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J. Biol. Chem., Vol. 277, Issue 11, 8866-8876, March 15, 2002

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Transcription Factors NF-Y and Sp1 Are Important Determinants of the Promoter Activity of the Bovine and Human Neuronal Nicotinic Receptor 4 Subunit Genes^*

Luis M. Valor^§¶, Antonio Campos-Caro^§, Carmen Carrasco-Serrano^§**, José A. Ortiz^§, Juan J. Ballesta^§§§, and Manuel Criado^§¶¶

From the Departments of  Biochemistry and Molecular Biology and ^§§ Pharmacology, and ^§ Instituto de Neurociencias, Universidad Miguel Hernández-C.S.I.C., 03550-San Juan, Alicante, Spain

Received for publication, October 31, 2001, and in revised form, December 11, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 4 subunit is a component of the neuronal nicotinic acetylcholine receptors which control catecholamine secretion in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was characterized. A proximal region (from 99 to 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 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 4 subunit promoter (Bigger, C. B., Casanova, E. A., and Gardner, P. D. (1996) J. Biol. Chem. 271, 32842-32848) except for the involvement of an Sp1 binding element, we propose that constitutive expression of the 4 subunit gene in these three close species may be controlled by the general transcription factor Sp1. Nevertheless, other components could determine species-specific 4 subunit expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 distribution 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 cis-elements 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation and Analysis of the 5'-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 5'-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 5'-untranslated 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)⁺ 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), [-³²P]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 5' 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 5'-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 5'-end of human 4 mRNA. In this case a 323-bp BamHI-SacI fragment whose 3'-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)⁺ 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 (5'-CTTTATGTTTTTGGCGTCTTCC-3') 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 99 to 64 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 p994LUC (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 74 to 44 of the human 4 promoter was based on the presence of a SacII site at the 3'-end of the region containing the elements to be mutated and an XmaI site (from pGL2-Basic) at the 5'-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.

Cell Culture and Reporter Assays-- Chromaffin cells were isolated from bovine adrenal glands as described (19) and cultured in 90% Dulbecco's modified Eagle's medium (Sigma, Madrid), 10% fetal calf serum, 10 µM cytosine arabinoside, and 10 µM 5-fluoro-2'-deoxyuridine (Sigma) to prevent fibroblast proliferation. SHSY-5Y human neuroblastoma cells were grown in 90% Eagle's minimal essential medium with Glutamax-1 (Invitrogen, Barcelona, Spain) and 10% fetal calf serum. COS cells were grown in 90% Dulbecco's modified Eagle's medium and 10% fetal calf serum. C2C12 cells were grown in 85% Dulbecco's modified Eagle's medium and 15% fetal calf serum.

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 × 10⁵ cells/well) were incubated with 0.75 µg of pGL2 vector or an equivalent amount (in molar terms) of the different constructs derived from this vector and with 0.75 µg of -galactosidase expression vector pCH110 (Amersham Biosciences, Inc.) as a control of transfection efficiency. SHSY-5Y (10⁵ cells/well), COS cells (5 × 10⁴ cells/well), or C2C12 cells (10⁴ cells/well) on 24-well plates, were incubated with 1.5 µg of the different 4 constructs and 1.5 µg 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 99 to +66 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 [-³²P]dATP. The human 4 probes were obtained by annealing complementary oligonucleotides corresponding to region 74 to 38 and end-labeled by Klenow filling with [-³²P]dCTP. The DNA-protein binding reaction volumes were 20 µl containing 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 5 µg of bovine serum albumin, 2 µg of poly(dA-dT)·poly(dA-dT) (Amersham Biosciences Inc.), 2 µg of nuclear extract protein, and 20,000 cpm of ³²P-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 µl 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Structure of the 5'-Flanking Region of the Bovine 4 Subunit Gene-- A bovine genomic library was screened and several overlapping clones were isolated. Clone bov4-11 contained ~13 kb of bovine genomic sequence including exon 1 and ~1.2 kb of 5'-flanking region. This region was further subcloned and sequenced (Fig. 1) revealing the lack of a TATA box. The 5'-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 ~309 bases that mapped transcription initiation to a thymine located 125 bp upstream of the initial ATG (arrowhead at position +1, 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.

View larger version (77K): [in this window] [in a new window]   Fig. 1.   The 5'-region of the bovine 4 subunit gene. A, the nucleotide sequence of a fragment of genomic clone bov4-11 carrying exon 1 (with the protein sequence indicated below in italics), the 5'-region of intron 1 (indicated in small letters) and ~1250 bp of 5'-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 GenBankTM is AF453876.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Determination of the bovine 4 subunit gene transcription initiation site(s). The 5'-end of the 4 subunit mRNA was mapped by RNase protection using two 4 probes whose structures are illustrated in the lower part of the figure. U, undigested probes, 496 (Probe 1, lane 2) and 352 (Probe 2, lane 5) bases (b). D, probes digested in the presence of yeast tRNA (lanes 1 and 6). C, probes digested in the presence of a sense cRNA control, yielding protected fragments of 215 (Probe 1, lane 3) and 234 (Probe 2, lane 7) bases. P, protected fragments using 7.5 µg of bovine adrenal medulla poly(A)+ mRNA (lanes 4 and 8). The sizes of several RNA fragments used for calibration of the gel are indicated to the left of the panel, whereas the sizes of the protected fragments are to the right.

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 (p814LUC) or more (up to 1256 bp) of 4 promoter sequence plus 66 bp of 5'-noncoding region showed similar activity. Two shorter constructs (p634LUC and p394LUC) showed a ~60% decrease in promoter activity, whereas a further 5' deletion (p44LUC) 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 p7874LUC, which showed a ~20-30% 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 353 and 787 with respect to the transcription initiation site may have a negative effect on 4 promoter activity. However, the largest construct tested (p12564LUC) also contains this region and exhibited increased activity (~100-120%) in the three cell types.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Deletion map analysis of bovine 4 gene promoter activity. Chromaffin, C2C12, and COS cells were transfected with each of the plasmids (named p4LUC with the number of promoter base pairs included in the construct) containing the luciferase reporter under the control of the different fragments of the 4 subunit promoter and pCH110/-galactosidase as a transfection efficiency control. Promoter activity was normalized to values obtained with the p994LUC construct because it contains elements important for transcription (see Fig. 4). Although construct p814LUC also contains these elements it was not chosen as reference because one of the elements is just at its 5'-end and it was not known how this would affect its activity. The mean ± S.E. (error bars) are given for at least two or three individual experiments, carried out in triplicate.

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 ~60% 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 p994LUC instead of p814LUC, to leave a few additional nucleotides at the 5'-end of one of the elements (numbered 2 in Fig. 4A). Otherwise, this region would be located just at the 5'-end of construct p814LUC. When the GC box was altered (site 1, Fig. 4A), 4 promoter activity in C2C12 and COS cells decreased to ~60% of that observed for the parent construct (p994LUC), 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 ~40% 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.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Sites 1 and 2 are functional elements required for 4 subunit gene expression. A, the proximal region of the 4 subunit promoter (nucleotides 99 to 64) is depicted with the putative regulatory elements boxed. This region contains putative binding sites for transcription factors Sp1 and NF-Y (denoted by numbers 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 p994LUC construct. Data are expressed as described in the legend to Fig. 3.

Characterization of the Regulatory Elements Present at 99/63 of the 4 Promoter by EMSA-- DNA fragments carrying the wild-type 99 to +66 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. 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).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Binding of cell nuclear proteins to the proximal region of the 4 subunit promoter. A, the DNA fragment corresponding to region 99 to +66 (WT 99/+66) or the analogous mutants of sites 1 (Mut 1) and 2 (Mut 2) were used as gel mobility shift probes in the presence of 2 µg of crude chromaffin (CR; lanes 2, 4, 5, 7, and 10) cell nuclear extracts or recombinant Sp1 (0.2 footprint units, 1 footprint unit/µl) (lanes 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 99/+66, 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 99 to +66 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).

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 p2-14LUC was 35, 52, and 42% of the one observed with the wild-type construct p994LUC 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 5'-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 ~20 kb of human genomic sequence including exon 1 and probably exon 2 as well as ~5 kb of 5'-flanking region. This region was further subcloned and about 720 bp located 5' 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 ~1200 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, ranging 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 5'-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 +1 (also indicated as arrowhead at position +1 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).

View larger version (55K): [in this window] [in a new window]   Fig. 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: , 60-75% identity; , 75-85% identity; , >85% identity. Non-homologous stretches are also indicated (= = =) 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 +1. 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 GenBankTM is AF453877.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Determination of the human 4 subunit gene transcription initiation site(s). The 5'-end of the human 4 subunit mRNA was mapped by RNase protection using a 4 probe whose structure is illustrated in the lower part of the figure. D, probe digested in the presence of yeast tRNA (lane 2). P, protected fragments using 2.5 µg of SHSY-5Y poly(A)+ mRNA (S, lane 3) and 1 µg of human brain (B, lane 4) or adrenal gland (A, lane 5) poly(A)+ mRNA. The sizes of fragments used for calibration of the gel are indicated to the left of the panel, whereas the sizes of some major protected fragments are to the right. Undigested probe is not shown given its large size.

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 +1 in Fig. 6 as reference for numbering) plus 97 bp of 5'-noncoding region (p74h4LUC) 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 p74h4LUC was 29 and 35% of the activity shown by pGL2Control in C2C12 and SHSY-5Y cells, respectively. When larger constructs were used (p255 and 960h4LUC) the relative luciferase activity decreased up to 60-70% of the activity observed with p74h4LUC. The activity of p74h4LUC in SHSY-5Y and C2C12 cells was about 90% reduced when 46 bp of the 4 promoter 5'-end were deleted further (p28h4LUC) and was barely detectable upon the additional deletion of 37 bp (p+9h4LUC). These results suggest that elements located between 74 and 28 bp of the transcription initiation site are essential for transcription.

View larger version (13K): [in this window] [in a new window]   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 ph4LUC with the number of promoter base pairs included in the construct (numbering considers in this case as +1 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 p74h4LUC construct. Data are expressed as described in the legend to Fig. 3.

Characterization of the Regulatory Elements Present at 74/44 of the Human 4 Promoter by Mutagenesis and Transient Transfections-- A search for transcription factors which could interact with elements at the proximal promoter region of the 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 p74h4LUC (Fig. 9B).

View larger version (19K): [in this window] [in a new window]   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 74 to 38) 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 p60h4LUC construct. Data are expressed as in Fig. 3.

Mutation of GC-box 1 had virtually no effect on functional activity relative to p74h4LUC. 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 p74h4LUC) 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 74/38 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 (dot) contributed by NF-Y was absent (lane 15). However, a 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).

View larger version (43K): [in this window] [in a new window]   Fig. 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 74 to 38 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 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 wild-type DNA fragment 74 to 38 (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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 + 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 5'-end deletion analysis (Fig. 3) of the bovine promoter, we determined that the region located between nucleotides 99 and +66 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 p994LUC 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 cell-specific expression are not included within the promoter fragments used in this study. Nevertheless, elements located between 353 and 787 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 p12564LUC regains activity despite containing the mentioned sequence. A large loss in promoter activity was observed when 36 bp were deleted from the 5'-end (compare p634LUC with respect to the larger construct p994LUC, Fig. 3). The most remarkable feature in the deleted region, between 64 and 99, 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 p994LUC, 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 (35-37). 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 63 and +66 may be required for optimal transcription, given that constructs p394LUC, p634LUC (Fig. 3), and p2-14LUC (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 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.

	ACKNOWLEDGEMENTS

The excellent technical assistance of Eva Martínez and Susana Gerber is appreciated. We thank Dr. J. Lindstrom for the human 4 cDNA.

	FOOTNOTES

^* This work was supported in part by Ministry of Education (DGICYT) Grants PB95-0690 and PM98-0104 of Spain, and the Generalitat Valenciana Grant GV-D-VS-20-158-96.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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/EBI Data Bank with accession number(s) AF453876 and AF453877.

^¶ Predoctoral fellow from Generalitat Valenciana.

Recipient of a CSIC-Bancaja postdoctoral fellowship.

^** Predoctoral fellow from Generalitat Valenciana.

Postdoctoral fellow from the Ministry of Education of Spain.

^¶¶ To whom correspondence should be addressed. Tel.: 34- 965919479; Fax: 34-965919484; E-mail: Manuel.Criado@umh.es.

Published, JBC Papers in Press, December 12, 2001, DOI 10.1074/jbc.M110454200

	ABBREVIATIONS

The abbreviations used are: nAChR, nicotinic acetylcholine receptor; EMSA, electrophoretic mobility shift assays.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES