Transcription Factors NF-Y and Sp1 Are Important Determinants of the Promoter Activity of the Bovine and Human Neuronal Nicotinic Receptor (cid:1) 4 Subunit Genes*

The (cid:1) 4 subunit is a component of the neuronal nicotinic acetylcholine receptors which control cate-cholamine secretion in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was characterized. A proximal region (from (cid:2) 99 to (cid:2) 64) was responsible for the transcriptional activity observed in chromaffin, C2C12, and COS cells. Within this region two cis -acting elements that bind transcription factors Sp1 and NF-Y were identified. Mutagenesis of the two elements indicated that they cooperate for the basal transcription activity of the promoter. The human (cid:1) 4 promoter, that was also characterized, shared structural and functional homologies with the bovine promoter. Thus, two adjacent binding elements for Sp1 and NF-Y were detected. Whereas the Sp1 site was an important determinant of the promoter activity, the NF-Y site may have cell-specific effects. Given that these promoters showed no structural or functional homology with the previously characterized rat (cid:1) 4 subunit promoter (Bigger, C. B., Casanova, E. A., and Gardner, P. D. (1996) selected from lysates of SHSY-5Y by oligo(dT)-Dyna- beads or purchased from CLONTECH brain and adrenal tissues). Plasmid Constructions— All (cid:2) 4 promoter-luciferase gene fusions were made in the pGL2-Basic vector (Promega, Madison, WI), introduc- ing in its polylinker, upstream of the luciferase gene, the suitable (cid:2) 4 promoter fragments. These fragments were generated with restriction enzymes and directly cloned into pGL2-Basic or subcloned first in pBluescript and then transferred to pGL2-Basic. The vector pGL2- Control, which express the luciferase gene under the regulation of the SV40 promoter and enhancer sequences, was used to check luciferase activity. Deletion analysis of the most promoter-proximal region was enzyme frag- and the incubation was continued for an additional 20-min period. For competition studies, the nuclear extract was incubated with the com- peting oligonucleotide prior to the labeled probe during 20 min. Supershift assays were performed by preincubating nuclear extracts with 1 (cid:3) l of antibodies against different transcription factors (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit IgG (Sigma) for 3 h on icebefore probe addition. The antibody against the B subunit of NF-Y was gen-erously provided by Drs. Mathis, Benoist, and Mantovani (Universite´ Louis Pasteur, Strasbourg, France). The equivalent anti-NF-Y b antibody ( CBF-A , C-20 ) from Santa Cruz Biotechnology was used in later experiments with the human (cid:2) 4 promoter.

Cloning of nicotinic acetylcholine receptor (nAChRs) 1 subunit cDNAs has revealed that the molecular heterogeneity of the gene families encoding the different receptor subunits is responsible for the pharmacological and functional diversity of nAChRs in the peripheral and central nervous systems (1,2). The varied tissue-, region-, and development-specific distribu-tion of nAChRs subunits (3) has suggested that complex transcriptional mechanisms direct nAChR expression. Moreover, potential changes in subunit transcription in response to modulation of synaptic function, might have important consequences on the signals transduced by nAChRs (4,5). For these reasons considerable effort has been dedicated to the elucidation of the molecular basis for the transcriptional regulation of neuronal nAChRs, and thus several cis-and trans-acting elements in the promoter of the different nAChRs subunits have been identified (6).
In our laboratory we have previously isolated and characterized the promoters of the bovine ␣5 (7) and ␣7 (8) subunits. These subunits are expressed in the chromaffin cells of the adrenal gland composing two different receptor subtypes, one of them formed by ␣7 subunits (9) and the other by ␣3, ␤4, and ␣5 subunits (10). Interestingly, the genes of the latter subunits are clustered in the vertebrate genome (11,12) and may have common patterns of regulation (13). We have analyzed here the bovine and human ␤4 promoters, finding that they are highly homologous in their proximal regions as well as in the ciselements governing basal transcriptional activity. Although their sequences differ from the one of the rat ␤4 promoter (14,15), the three promoters have in common their regulation by the ubiquitous transcription factor Sp1. Binding motifs for Sp1 have been also located in close proximity in the promoters of rat ␣3 (16) and bovine (7) and human (17) ␣5 subunits, suggesting that this transcription factor plays a fundamental role in the expression of several nAChRs subunit genes.

EXPERIMENTAL PROCEDURES
Isolation and Analysis of the 5Ј-Flanking Sequence of the ␤4 Subunit-For the bovine promoter a cDNA probe corresponding to 218 bp at the beginning of the coding sequence and the contiguous 38 bp of 5Ј-untranslated region (10) was used to screen a genomic library. For the human promoter, a cDNA probe corresponding to 87 bp at the beginning of the coding sequence and the contiguous 99 bp of 5Јuntranslated region (18) was used to screen a genomic library. Both libraries were constructed in EMBL-3 SP6/T7 (CLONTECH, Heidelberg, Germany) and tested as previously described (9). In both cases several overlapping bacteriophage clones were purified and characterized.
RNase Protection-Poly(A) ϩ RNA was directly selected from lysates of several bovine adrenal medullas by oligo(dT)-Dynabeads (Dynal, Oslo, Norway) and used in the RNase protection experiments. Probes were generated with SP6 and T7 polymerases (Roche Molecular Biochemicals, Barcelona, Spain), [␣-32 P]CTP (Amersham Biosciences, Inc., Madrid, Spain) and the corresponding linearized templates (in the pSPT18 vector, Roche Molecular Biochemicals). A 462-bp RsaI-PstI fragment of the bovine ␤4 gene that included 286 bp 5Ј to the beginning of the signal peptide sequence and 176 bp corresponding to the rest of the first exon and part of the second one was subcloned into the SmaI and PstI sites of pSPT18. After linearization of the plasmid with EcoRI, an antisense probe of 496 nucleotides was synthesized with SP6 RNA polymerase. To control protection efficiency a sense cRNA was synthesized by SP6 RNA polymerase transcription of a ␤4 cDNA construct linearized with XbaI. This cRNA should protect a fragment of 215 nucleotides when used in combination with the antisense probe. Parallel experiments were carried out with a smaller antisense probe which overlapped the 5Ј-end of the first one. For this purpose a RsaI-AvaII fragment of 310 bp was subcloned into the HincII site of pSPT18 previous filling-in with Klenow enzyme. An antisense probe of 352 nucleotides was obtained upon linearization with EcoRI and transcription with SP6 polymerase. As above, a cRNA sense fragment was used to control protection. In this case it was obtained by T7 polymerase transcription of the DNA used to obtain the large probe, previous linearization with XhoI and produced a protected fragment of 234 nucleotides (see Fig. 2 for further explanations). RNase protection experiments were performed using an RNase Protection Kit (Roche Molecular Biochemicals) as indicated by the manufacturer. Protected fragments were separated on a 7 M urea and 6% acrylamide gel along with several other labeled RNAs of known size which were also synthesized and used for calibration. Similar RNase protection experiments were carried out to find the 5Ј-end of human ␤4 mRNA. In this case a 323-bp BamHI-SacI fragment whose 3Ј-end was at 30 bp from the initial ATG, was cloned into the pSPT19 vector to generate a 362-nucleotide probe which was labeled according to the instructions of the MaxiScript SP6 kit (Ambion). The same fragment was also cloned into pSPT18 to synthesize a control RNA with SP6 RNA polymerase. This RNA generated a 325-nucleotide protected fragment in the presence of the labeled probe. In the case of the human promoter the RNase protection experiments were performed using a RPA III kit from Ambion and poly(A) ϩ RNA directly selected from lysates of SHSY-5Y cells by oligo(dT)-Dynabeads or purchased from CLONTECH (human brain and adrenal tissues).
Plasmid Constructions-All ␤4 promoter-luciferase gene fusions were made in the pGL2-Basic vector (Promega, Madison, WI), introducing in its polylinker, upstream of the luciferase gene, the suitable ␤4 promoter fragments. These fragments were generated with restriction enzymes and directly cloned into pGL2-Basic or subcloned first in pBluescript and then transferred to pGL2-Basic. The vector pGL2-Control, which express the luciferase gene under the regulation of the SV40 promoter and enhancer sequences, was used to check luciferase activity. Deletion analysis of the most promoter-proximal region was performed by generating either appropriate restriction enzyme fragments or polymerase chain reaction fragments with suitable sense oligonucleotides and an antisense primer (5Ј-CTTTATGTTTTTG-GCGTCTTCC-3Ј) that anneals to the pGL2-Basic vector, downstream of the site of transcription initiation.
The basic strategy for site-directed mutagenesis of the different elements in region Ϫ99 to Ϫ64 of the bovine ␤4 promoter (see Fig. 4) consisted of the following steps. (a) We performed polymerase chain reaction (25 cycles at 94°C for 10 s, 50°C for 30 s, 72°C for 45 s) amplification of p99␤4LUC (or its single mutant when the double mutant was desired) with appropriate mutagenic primers in the sense orientation, which generated restriction sites useful to confirm mutagenesis. We used the same oligonucleotide mentioned above, as antisense primer. (b) Polymerase chain reaction products were cloned into pBluescript, sequenced, and transferred to the appropriate construct into the pGL2-Basic vector. The introduced mutations are indicated in lowercase letters in Fig. 4A (sites 1 and 2). The strategy for mutagenesis of the elements in region Ϫ74 to Ϫ44 of the human ␤4 promoter was based on the presence of a SacII site at the 3Ј-end of the region containing the elements to be mutated and an XmaI site (from pGL2-Basic) at the 5Ј-end. Complementary oligonucleotides carrying the desired mutations and the mentioned restriction sites were annealed and cloned into the corresponding construct in place of the original sequences.
Plasmids were purified by Concert columns (Invitrogen). All cell types were transfected by the calcium phosphate procedure (20). Chromaffin cells on 48-well plates (5 ϫ 10 5 cells/well) were incubated with 0.75 g of pGL2 vector or an equivalent amount (in molar terms) of the different constructs derived from this vector and with 0.75 g of ␤-galactosidase expression vector pCH110 (Amersham Biosciences, Inc.) as a control of transfection efficiency. SHSY-5Y (10 5 cells/well), COS cells (5 ϫ 10 4 cells/well), or C2C12 cells (10 4 cells/well) on 24-well plates, were incubated with 1.5 g of the different ␤4 constructs and 1.5 g of pCH110 per well. Cells were harvested after 48 h and lysed with reporter lysis buffer (Promega). ␤-Galactosidase and luciferase activities were then determined in the lysates with the corresponding assay systems (Promega). Luciferase activity was normalized to values obtained with constructs representative of each ␤4 subunit. They are indicated in the corresponding figure legends.
Electrophoretic Mobility Shift Assay-Crude nuclear extracts were prepared from cultured cells as described by Schreiber et al. (21). The DNA fragment corresponding to region Ϫ99 to ϩ66 of the bovine ␤4 promoter was obtained by digesting pBluescript subclones either with XbaI and EcoRI (wild probe) or SacI and EcoRI (mutant probes) and end-labeled by Klenow filling with [␣-32 P]dATP. The human ␤4 probes were obtained by annealing complementary oligonucleotides corresponding to region Ϫ74 to Ϫ38 and end-labeled by Klenow filling with [␣-32 P]dCTP. The DNA-protein binding reaction volumes were 20 l containing 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 5 g of bovine serum albumin, 2 g of poly(dA-dT)⅐poly(dA-dT) (Amersham Biosciences Inc.), 2 g of nuclear extract protein, and 20,000 cpm of 32 P-labeled probe. Reactions were incubated for 10 min at room temperature; the labeled probe was added; and the incubation was continued for an additional 20-min period. For competition studies, the nuclear extract was incubated with the competing oligonucleotide prior to the labeled probe during 20 min. Supershift assays were performed by preincubating nuclear extracts with 1 l of antibodies against different transcription factors (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit IgG (Sigma) for 3 h on ice before probe addition. The antibody against the B subunit of NF-Y was generously provided by Drs. Mathis, Benoist, and Mantovani (Université Louis Pasteur, Strasbourg, France). The equivalent anti-NF-Y b antibody (CBF-A, C-20) from Santa Cruz Biotechnology was used in later experiments with the human ␤4 promoter.

Structure of the 5Ј-Flanking Region of the Bovine ␤4 Subunit
Gene-A bovine genomic library was screened and several overlapping clones were isolated. Clone bov␤4 -11 contained ϳ13 kb of bovine genomic sequence including exon 1 and ϳ1.2 kb of 5Ј-flanking region. This region was further subcloned and sequenced ( Fig. 1) revealing the lack of a TATA box. The 5Ј-end of ␤4 mRNA was mapped by RNase protection analyses (Fig. 2). A 496-residue antisense riboprobe (Fig. 2, Probe 1) yielded a protected fragment of ϳ309 bases that mapped transcription initiation to a thymine located 125 bp upstream of the initial ATG (arrowhead at position ϩ1, in Fig. 1). Other protected fragments of smaller size and similar intensity were also observed, suggesting that alternative initiation sites exist. They are also indicated in Fig. 1 (arrowheads and small squares). To improve precision in the determination of the transcription initiation sites, a second overlapping probe was used (Fig. 2, Probe 2). In this case several protected fragments of 156, 155, 134, and 133 bp were observed and mapped transcription initiation to the same sites that the larger probe.
Functional Analysis of the Bovine ␤4 Subunit Promoter-A series of constructs was generated to determine the regions of the bovine ␤4 subunit promoter that contributed to its maximal activity (Fig. 3). These constructs were introduced into chromaffin, C2C12, and COS cells. Constructs containing 81 bp (p81␤4LUC) or more (up to 1256 bp) of ␤4 promoter sequence plus 66 bp of 5Ј-noncoding region showed similar activity. Two shorter constructs (p63␤4LUC and p39␤4LUC) showed a ϳ60% decrease in promoter activity, whereas a further 5Ј deletion (p4␤4LUC) was even less active. The pattern of promoter activity was similar in the three cell types mentioned above, indicating that the tested constructs lacked elements able to confer cell-specific transcription. The only exception was construct p787␤4LUC, which showed a ϳ20 -30% decrease in activity in C2C12 and COS cells but not in chromaffin cells.
Therefore, in C2C12 and COS but not chromaffin cells, elements predominantly located between Ϫ353 and Ϫ787 with respect to the transcription initiation site may have a negative effect on ␤4 promoter activity. However, the largest construct tested (p1256␤4LUC) also contains this region and exhibited increased activity (ϳ100 -120%) in the three cell types.
Elements in the minimal promoter, between 81 and 63 bp upstream of the start site of transcription, appear to be critical for basal transcription of the ␤4 subunit gene in transient transfection assays, since: (a) their deletion produced ϳ60% decrease in transcriptional activity, and (b) additional upstream sequences did not significantly increase activity. In this region the presence of inverted GC and CCAAT boxes was detected (see Fig. 4A) and, therefore, they were chosen for mutagenic analysis. These analysis were performed, however, in the context of p99␤4LUC instead of p81␤4LUC, to leave a few additional nucleotides at the 5Ј-end of one of the elements (numbered 2 in Fig. 4A). Otherwise, this region would be located just at the 5Ј-end of construct p81␤4LUC. When the GC box was altered (site 1, Fig. 4A), ␤4 promoter activity in C2C12 and COS cells decreased to ϳ60% of that observed for the parent construct (p99␤4LUC), whereas in chromaffin cells the decrease was less pronounced (Fig. 4B). The mutant of the CCAAT box (site 2, Fig. 4B) affected promoter activity in chromaffin and C2C12 cells (about 25% decrease) but did not have any effect in COS cells (Fig. 4B). Finally, the double mutant of sites 1 and 2 produced a stronger decrease (to ϳ40% of the parent construct) than the sum of the single mutations ( Fig.  4B) in chromaffin and COS cells, whereas in C2C12 cells the effects were additive. Thus, in chromaffin cells a mere addition of the single mutant effects would produce a decrease of about 33% in activity, whereas the double mutant yielded a 65% decrease. In COS cells a decrease of 35% in activity would be expected upon the addition of the single mutant effects, however, the double mutant yielded a 58% decrease. These results suggest that sites 1 and 2 do integrate a whole synergistic mechanism required for basal promoter activity in chromaffin and COS cells.
Characterization of the Regulatory Elements Present at Ϫ99/ Ϫ63 of the ␤4 Promoter by EMSA-DNA fragments carrying the wild-type Ϫ99 to ϩ66 promoter region and the corresponding site 1 and site 2 mutants from the previous functional studies (Fig. 4) were labeled and incubated with nuclear extracts from chromaffin cells (Fig. 5). Two retarded bands were observed (Fig. 5A, lane 2, labeled as circle and arrowhead) when using the wild-type fragment. Both bands were competed with increasing amounts of unlabeled fragment (Fig. 5A, lanes  4 and 5). Recombinant Sp1 produced a main retarded complex (Fig. 5A, lane 3), coincident in position with one of those observed with nuclear extracts (arrowhead). By contrast, when the site 1 mutant was used as probe, neither the upper complex was formed with chromaffin nuclear extracts (Fig. 5A, lane 7) nor recombinant Sp1 retarded the probe (Fig. 5A, lane 8). This suggests that a protein from chromaffin extracts, which could be Sp1, is binding to the probe at site 1. When the site 2 mutant was used as probe the formation of the lower complex was abolished (Fig. 5A, lane 10) and Sp1 was able to form a complex (Fig. 5A, lane 11), suggesting that a protein from chromaffin extracts, which is not Sp1, is binding to the probe at site 2. Antibody supershift analysis was employed in an attempt to identify the proteins producing the retarded bands. The upper complex (arrowhead) observed with both the wild and the site 2 mutant probes was retarded by an anti-Sp1 antibody (Fig.  5B, lanes 15 and 22, respectively), whereas no supershift was observed with antibodies against Sp3 (Fig. 5B, lane 16). The lower complex (circle) was shifted by an anti-NF-Y b antibody FIG. 1. The 5-region of the bovine ␤4 subunit gene. A, the nucleotide sequence of a fragment of genomic clone bov␤4-11 carrying exon 1 (with the protein sequence indicated below in italics), the 5Ј-region of intron 1 (indicated in small letters) and ϳ1250 bp of 5Ј-flanking sequence is indicated. The translation start codon is underlined, and the major transcription initiation sites are denoted by the arrowheads. Minor transcription initiation sites are also indicated (small squares). The accession number of this sequence in GenBank TM is AF453876. (Fig. 5B, lane 19). These results suggest that transcription factors Sp1 and NF-Y are binding to the probe. When using C2C12 and COS cell nuclear extracts a similar pattern of retarded bands was observed (Fig. 5C, lanes 24 and 27), although faster migrating complexes were also present with C2C12 extracts and the Sp1 upper band was less prominent. Again, the major band was supershifted with an anti-NF-Y b antibody (Fig. 5C, lanes 26 and 29).
Given that Sp1 and NF-Y bind to the GC and CCAAT boxes at sites 1 and 2, respectively (Fig. 5), and that the simultaneous alteration of these boxes produced a significant decrease of the transcriptional activity in luciferase reporter experiments (i.e. the activity of the double mutant p2-1␤4LUC was 35, 52, and 42% of the one observed with the wild-type construct p99␤4LUC in chromaffin, C2C12, and COS cells, respectively), we suggest that both, Sp1 and NF-Y, are involved in the transcriptional regulation of the bovine ␤4 promoter.

Structure of the 5Ј-Flanking Region of the Human ␤4 Subunit
Gene-Comparison of the ␤4 bovine promoter to its rat counterpart previously published (14, 15) did not show significant sequence homology. In an attempt to know whether this heterogeneity is extended to other species, we decided to isolate and characterize the human ␤4 promoter. A human genomic library was screened and several overlapping clones were isolated. Clone 101 contained ϳ20 kb of human genomic sequence including exon 1 and probably exon 2 as well as ϳ5 kb of 5Ј-flanking region. This region was further subcloned and about 720 bp located 5Ј from the initial ATG were sequenced. This sequence was identical to the one deposited in the NCBI data base with accession number NT_010218 Region 389312-390032. A comparison of ϳ1200 bp of bovine and human ␤4 promoter sequences adjacent to the transcription initiation site was performed with the Blast 2 program (22) and the result is shown in Fig. 6. Several homology regions were detected, rang- ing from 60% to more than 85% identity. These highly similar regions accounted for more than half of the whole sequences. As indicated in the diagram of Fig. 6 (lower panel), the structure of both promoters is similar, revealing regions of high or moderate homology flanked by stretches (ranging from 50 to 165 bp) of dissimilar sequences. By contrast, comparisons performed with the rat and either the bovine or human promoters did not yield significant homologies. The 5Ј-end of human ␤4 mRNA was also mapped by RNase protection analyses (Fig. 7). An antisense riboprobe incubated with mRNA from SHSY-5Y cells yielded several protected fragments that mapped transcription initiation to sites located between 91 and 125 bp upstream of the initial ATG (indicated by arrowheads for major fragments and small squares for minor ones in Fig. 6). One of them was also predominant in experiments performed with human brain and adrenal mRNAs (Fig. 7) and for this reason we have numbered this position as ϩ1 (also indicated as arrowhead at position ϩ1 in Fig. 6). The multiple initiation sites for the human and bovine ␤4 subunit genes are approximately located in the same area (indicated as vertical boxes in lower panel of Fig. 6).
Deletion Analysis of the Promoter for the Human nAChR ␤4 Subunit-A series of constructs was generated to determine the regions of the ␤4 subunit proximal promoter (Fig. 8) that contributed to its maximal activity. These constructs were introduced into SHSY-5Y cells, a human neuroblastoma cell line that express the ␤4 subunit endogenously (23) as well as in mouse muscle C2C12 cells, that express the muscular-type nAChR (24,25). In both cell lines, the construct containing 74 bp of ␤4 promoter sequence (considering the initiation site labeled as ϩ1 in Fig. 6 as reference for numbering) plus 97 bp of 5Ј-noncoding region (p74h␤4LUC) showed the maximal activity. When the luciferase activity of this construct was normalized for transfection efficiency and compared in these two cell lines it was about 8 times higher in SHSY-5Y cells than in C2C12 cells. On the other hand, the activity of p74h␤4LUC was 29 and 35% of the activity shown by pGL2Control in C2C12 and SHSY-5Y cells, respectively. When larger constructs were used (p255 and 960h␤4LUC) the relative luciferase activity decreased up to 60 -70% of the activity observed with p74h␤4LUC. The activity of p74h␤4LUC in SHSY-5Y and C2C12 cells was about 90% reduced when 46 bp of the ␤4 promoter 5Ј-end were deleted further (p28h␤4LUC) and was barely detectable upon the additional deletion of 37 bp (pϩ9h␤4LUC). These results suggest that elements located between 74 and 28 bp of the transcription initiation site are essential for transcription.  Fig. 4). Although construct p81␤4LUC also contains these elements it was not chosen as reference because one of the elements is just at its 5Ј-end and it was not known how this would affect its activity. The mean Ϯ S.E. (error bars) are given for at least two or three individual experiments, carried out in triplicate.  1 and 2, respectively). Several nucleotides of each potential element were mutated as indicated below the sequence to yield constructs analyzed in transfection experiments (B). B, the name of each mutant construct indicates the element(s) that have been altered. Plasmids were transfected into chromaffin, C2C12, and COS cells and activities were measured. Luciferase activity was normalized to values obtained with the p99␤4LUC construct. Data are expressed as described in the legend to Fig. 3.

Characterization of the Regulatory Elements Present at
␤4 subunit revealed the existence of two GC-boxes (labeled 1 and 2 in Fig. 9A) and an inverted CCAAT box with one mismatch in the core motif (labeled 3 in Fig. 9A). A systematic analysis of these putative regulatory elements was carried out, by looking at the functional effects produced by their mutagenesis in the context of p74h␤4LUC (Fig. 9B).
Mutation of GC-box 1 had virtually no effect on functional activity relative to p74h␤4LUC. However, mutation of GC-box 2 resulted in 75, 84, and 83% decrease in transcriptional activity in SHSY-5Y, C2C12, and COS cells, respectively (Fig. 9B). Mutation of the CCAAT box (element 3 in Fig. 8A) did not affect promoter activity in SHSY-5Y cells. By contrast, it induced increased activity (191% of p74h␤4LUC) in C2C12 cells. Also in COS cells, this construct was more active (159%) than the control (Fig. 9B). Interestingly, the decrease observed upon mutation of the GC-box 2 was further enhanced when the latter and the CCAAT box were simultaneously mutated (18,11, and 17% of the non-mutated control, in SHSY-5Y, C2C12, and COS cells, respectively). Therefore, it appears that GC-box 2 plays a determinant role in the transcriptional activity of the human ␤4 promoter, regardless of the effect that the inverted CCAAT box may play in certain cell types.
Characterization of the Regulatory Elements Present at the Human ␤4 Promoter by EMSA-DNA fragments carrying the wild-type Ϫ74/Ϫ38 promoter region and the corresponding mutants of the previously mentioned boxes were labeled and incubated with nuclear extracts from SHSY-5Y cells (Fig. 10A). Two main retarded bands (lane 1, dot and arrowhead) were observed when using the wild-type fragment. Formation of both complexes was competed by an excess of the same probe used in the EMSA experiments (lanes 4 and 5). An excess of double stranded oligonucleotides containing consensus binding sites for transcription factors NF-Y (lane 2) and Sp1 (lane 3) were also used in competition experiments. They abolished the formation of the lower (dot) and upper (arrowhead) complexes, respectively, suggesting that they may result from interactions with the mentioned transcription factors. Antibody supershift analysis was employed to test this hypothesis. Thus, the upper complex (arrowhead) was shifted by antibodies against Sp1 (lane 9) but not with anti-Sp3 antibodies (lane 10), confirming that Sp1 is binding to the probe. Likewise, the lower band (dot) was shifted by a NF-Y antibody (lane 11). Moreover, under conditions in which the two GC boxes (elements 1 and 2, Fig.  9A) and the inverted CCAAT box (element 3, Fig. 9A) had been disrupted, it was possible to identify the elements to which these transcription factors were binding (Fig. 10B). Accordingly, when the CCAAT box was mutated, the lower complex  3, 8, and 11). Lanes 1, 6, and 9 are probes run in the absence of protein extracts. Two prominent bands (filled dot and arrowhead) were observed with chromaffin nuclear extracts. One of them (arrowhead) was also observed with recombinant Sp1. Competition with unlabeled probe added in 10-(lane 4) and 100-fold (lane 5) excess, decreased significantly the amount of retarded complexes. B, probes WT Ϫ99/ϩ66, Mut1, and Mut 2 were used with chromaffin nuclear extracts in a supershift assay. The band labeled with an arrowhead was supershifted by Sp1 antibodies (lanes 15 and 22) but not by Sp3 antibodies (lane 16). The band labeled with a filled dot was supershifted by NF-Y antibodies as deduced by the lower intensity of the NF-Y band in the presence of antibody (compare lanes 18 and 19). Lanes 12,17, and 20 (F) are probes run in the absence of protein extracts C, the gel mobility assay was run using DNA fragment Ϫ99 to ϩ66 as the labeled probe and nuclear extracts from C2C12 (lanes 24 -26) and COS (lanes 27-29) cells. The prominent band, indicated by a filled dot, was displaced with NF-Y antibodies (lanes 26 and 29). 6. Comparison of the nucleotide sequence of the 5-flanking regions of the bovine and human nAChR ␤4 subunit genes. Upper panel, the nucleotide sequences of the bovine (BOV) and human (HUM) proximal regions of the ␤4 subunit promoters are depicted, including the initial ATG (bold) and the signal peptide sequences. Comparisons were performed with the Blast 2 program (22) and the degree of identity is indicated by bars located above the sequences according to the following code: Ⅺ, 60-75% identity; u, 75-85% identity; f, Ͼ85% identity. new band located slightly below the NF-Y complex appeared (square). In the case of the GC boxes, only disruption of GC-box 2 abolished the binding of Sp1 (lane 15) whereas the mutant of GC-1 (lane 17) produced a pattern similar to the wild probe. Therefore, NF-Y binds to the inverted CCAAT box and Sp1 to the adjacent GC box. In fact, when both boxes were simultaneously mutated none of these transcription factors were able to produce retarded complexes (lane 19). Similar results were obtained with nuclear extracts from C2C12 cells (not shown).

DISCUSSION
Two classes of nAChRs have been identified on bovine chromaffin cells. One class, probably formed by ␣3, ␣5, and ␤4 subunits, is present in all chromaffin cells of the adrenal medulla (10) and appears to be representative of many nAChRs present in the peripheral nervous system. The other binds ␣-bungarotoxin, is expressed only in adrenergic cells (26) and contains ␣7 subunits (9). In our laboratory we have previously isolated and characterized the bovine ␣7 (8, 26) and ␣5 (7) subunits promoters. In this article we describe the characterization of the ␤4 subunit promoter in the bovine and human species and compare them with its rat counterpart, that has been previously studied in great detail (14,15,27,28).
The core promoter regions of both the bovine and human ␤4 subunits do not contain TATA boxes, but do have G ϩ C-rich domains. This characteristic is also found in the rat ␤4 promoter (14) as well as in the promoters of the ␣2 (29), ␣3 (30, 31), ␣5 (7,17), and ␣7 (26, 32-34) subunits. From 5Ј-end deletion analysis (Fig. 3) of the bovine promoter, we determined that the region located between nucleotides Ϫ99 and ϩ66 was necessary for the basal promoter activity detected in chromaffin, C2C12, and COS cells. A comparison of normalized (for transfection efficiency) luciferase activity values of p99␤4LUC in the three mentioned cell types, indicated that promoter activity was similar in chromaffin and COS cells whereas in C2C12 was about 40% lower. Since COS cells do not endogenously produce ␤4 subunits, it is possible that promoter elements needed for cellspecific expression are not included within the promoter fragments used in this study. Nevertheless, elements located between Ϫ353 and Ϫ787 appear responsible for a slight decline in activity in C2C12 and COS cells and could be involved in a silencing mechanism. However, this mechanism might not be totally effective or may need additional elements, since the largest construct p1256␤4LUC regains activity despite containing the mentioned sequence. A large loss in promoter ac- Non-homologous stretches are also indicated (ϭ ϭ ϭ) with the number of bases they contain. The putative regulatory elements are in capital letters and underlined in the two promoter sequences. Numbering in both sequences considers the initial ATG site as ϩ1. The multiple sites of transcription initiation in the human promoter are indicated below the sequence with arrowheads (main sites) and small squares (secondary sites). Lower panel, an schematic diagram of the two proximal promoter regions depicting the homologous fragments (same code as above), the non-related stretches (-), the approximate location of the regulatory elements identified in the bovine promoter (NF-Y/Sp1), and the location of the regions protected in the RNase protection analysis (vertical boxes). The accession number of the human sequence in GenBank TM is AF453877. tivity was observed when 36 bp were deleted from the 5Ј-end (compare p63␤4LUC with respect to the larger construct p99␤4LUC, Fig. 3). The most remarkable feature in the deleted region, between Ϫ64 and Ϫ99, was the presence of sites for Sp1 and NF-Y (labeled 1 and 2, respectively, in Fig. 4A). These transcription factors, or other proteins closely related inmunologically with them, were able to bind to the mentioned elements (Fig. 5). Therefore, these elements appeared to be suitable candidates for controlling promoter activity. Consequently, when they were simultaneously mutated in the context of p99␤4LUC, promoter activity was strongly reduced (Fig.  4B). The sum of effects due to the single alteration of these sites was lower than the effect of mutating them simultaneously, suggesting that Sp1 and NF-Y act cooperatively to play a crucial role in the transcriptional regulation of the bovine ␤4 gene. Both, Sp1 and NF-Y, are ubiquitously expressed transcription factors that play a major role in the transcription of many genes (35)(36)(37). Moreover, there are abundant examples of promoters that require the concerted action of Sp1 and NF-Y (see Refs. 38 -42 for recent studies), and in two cases a physical interaction between these factors has been demonstrated (43,44). In addition, other positive elements, located between Ϫ63 and ϩ66 may be required for optimal transcription, given that constructs p39␤4LUC, p63␤4LUC (Fig. 3), and p2-1␤4LUC (Fig. 4) that do not contain the Sp1 and NF-Y sites, still exhibited about 30 -40% of the maximal promoter activity. However, if these additional factors exist, they were not detected with the standard conditions used in our EMSA experiments.
Albeit the high sequence identity (84.4%) between the coding regions of the bovine and rat ␤4 subunits (10), their proximal promoter regions did not show significant sequence homology. In an attempt to clarify the significance of this heterogeneity we decided to isolate and characterize another ␤4 subunit promoter, the one from the human species. As shown in Fig. 6, the structures of the human and bovine promoters are highly homologous, containing regions of 60 -95% identity separated by non-related stretches of 50 -165 bp, and indicating that there may be common regulatory mechanisms leading to their expression. This was confirmed upon transfection and EMSA experiments that demonstrated the involvement of Sp1 and NF-Y in the regulation of the human ␤4 promoter. However, the contribution of these factors was different from the one exhibited in the bovine promoter for several reasons. First, the situation of the elements to which these factors bind is different in the two promoters and despite the existence of several regions of moderate to high homology, they are located in sequence stretches that are not homologous. Second, in the absence of the Sp1 site, the promoter activity that remains is clearly lower in the human promoter ( Fig. 9) than in its bovine counterpart (Fig. 4), suggesting a different action mechanism for this transcription factor in the two promoters. And finally, the role of NF-Y also appears to be different in the two cases. Thus, in the bovine promoter NF-Y acts in a concerted manner with Sp1, whereas the role of NF-Y in the human promoter appears to depend on the cell context where promoter activity is being tested. Thus, in SHSY-5Y cells appears to be irrelevant, while in C2C12 and COS cells could be involved in a repressing mechanism as it is suggested by the increase of transcriptional activity observed upon its mutation (Fig. 9). A possible explanation for this effect would be that the absence of NF-Y facilitates the binding of an activating factor. In fact, EMSA experiments performed with a probe in which the NF-Y site had been altered (Fig. 10B, lane 15, small square) revealed the formation of a complex that was not observed with the wild probe. However, this shift pattern was observed not only with C2C12 extracts (not shown) but also with nuclear extracts from SHSY-5Y cells (Fig. 10), in which promoter activity was not modified by the NF-Y mutation. Alternatively, and given that there is a 3-bp overlap within the Sp1 and NF-Y sites, the absence of NF-Y would alleviate steric restrictions and increase the binding of Sp1, thus helping to enhance promoter activity. Such a situation, however, would be expected to occur in all the tested cells lines and one would anticipate the same effect of the NF-Y mutant in the three cell lines analyzed, what was not the case. Therefore, it is possible that more complex mechanisms are implicated in the differential effect of the NF-Y FIG. 8. Deletion map analysis of human ␤4 gene promoter activity. SHSY-5Y and C2C12 cells were transfected with each of the plasmids, which were named ph␤4LUC with the number of promoter base pairs included in the construct (numbering considers in this case as ϩ1 one of the major protected fragments in the RNase experiments) and contained the luciferase reporter under the control of the different fragments of the human ␤4 subunit promoter and pCH110/␤-galactosidase as a transfection efficiency control. Promoter activity was normalized to values obtained with the p74h␤4LUC construct. Data are expressed as described in the legend to Fig. 3.   FIG. 9. Site 2 is the major determinant of the human ␤4 subunit promoter activity. A, the proximal region of the human ␤4 subunit promoter (nucleotides Ϫ74 to Ϫ38) is depicted with the putative regulatory elements boxed. This region contains putative binding sites for transcription factors Sp1 (boxes of solid lines, elements 1 and 2) and NF-Y (dashed box, element 3). Several nucleotides of each potential element were mutated as indicated below the sequence to yield constructs analyzed in transfection (panel B) experiments. B, the name of each mutant construct indicates the element(s) that have been altered. Plasmids were transfected into SHSY-5Y, C2C12, and COS cells, and their activities were measured. Luciferase activity was normalized to values obtained with the p60h␤4LUC construct. Data are expressed as in Fig. 3. mutation in the cell types used, perhaps involving different protein modifications, protein-protein interactions, and/or a distinct balance between NF-Y and Sp1 in the three cells lines analyzed. In any case, the dominant role of the Sp1 element in the human promoter was further demonstrated by the fact that the double mutant of the Sp1 and NF-Y elements showed very low promoter activity in C2C12 and COS cells and, therefore, the activating effect of the NF-Y mutation was overcome by the detrimental effect of the Sp1 mutation. This predominant role of Sp1 has also been demonstrated for the rat promoter, since the alteration of the Sp1 element (CA box) produced a 90% decrease in promoter activity (14). In this case, additional factors appear to modulate promoter activity, such as the transcription factors Pur␣ (27), Sox10 (45,46), and the heterogeneous nuclear ribonucleoprotein K (28), the latter repressing Sp1 function totally. Taking into account all these data, we propose a general model of transcriptional regulation for the ␤4 subunit in different species, in which Sp1 would play a common and critical role through its multiple interactions, some of them with the basic transcriptional machinery and others with additional transcription factors, that could themselves constitute further interacting platforms (47). The latter could contribute to the necessary cell, tissue, and species specificity. This proposition could be also extended to the ␣3 (16, 31) and ␣5 (7) nAChR subunits that are also regulated by Sp1. retarded complexes. The complex labeled with a dot was competed with a NF-Y oligonucleotide and displaced by a NF-Y antibody (lane 11), whereas the band labeled with an arrowhead was competed with a Sp1 oligonucleotide and shifted by a Sp1 antibody (lane 9). B, labeled wildtype DNA fragment Ϫ74 to Ϫ38 (lanes 12 and 13) and the corresponding fragments mutated at sites 3 (lanes 14 and 15), 2 (lanes 16 and 17),  1 (lanes 18 and 19), or 2 and 3 simultaneously (lanes 20 and 21) were used as gel mobility shift probes in the presence (lanes 13, 15, 17, 19, and 21) or absence (lanes 12, 14, 16, 18, and 20) of SHSY-5Y cell nuclear extracts. When site 3 was altered, the formation of a new complex, labeled with a square, was observed (lanes 15 and 21).  10. Identification of Sp1 and NF-Y as proteins binding to the cis-elements 2 and 3 of the proximal region of the human ␤4 subunit promoter. A, labeled wild-type DNA fragment corresponding to region from Ϫ74 to Ϫ38 was used as gel mobility shift probe in the presence of crude SHSY-5Y cell nuclear extracts. Lane 1 is the probe run in the presence of protein extracts. Lanes 2 and 3 represent complexes obtained with nuclear extracts in the presence of a 100-fold excess of competitor oligonucleotides with consensus sites for NF-Y and Sp1, respectively. Lanes 4 and 5 represent complexes obtained with nuclear extracts in the presence of a 100-and 10-fold excess of unlabeled probe, respectively. Lane 6 is the probe run in the absence of protein extracts. In lanes 7-11, antibodies specific for the indicated transcription factors were used to identify the proteins producing the