (Gao and Kunos, 1994). The 3.3-kb species is preferentially expressed in liver, whereas the 2.7-kb species is widely expressed in many tissues (McGehee et al., 1990; McGehee and Cornett, 1991; Lomasney et al., 1991). The low abundance 2.3-kb species is difficult to detect and has only been reported in our earlier study in rat liver (Gao and Kunos, 1994), and by Hu et al.(1993) in hamster DDT(1) MF-2 cells. We have further found that these three alphaAR mRNAs in rat liver are transcribed from three distinct promoters (Gao and Kunos, 1994). The proximal promoter (P1), likely involved in generating the low abundance 2.3-kb mRNA species, does not contain homologies with the consensus sequences of known transcription factors. The middle promoter (P2), responsible for generating the major 2.7-kb mRNA species, is G+C-rich, lacks a TATA box, and contains binding sites for several trans-acting factors. The distal promoter (P3), responsible for generating the 3.3-kb species, contains a putative TATA and CCAAT box and has in its vicinity recognition sites for liver-specific transcription factors. In order to further characterize the functional organization of the major P2 promoter and its associated trans-acting factors, we have employed DNase I footprinting to identify the sites of DNA-protein interactions, and DNA mobility shift assays (DMSA) to analyze the nature of the proteins that bind to these sites.


MATERIALS AND METHODS

Oligonucleotides

The synthetic oligodeoxyribonucleotides (oligos) shown in Fig. 4A were prepared on a Cyclone Plus DNA synthesizer (Milligen). After ammonium hydroxide deprotection, oligos were evaporated to dryness by vacuum centrifugation (Savant Speed-Vac) and purified by electrophoresis on a 10% polyacrylamide, 8 M urea gel (Sambrook et al., 1989). The following additional consensus oligos were used: NF-I, 5`-TAT TTT GGA TTG AAG CCA ATA TGA TAA TGA-3` (from adenovirus 2; Rawlins et al., 1984); AlbD, 5`-AAA GAT GGT ATG ATT TTG TAA TGG GGT AGG A-3` (-121 to -90 in the rat albumin gene promoter, which binds the C/EBP-related proteins; Lichsteiner et al., 1987); CP1, 5`-GCC ACA AAC CAG CCA ATG AGT AAC TGC TCC AAG-3` (-99 to -71 in the murine globin gene promoter; Dusserre and Mermod, 1992); BTE, 5`- GAG AAG GAG GCG TGG CCA AC-3` (-59 to -40 in the rat P450C gene promoter; Yanagida et al., 1990); M-CAT, 5`-CGT GTT GCA TTC CTC TCT GGA TC-3` (-102 to -80 in the cardiac troponin T gene; Mar and Ordahl, 1990); Sp1, 5`-ATT CGA TCG GGG CGG GGC GAG C-3`; AP1, 5`-CGC TTG ATG AGT CAG CCG GAA-3`; AP2, 5`-GAT CGA ACT GAC CGC CCG CGG CCC GT-3`; AP3, 5`-CTA CTG GGA CTT TCC ACA CAT C-3`; TFIID, 5`-GCA GAG CAT ATA AGG TGA GGT AGG A-3`; GRE, 5`-TCG ACT GTA CAG GAT GTT CTA GCT ACT-3`; CRE, 5`-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3` and NF-kappaB, 5`-AGT TGA GGG GAC TTT CCC AGG C-3`.


Figure 4: DNA mobility shift analysis of the specific proteins interacting with the footprint II region. A, DNA sequence of footprint II region between -483 and -543. Lines represent oligos used in DMSA. B-F, DMSA performed with labeled oligos II, IIb, IIa, IIa1, and IIa2, respectively. Lanes 1, labeled oligo alone; lanes 2, 1 ng of labeled oligo was incubated with 10 µg of liver nuclear extract; lanes 3 and up, 1 ng of labeled oligo was incubated with 10 µg of liver nuclear extract and 100 ng of different competitor oligos, as indicated above the lanes. Various amounts of CP1 oligo used are indicated in the panelsE and F.



Nuclear Extracts

Nuclear extracts used in DNase I footprinting experiments were prepared from rat liver as described by Gorski et al.(1986), with modifications. Liver tissue (10-15 g) from 120-day-old rats was minced with scissors into approximately 3-mm cubes and suspended in 30 ml of homogenization buffer (2 M sucrose, 10 mM Hepes (pH 7.6), 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 10% glycerol, 1 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)). The suspension was homogenized using a motor-driven 30-ml Teflon-glass homogenizer until more than 90% of the cells were broken. The homogenate was diluted to 85 ml with homogenization buffer, layered in triplicate 27-ml aliquots over 10-ml cushions of the same buffer in 38-ml polyallomer centrifuge tubes (Beckman), and spun at 24,000 rpm for 30 min at 0 °C in an SW 28 rotor (Beckman). The combined nuclear pellets were resupended in 50 ml of a 9:1 (v/v) mixture of homogenization buffer and glycerol, and rehomogenized and centrifuged under the same conditions as described above. The pelleted nuclei were resuspended in 20 ml of nuclear lysis buffer (10 mM Hepes (pH 7.6), 100 mM KCl, 3 mM MgCl(2), 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 0.5 mM PMSF) using an all-glass Dounce homogenizer. The nuclear homogenate was diluted to 40 ml with nuclear lysis buffer, and 4 ml of 4 M ammonium sulfate was added dropwise. The samples were mixed by gentle rocking at 4 °C for 30 min before being spun again at 35,000 rpm for 1 h in a Ti 70 rotor. Ammonium sulfate powder, 0.33 g, was added into each ml of supernatant and was dissolved by slow stirring at 4 °C. The precipitated protein was pelleted by centrifugation at 36,000 rpm for 25 min at 0 °C in a Ti 70 rotor. The pellet was resuspended in 500 µl of nuclear dialysis buffer (20 mM Hepes (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, and 0.5 mM PMSF) and dialyzed twice against 200 ml of nuclear dialysis buffer for 2 h at 4 °C. The nuclear extract was then centrifuged in a microcentrifuge tube at 4 °C for 10 min to remove the precipitated forms, aliquoted, quick-frozen in dry ice, and stored at -70 °C prior to be used in the assays. Nuclear extracts used in DMSA were prepared as described by Roy et al.(1991).

DNase I Footprinting

The fragments used in the DNase I footprinting analysis represent various parts of the alphaAR gene P2 promoter region, and are shown in Fig. 1. The sense strands from p(-813,-432)CAT or p(-485,-432)CAT were phosphorylated at the unique BamHI site(-612) of the P2 promoter region, or at the unique HindIII site of the pCAT vector with T(4) polynucleotide kinase and [-P] ATP. Subsequent digestion with EcoRI yielded the end-labeled sense strand probes A and B, respectively. The antisense strand from p(-813,-432)CAT was phosphorylated at the unique XbaI or BamHI sites as described above, and subsequent digestion with HindIII yielded the end-labeled antisense strand probes C and D, respectively. These fragments were isolated by agarose gel electrophoresis and purified by Geneclean (BIO 101, CA). All phosphorylating and labeling reactions were performed as described by Sambrook et al.(1989).


Figure 1: Diagrammatic representation of sense (A and B) and antisense (C and D) DNA fragments used in the footprinting experiments. End labeling of the fragments was described under ``Materials and Methods.'' The top line with negative numbers represents the rat alphaAR gene middle promoter (P2) region. Asterisks represent the P end label.



The DNase I footprinting standard reaction was performed according to Galas and Schmitz(1978), with some modifications. The binding reaction was performed in a final volume of 100 µl, containing 20 mM Hepes (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, and 0.5 mM PMSF. Twenty to 80 µg of nuclear extract or 1 µg of purified AP2 protein (Promega) were preincubated with 2 µg of poly(dI-dC) (U. S. Biochemical) for 30 min at 0 °C. Then, about 1 ng of labeled fragment (20,000 counts/min) was added, and the incubation was continued for 30 min at 25 °C. The reaction was then diluted 2-fold with a solution of 5 mM CaCl(2)/10 mM MgCl(2) and digested with 0.15-0.075 units of RQ1 RNase-free DNase (Promega) for 1 min at 25 °C. The reaction was stopped by the addition of 180 µl of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, 100 µg/ml yeast tRNA), and DNase I was removed by proteinase K. The reaction product was extracted by phenol/chlorform, precipitated with ethanol, and analyzed on a 8% polyacrylamide, 8 M urea sequencing gel.

To determine the specific nucleotides protected from DNase I digestion, a sequence ladder derived for each fragment by the method of Maxam and Gilbert(1980) was electrophoresed alongside each set of protected and unprotected DNase I-digested sample.

DNA Mobility Shift Assays (DMSA)

Samples used in DMSA contained 1 ng of P-labeled probe and 10 µg of nuclear extract in 20 mM Tris-HCl, pH 7.9, 1.5% glycerol, 50 µg/ml bovine serum albumin, 1 mM DTT, 0.5 mM PMSF, and 2 µg of poly(dI-dC) in a volume of 20 µl. In competition experiments, radioactive probe and competitor were mixed prior to addition of nuclear extract. Reactions were incubated at 25 °C for 20 min and subsequently analyzed by electrophoresis through nondenaturing 10% polyacrylamide gels in 0.5 times TBE buffer containing 44.5 mM Tris-HCl, pH 8.2, 44.5 mM boric acid, and 1 mM EDTA. After prerunning the gel at 100 V for 2 h, electrophoresis was performed at 270 V for 2 h at 4 °C. The gels were analyzed by phosphorimaging (PhosphorImager(TM)) using ImageQuant(TM) software (Molecular Dynamics).

Construction of Plasmids

Footprint II was removed from the P2 promoter region to generate the internal deletion construct P2-II. This construct was obtained by generating the fragments -813 to -540 and -490 to -432 of the rat alphaAR 5`-flanking region via PCR, and subcloning them into a pCAT enhancer reporter vector (Promega). All other techniques were performed as described previously (Gao and Kunos, 1994). The same strategy was used to construct P2-IIb and P2-IIa, which had internal deletions between -540 and -517 and between -517 and -490, respectively.

Transient Transfections and CAT Assays

Transient transfections and CAT assays were performed as described previously (Gao and Kunos, 1994). DDT(1) MF-2 hamster smooth muscle cells were obtained from ATCC (Rockville, MD) and cultured as described (Gao and Kunos, 1994).


RESULTS

The alphaAR P2 Promoter Contains Multiple Binding Sites for Liver Nuclear Proteins

Our previous transfection experiments have shown that the full activity of the P2 promoter of the alphaAR gene is contained within 381 bp upstream of and around tsp2. To further delineate the functional cis elements in this region, we searched for the potential protein-binding sites using DNase I footprinting analysis. In order to identify protected regions more clearly, the 381-bp alphaAR P2 promoter region was subdivided into various fragments as shown in Fig. 1. Footprints on these fragments using rat liver nuclear extract or purified AP2 protein (Promega) are shown in Fig. 2. The footprint patterns on the sense strand fragments A and B (Fig. 2, A and B) and antisense strand fragments C and D (Fig. 2, C and D) yielded three protected regions: footprints I (-432 to -452), II (-490 to -540), and III (-609 to -690). A summary of the footprinting data on the P2 promoter is presented in Fig. 3. Footprint I contains a putative CRE with one mismatch (Roesler et al., 1988), and a putative inverted GC box that has 8/10 match to the consensus sequence (Briggs et al., 1986). Footprint II, which is interrupted by several DNase I-hypersensitive sites (filled arrows in Fig. 2, A and C), is the strongest protected region and contains four binding sites (three TGGC: -504 to -507, -525 to -528, and -531 to -534; one TGGA: -500 to -503) for half of the NF-I palindrome (Paonessa etal., 1988), with no recognition sequences for any other known transcription factor. However, gel shift analyses showed that the proteins binding this region were not competed by the NF-I consensus sequence, suggesting that some novel transcription factors may be involved in this protected region (see below). Footprint III contains a perfect match of an AP2 binding site (Imagawa et al., 1989; Mitchell et al., 1989), which is protected by purified AP2 protein (Fig. 2, C and D). The region immediately upstream from the protected AP2 site displays DNase I hypersensitivity when using liver nuclear extract with the longer (C) but not the shorter DNA probe (D). Since DNase I hypersensitivity is thought to reflect bending of the DNA strand due to complex interactions with binding proteins, this difference may be due to the presence of the additional footprint II in probe C but not in probe D. Footprint III also contains one mismatch consensus sequences for HNF-1 (Cereghini et al., 1988), HNF-5 (Grange et al., 1991), and C/EBP (Johnson et al., 1987; Costa et al., 1988). The region (-569 to -545) between footprints II and III is G+C-rich and is weakly protected on both strands. However, further DMSA analysis of this region showed no specific mobility shift bands (data not shown), suggesting that the weak protection is nonspecific. Next, we analyzed footprint II by DMSA.


Figure 2: DNase I footprint analysis of the rat alphaAR P2 promoter. PanelsA-D illustrate the footprints (marked by brackets and Roman numerals) obtained on DNA fragments A-D, respectively (fragments labeled as in Fig. 1). DNase I-hypersensitive sites are marked by filled arrows. G + A, Maxam-Gilbert G + A sequencing reaction; NE, rat liver nuclear extract.




Figure 3: DNA sequence of regions of protein-DNA interactions in the rat alphaAR gene P2 promoter. Footprints are underlined. The filled arrows mark sites of DNase I hypersensitivity. Consensus DNA binding sites are boxed. Negativenumbers on the right indicate nucleotide position relative to the translation start codon (+1). The ellipses above footprint II represent interactions with CP1 and alphaARTF.



Binding of a Novel Transcription Factor to Footprint II

Footprint II was distinctly circumscribed by multiple DNase I hypersensitive sites (filled arrows in Fig. 2, A and C). This type of DNase I footprint has been described previously for complex promoters and enhancers containing multiple, closely spaced cis-acting elements, and may reflect the bending of DNA adjacent to these sites (Gottschalk and Leiden, 1990; Iannello et al., 1993). It is interesting to note that the protected region on the sense strand contains three DNase I-hypersensitive sites at positions -518, -519, and -520 (filled arrows in Fig. 2A and Fig. 3), and corresponding DNase I-hypersensitive sites are also located on the antisense strand (filled arrows in Fig. 2C and 3). This suggests that footprint II is a composite of at least two independent protein-binding domains. To further characterize the transcription factors interacting with this region, we did DNA mobility shift assays using oligo II (-484 to -543), which covers the entire footprint II, and oligos IIb (-513 to -543) and IIa (-484 to -519), which are overlapping and correspond to the 5` and 3` halves of the footprint, respectively (see Fig. 4A). DMSA analysis using oligo II (Fig. 4B) revealed two major complexes and one minor one (lane 2), all three of which are specific since they are competed away by unlabeled oligo II (lane 3). The two major complexes are also abolished by oligo IIb (lane 4) and reduced by oligo IIa (lane 5), but not affected by oligos containing binding sites for NF-I (lane6), CP1 (lane 7), SP1, AP1, AP2, AP3, TFIID, GRE, CREB, or NF-kappaB (data not shown). This suggests that the protein(s) generating the strong bands bind to two adjacent sites centered in the 5` half of oligo II. The minor complex is abolished by oligos IIa (lane5) and CP1 (lane7), but less effectively by oligo IIb (lane4) and not at all by NF-I (lane6). This suggests that the minor complex contains a CP1-related factor, with its binding site centered in the 3` half of oligo II, in the region corresponding to oligo IIa.

In order to obtain better resolution of multiple binding proteins, we performed DMSA using subregions of oligo II as radiolabeled probes. Fig. 4C illustrates the results obtained with oligo IIb. Incubation of the labeled oligo IIb with liver nuclear extract generated a single shifted band (lane2), effectively competed by a 100-fold excess of unlabeled oligo IIb (lane3). This complex is also efficiently inhibited by an excess of oligo IIa (lane 4), suggesting that the factor that binds oligo IIb also binds oligo IIa. The oligos containing consensus binding sites for SP1, AP2, CP1, C/EBP, CTF/NF-I (lanes 5-9), and AP1, AP3, BTE, TFIID, GRE, CREB, NF-kappaB (data not shown), did not compete this complex. A computer-based search of the Sitedata data base of several thousand sequence-specific response elements yielded no homologies with sequences in oligos IIb and IIa, suggesting that the protein binding to these oligos may be a novel transcription factor. We tentatively name this factor alphaARTF, for alpha-Adrenergic Receptor Transcription Factor. When P-labeled oligo IIa was used as a probe, the DMSA (Fig. 4D) yielded two DNA-protein complexes (lane 2), which are specific as they are competed away by an excess of unlabeled oligo IIa (lane 3). The top (major) complex, but not the bottom (minor) complex, is abolished by an excess of oligo IIb (lane 4), in agreement with the DMSA analysis of oligo IIb. This suggests that the protein in the top complex is the same as the one in the complex revealed with oligo IIb, i.e. alphaARTF. The bottom, but not the top, complex is competed away by an excess of a consensus oligo for the transcription factor CP1 (lane 5). Neither complex is competed by oligos containing consensus binding sites for C/EBP, NF-I (lanes 6 and 7), or AP1, AP2, AP3, SP1, TFIID, GRE, CREB, or NF-kappaB (data not shown). Taken together, these findings can be interpreted to indicate that oligos IIb and IIa each contain a binding site for the same protein, alphaARTF, while oligo IIa contains an additional protein binding site for a CP1-related factor.

To further delimit the binding domains of oligo IIa for alphaARTF and CP1, we synthesized two overlapping oligos, IIa1 and IIa2, which encompass oligo IIa, and employed them in DMSA with rat liver nuclear extract. As shown in Fig. 4E, P-labeled oligo IIa1 binds a complex specifically (lane 2), as it is competed away by unlabeled oligo IIa1 (lane 3). This complex is also abolished by unlabeled oligo IIb (lane 5) but not by oligo IIa2 (lane 4) or the consensus oligo for CP1 (lanes 6-9), suggesting that the factor that binds oligo IIa1 is alphaARTF. Fig. 4F shows that oligo IIa2 binds two specific proteins (lane 2), which is abolished by self competition (data not shown). The major protein in the top complex is the CP1-related factor, as it is competed away dose-dependently by the consensus oligo for CP1 (lanes 5-8) or by oligo IIa (lane 4), but not by oligo IIb (lane 3). The factor in the bottom complex is alphaARTF, which is competed away by oligo IIb (lane 3), but not by CP1 (lanes 5-8). In summary, footprint II appears to contain two binding sites for alphaARTF and one for CP1, as schematically illustrated in Fig. 3.

alphaARTF Is a Ubiquitous Transcription Factor

Unlike the liver-specific P3 promoter, the rat alphaAR gene P2 promoter is responsible for the widespread expression of the 2.7-kb mRNA species. It was of interest, therefore, to test whether alphaARTF, the major transcription factor binding to the P2 promoter, is also ubiquitous. To quantify the relative tissue concentration of the alphaARTF, we did DMSA using oligos IIb and IIa1 as the labeled probes and nuclear extracts from different tissues. As shown in Fig. 5A, the relative intensity of the shifted bands in different tissues is similar for oligos IIb and IIa1, which is compatible with the same factor, such as alphaARTF, binding to both probes. Quantitation of the radioactivity of the bands in various tissues indicates that the protein in the complex is ubiquitous and most prominent in brain, followed by liver, spleen, kidney, and lung, with no significant amount detected in heart. The absence of alphaARTF activity in heart could not be due to possible technical problems with the cardiac nuclear extract, because using the same nuclear extract with a labeled consensus oligonucleotide for the heart-specific factor M-CAT, the detected band is much stronger in heart than in brain or liver (Fig. 5B), similar to earlier published findings (Mar and Ordahl, 1990). Fig. 5A also shows that the tissue distribution of CTF/NF-I is different from that of alphaARTF, CTF/NF-I being less abundant in brain and more abundant in heart than alphaARTF, which also indicates that the absence of alphaARTF in the heart is real. The relatively low amount of CTF/NF-I in the heart compared to other tissues is in good agreement with findings reported by others (Paonessa et al., 1988). These findings further support the notion that CTF/NF-I and alphaARTF are distinct entities.


Figure 5: alphaARTF is a ubiquitous factor. P-Labeled oligos IIb, IIa1, and NF-I (A) or M-CAT (B) were incubated with 10 µg of nuclear extract prepared from different rat tissues and subjected to DMSA, as described under ``Materials and Methods.''



Deletions of alphaARTF Binding Sites Significantly Reduce P2 Promoter Activity

Previous transient transfection experiments showed that deletions of regions -432 to -460 and -675 to -813, which contain footprints I and III, respectively, abolished the P2 promoter activity, suggesting that trans-acting factors binding to these two footprints are positive constitutive regulators of the P2 promoter. To assess the functional importance of footprint II, we prepared a series of internal deletion constructs and transfected them into DDT(1) MF-2 cells. As illustrated in Fig. 6, deletion of the entire footprint II, or footprint IIa or IIb separately, abolished the promoter activity of P2, as determined in CAT assays. The figure also illustrates the similar effect of deleting footprints I or III.


Figure 6: The effects of footprint deletions on the activity of the P2 promoter of the rat alphaAR gene. The left side is the schematic representation of the pCAT constructs, containing the intact P2 promoter or its variants with deletions of footprints I, II, III, IIa, or IIb, used in cell transfection experiments. The right side shows CAT activities measured in DDT(1) MF-2 cells, expressed as percent of the positive control. CAT activities were corrected for transfection efficiencies, as described (Gao and Kunos, 1994). Means ± S.E. from three experiments are shown.




DISCUSSION

The experiments reported in this paper suggest a complex interaction of specific DNA-binding proteins with the major, middle promoter (P2) of the rat alphaAR gene. We have identified three groups of binding sites (footprints I-III) for potential trans-acting factors within the 381-bp segment of the 5`-flanking region, which was found necessary and sufficient for maintaining maximal transcriptional activity of P2 (Gao and Kunos, 1994).

Footprint I contains a CRE and a GC box, which may play positive roles in the transcription of the rat alphaAR gene, since deletion of the region -432 to -460, which contains footprint I, abolished the P2 promoter activity (see Fig. 6). In some genes with TATA-less promoters containing multiple GC boxes, binding of Sp1 has been shown to be critical for transcription initiation (Pugh and Tjian, 1990, 1991). It remains to be determined whether mutation of the GC box in footprint I rather than deleting the entire footprint is sufficient to eliminate P2 promoter activity.

Footprint III contains an AP2 binding site, but the footprint is much larger than the region protected by the purified AP2 protein, which suggests the binding of some additional factors to this footprint. Sequence analysis reveals that this region contains putative binding sites for the liver specific factors HNF-1, HNF-5, and C/EBP (Fig. 3). If these binding sites are functional, they may, in part, account for the much stronger expression of the 2.7-kb alphaAR mRNA in the liver, than in other tissues of the rat. Deletion of footprint III also abolishes P2 promoter activity (Fig. 6), but it is not clear which factor or combination of factors plays the critical role.

Footprint II contains four binding sites for half of the NF-I palindrome. However, several lines of evidence suggest that the major factor that binds to footprint II is distinct from NF-I. (a) In DMSA, the NF-I consensus oligo did not compete with oligos II, IIb, and IIa1 (Fig. 4, B-D) and, conversely, oligos IIb and IIa1, which contain half of the NF-I palindrome, did not compete with the labeled NF-I oligo (data not shown). (b) The factors binding to footprint II and NF-I have distinct DNA contact points identified by methylation interference assays. (^2)(c) The tissue distribution pattern of this factor is different from that of NF-I (Fig. 5). It is interesting to note that the protein BTEB, which is distinct from NF-I, has been identified as the factor binding to the half-NF-I consensus sequence TGGC in the regulatory domains of the rat P-450c gene (Yanagida et al., 1990; Imataka et al., 1992). However, the major factor that binds to footprint II is not competed by a consensus oligo for the BTE binding sequence, suggesting that it is not BTEB. Since computer-based sequence analysis of footprint II revealed no apparent homologies with the consensus binding sites for any other known transcription factors, we have tentatively named the major factor binding to footprint II alphaARTF.

The results of DNA mobility shift assays indicated that oligos IIb and IIa1, which cover adjacent, partially overlapping segments of footprint II, were able to cross-compete. This suggests that the same factor, most likely alphaARTF, binds to both oligos. Examination of the sequence of oligos IIb and IIa1 reveals some similarities, such as the presence of the motif GCTGG in both (IIb: -513 to -520; IIa1: -501 to -505). Furthermore, in DNA mobility shift assays both oligo II and IIa bound alphaARTF strongly and CP1 weakly, whereas oligo IIa2 showed strong CP1 binding. This suggests that alphaARTF can interfere with CP1 binding, probably as a result of adjacent or partially overlapping binding domains. Oligo IIa2 contains an inverted GCAAT sequence which has been shown to bind the heat-resistant protein C/EBP with high affinity (Graves et al., 1986; Johnson et al., 1987). However, the protein binding of oligo IIa2 is abolished by heating the nuclear extract at 90 °C for 5 min (data not shown). Furthermore, binding was not competed by the oligo AlbD, which binds the C/EBP protein (Lichtsteiner et al., 1987), but it was competed by the CP1 oligo. These observations suggest that the protein that binds to oligo IIa2 is a CP1-related protein and not C/EBP. In summary, the footprint II region of P2 contains two binding sites for alphaARTF and one for CP1, and alphaARTF can interfere with CP1 binding. These complex protein interactions may contribute to the formation of the multiple DNase hypersensitity sites in footprint II. The critical role of alphaARTF in the transcription of the alphaAR gene is indicated by the finding that deletion of either footprint IIa or IIb abolishes the activity of the major, P2 promoter (see Fig. 6). The results of the deletion studies thus indicate that each of the three footprint domains is essential for P2 promoter activity and they do not have a simple additive effect. This could suggest that the multiple proteins that bind to these elements also interact with each other to form the primary transcription complex, where removal of any one component may have a ``domino'' effect.

The tissue distribution of alphaARTF generally fits the relative abundance of the 2.7-kb alphaAR mRNA species, with the exception of the heart. The rat heart expresses high levels of alphaAR binding sites and of the 2.7-kb alphaAR mRNA (Lomasney et al., 1991; McGehee et al., 1992), but little if any alphaARTF was detected (see Fig. 5A). This suggests that transcription of the alphaAR gene in the rat heart involves some alternative mechanisms, such as the binding of heart-specific transcription factor(s). Indeed, sequence analysis reveals that the 5`-flanking region of the rat alphaAR gene contains several putative consensus sequences for cardiac myocyte nuclear factors; the region between -736 to -730 contains one mismatch (CATGGCT) to the M-CAT consensus sequence (CATNC(C/T)(T/A)), which is involved in the expression of several cardiac-specific genes (Iannello et al., 1991; Farrance et al., 1992). The region -763 to -768 (CAGTTG) contains an E-box (Blackwell and Weintraub, 1990), which has also been implicated in the control of cardiac-specific gene expression (Thompson et al., 1991; French et al., 1991). The possible role of these cardiac-specific transcription factors in the expression of the alphaAR gene in the heart remains to be determined. Also, in preliminary experiments we have found that the transcription start point corresponding to the 2.7-kb alphaAR mRNA is located at different sites in heart and liver.^2 Regardless of the specific mechanisms involved, tissue-dependent differences in the transcription of the alphaAR gene have important physiological implications; they may provide the molecular basis for opposite changes in alphaAR gene expression in heart and liver under certain conditions, such as hypothyroidism (Lazar-Wesley et al., 1991). Whether there is a direct correlation between the transcription of the rat alphaAR gene in different tissues (except in heart) and the levels of alphaARTF awaits further purification and characterization of this factor.

In summary, we have identified multiple protein-binding sites and complex DNA-protein interactions in the major promoter of the rat alphaAR gene, including the involvement of a novel protein factor in its transcription. Taken together with our previous demonstration of three different promoters, the present findings further document the complex mechanisms involved in controlling the transcription of the alphaAR gene.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL49938 (to G. K.) 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.

§
To whom correspondence should be addressed: Box 980613, Richmond, VA 23298. Tel.: 804-828-2073; Fax: 804-828-2117.

(^1)
The abbreviations used are: AR, adrenergic receptor; CRE, cAMP response element(s); GRE, glucocorticoid response element; NF-I, nuclear factor I; HNF-1 and HNF-5, hepatocyte nuclear factor 1 and 5; alphaARTF, alpha-adrenergic receptor transcription factor; C/EBP, CCAAT/enhancer-binding protein; CAT, chloramphenicol acetyltransferase; bp, base pair(s); kb, kilobase(s);, oligo, oligodeoxyribonucleotide; DMSA, DNA mobility shift assay; PMSF, phenylmethylsulfonyl fluoride.

(^2)
B. Gao, M. S. Spector, and G. Kunos, unpublished observations.


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