Transcription Factors NF-Y and Sp1 Are Important Determinants of the Promoter Activity of the Bovine and Human Neuronal Nicotinic Receptor β4 Subunit Genes
2002; Elsevier BV; Volume: 277; Issue: 11 Linguagem: Inglês
10.1074/jbc.m110454200
ISSN1083-351X
AutoresLuis M. Valor, Antonio Campos‐Caro, Carmen Carrasco-Serrano, J A Ortíz, Juan J. Ballesta, Manuel Criado,
Tópico(s)Receptor Mechanisms and Signaling
ResumoThe β4 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 twocis-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 β4 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 β4 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 β4 subunit gene in these three close species may be controlled by the general transcription factor Sp1. Nevertheless, other components could determine species-specific β4 subunit expression. The β4 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 twocis-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 β4 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 β4 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 β4 subunit gene in these three close species may be controlled by the general transcription factor Sp1. Nevertheless, other components could determine species-specific β4 subunit expression. nicotinic acetylcholine receptor electrophoretic mobility shift assays 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 (1Boyd R.T. Crit. Rev. Toxicol. 1997; 27: 299-318Crossref PubMed Scopus (81) Google Scholar, 2Changeux J.P. Bertrand D. Corringer P.J. Dehaene S. Edelstein S. Lena C. Le Novere N. Marubio L. Picciotto M. Zoli M. Brain Res. Rev. 1998; 26: 198-216Crossref PubMed Scopus (263) Google Scholar). The varied tissue-, region-, and development-specific distribution of nAChRs subunits (3Sargent P.B. Annu. Rev. Neurosci. 1993; 16: 403-443Crossref PubMed Scopus (935) Google Scholar) 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 (4McGehee D.S. Role L.W. Annu. Rev. Physiol. 1995; 57: 521-546Crossref PubMed Scopus (898) Google Scholar, 5Role L.W. Berg D.K. Neuron. 1996; 16: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar). For these reasons considerable effort has been dedicated to the elucidation of the molecular basis for the transcriptional regulation of neuronal nAChRs, and thus severalcis- and trans-acting elements in the promoter of the different nAChRs subunits have been identified (6Fornasari D. Battaglioli E. Flora A. Terzano S. Clementi F. Arneric S.P. Brioni J.D. Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities. Wiley-Liss, New York1998: 25-42Google Scholar). In our laboratory we have previously isolated and characterized the promoters of the bovine α5 (7Campos-Caro A. Carrasco-Serrano C. Valor L.M. Viniegra S. Ballesta J.J. Criado M. J. Biol. Chem. 1999; 274: 4693-4701Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and α7 (8Carrasco-Serrano C. Campos-Caro A. Viniegra S. Ballesta J.J. Criado M. J. Biol. Chem. 1998; 273: 20021-20028Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) subunits. These subunits are expressed in the chromaffin cells of the adrenal gland composing two different receptor subtypes, one of them formed by α7 subunits (9Garcı́a-Guzmán M. Sala F. Sala S. Campos-Caro A. Stühmer W. Gutiérrez L.M. Criado M. Eur. J. Neurosci. 1995; 7: 647-655Crossref PubMed Scopus (96) Google Scholar) and the other by α3, β4, and α5 subunits (10Campos-Caro A. Smillie F. Domı́nguez del Toro E. Rovira J.C. Vicente-Agulló F. Chapuli J. Juı́z J.M. Sala S. Sala F. Ballesta J.J. Criado M. J. Neurochem. 1997; 68: 488-497Crossref PubMed Scopus (113) Google Scholar). Interestingly, the genes of the latter subunits are clustered in the vertebrate genome (11Couturier S. Erkman L. Valera S. Rungger D. Bertrand S. Boulter J. Ballivet M. Bertrand D. J. Biol. Chem. 1990; 265: 17560-17567Abstract Full Text PDF PubMed Google Scholar, 12Boulter J. O'Shea-Greenfield A. Duvoisin R.M. Connolly J.G. Wada E. Jensen A. Gardner P.D. Ballivet M. Deneris E.S. McKinnon D. Heinemann S. Patrick J. J. Biol. Chem. 1990; 265: 4472-4482Abstract Full Text PDF PubMed Google Scholar) and may have common patterns of regulation (13McDonough J. Francis N. Miller T. Deneris E.S. J. Biol. Chem. 2000; 275: 28962-28970Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). We have analyzed here the bovine and human β4 promoters, finding that they are highly homologous in their proximal regions as well as in thecis-elements governing basal transcriptional activity. Although their sequences differ from the one of the rat β4 promoter (14Bigger C.B. Casanova E.A. Gardner P.D. J. Biol. Chem. 1996; 271: 32842-32848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15Bigger C.B. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1997; 272: 25976-25982Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), 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 α3 (16Yang X. Fyodorov D. Deneris E.S. J. Biol. Chem. 1995; 270: 8514-8520Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and bovine (7Campos-Caro A. Carrasco-Serrano C. Valor L.M. Viniegra S. Ballesta J.J. Criado M. J. Biol. Chem. 1999; 274: 4693-4701Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and human (17Flora A. Schulz R. Benfante R. Battaglioli E. Terzano S. Clementi F. Fornasari D. J. Neurochem. 2000; 75: 18-27Crossref PubMed Scopus (41) Google Scholar) α5 subunits, suggesting that this transcription factor plays a fundamental role in the expression of several nAChRs subunit genes. 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 (10Campos-Caro A. Smillie F. Domı́nguez del Toro E. Rovira J.C. Vicente-Agulló F. Chapuli J. Juı́z J.M. Sala S. Sala F. Ballesta J.J. Criado M. J. Neurochem. 1997; 68: 488-497Crossref PubMed Scopus (113) Google Scholar) 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 (18Kormelink P.J.G. Luyten W.H.M.L. FEBS Lett. 1997; 400: 309-314Crossref PubMed Scopus (45) Google Scholar) was used to screen a genomic library. Both libraries were constructed in EMBL-3 SP6/T7 (CLONTECH, Heidelberg, Germany) and tested as previously described (9Garcı́a-Guzmán M. Sala F. Sala S. Campos-Caro A. Stühmer W. Gutiérrez L.M. Criado M. Eur. J. Neurosci. 1995; 7: 647-655Crossref PubMed Scopus (96) Google Scholar). In both cases several overlapping bacteriophage clones were purified and characterized. 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), [α-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 β4 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 β4 cDNA construct linearized withXbaI. 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 aRsaI-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 withXhoI 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 β4 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). All β4 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 β4 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 β4 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 p99β4LUC (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. 4 A (sites 1 and 2). The strategy for mutagenesis of the elements in region −74 to −44 of the human β4 promoter was based on the presence of a SacII site at the 3′-end of the region containing the elements to be mutated and anXmaI 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. Chromaffin cells were isolated from bovine adrenal glands as described (19Gandı́a L. Casado L.F. López M.G. Garcı́a A.G. Br. J. Pharmacol. 1991; 103: 1073-1078Crossref PubMed Scopus (42) Google Scholar) 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 (20Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6495) Google Scholar). 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 β-galactosidase expression vector pCH110 (Amersham Biosciences, Inc.) as a control of transfection efficiency. SHSY-5Y (105 cells/well), COS cells (5 × 104cells/well), or C2C12 cells (104 cells/well) on 24-well plates, were incubated with 1.5 μg of the different β4 constructs and 1.5 μg of pCH110 per well. Cells were harvested after 48 h and lysed with reporter lysis buffer (Promega). β-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 β4 subunit. They are indicated in the corresponding figure legends. Crude nuclear extracts were prepared from cultured cells as described by Schreiber et al. (21Schreiber E. Matthias P. Müller M.M. Schaffner W. Nucleic Acids Res. 1989; 176419Crossref PubMed Scopus (3917) Google Scholar). The DNA fragment corresponding to region −99 to +66 of the bovine β4 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 [α-32P]dATP. The human β4 probes were obtained by annealing complementary oligonucleotides corresponding to region −74 to −38 and end-labeled by Klenow filling with [α-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 mmdithiothreitol, 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-Yb antibody (CBF-A, C-20) from Santa Cruz Biotechnology was used in later experiments with the human β4 promoter. A bovine genomic library was screened and several overlapping clones were isolated. Clone λbovβ4–11 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 β4 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. A series of constructs was generated to determine the regions of the bovine β4 subunit promoter that contributed to its maximal activity (Fig. 3). These constructs were introduced into chromaffin, C2C12, and COS cells. Constructs containing 81 bp (p81β4LUC) or more (up to 1256 bp) of β4 promoter sequence plus 66 bp of 5′-noncoding region showed similar activity. Two shorter constructs (p63β4LUC and p39β4LUC) showed a ∼60% decrease in promoter activity, whereas a further 5′ deletion (p4β4LUC) 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 p787β4LUC, 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 β4 promoter activity. However, the largest construct tested (p1256β4LUC) also contains this region and exhibited increased activity (∼100–120%) 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 β4 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.4 A) and, therefore, they were chosen for mutagenic analysis. These analysis were performed, however, in the context of p99β4LUC instead of p81β4LUC, to leave a few additional nucleotides at the 5′-end of one of the elements (numbered2 in Fig. 4 A). Otherwise, this region would be located just at the 5′-end of construct p81β4LUC. When the GC box was altered (site 1, Fig. 4 A), β4 promoter activity in C2C12 and COS cells decreased to ∼60% of that observed for the parent construct (p99β4LUC), whereas in chromaffin cells the decrease was less pronounced (Fig. 4 B). The mutant of the CCAAT box (site 2, Fig. 4 B) affected promoter activity in chromaffin and C2C12 cells (about 25% decrease) but did not have any effect in COS cells (Fig. 4 B). 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. 4 B) 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. 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. 5 A, lane 2, labeled as circleand arrowhead) when using the wild-type fragment. Both bands were competed with increasing amounts of unlabeled fragment (Fig.5 A, lanes 4 and 5). Recombinant Sp1 produced a main retarded complex (Fig. 5 A, 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. 5 A, lane 7) nor recombinant Sp1 retarded the probe (Fig. 5 A, 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. 5 A, lane 10) and Sp1 was able to form a complex (Fig. 5 A, 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.5 B, lanes 15 and 22, respectively), whereas no supershift was observed with antibodies against Sp3 (Fig.5 B, lane 16). The lower complex (circle) was shifted by an anti-NF-Yb antibody (Fig. 5 B, 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. 5 C, lanes 24 and27), 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-Yb antibody (Fig. 5 C, 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 p2-1β4LUC was 35, 52, and 42% of the one observed with the wild-type construct p99β4LUC in chromaffin, C2C12, and COS cells, respectively), we suggest that both, Sp1 and NF-Y, are involved in the transcriptional regulation of the bovine β4 promoter. Comparison of the β4 bovine promoter to its rat counterpart previously published (14Bigger C.B. Casanova E.A. Gardner P.D. J. Biol. Chem. 1996; 271: 32842-32848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15Bigger C.B. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1997; 272: 25976-25982Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) 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 β4 promoter. A human genomic library was screened and several overlapping clones were isolated. Clone λ101 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 β4 promoter sequences adjacent to the transcription initiation site was performed with the Blast 2 program (22Tatusova T.A. Madden T.L. FEMS Microbiol. Lett. 1999; 174: 247-250Crossref PubMed Google Scholar) 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 β4 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 β4 subunit genes are approximately located in the same area (indicated as vertical boxes in lower panel of Fig. 6).Figure 7Determination of the human β4 subunit gene transcription initiation site(s). The 5′-end of the human β4 subunit mRNA was mapped by RNase protection using a β4 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 theleft of the panel, whereas the sizes of some major protected fragments are to the right. Undigested probe is not shown given its large size.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A series of constructs was generated to determine the regions of the β4 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 β4 subunit endogenously (23Lukas R. Norman S. Lucero L. Mol. Cell Neurosci. 1993; 4: 1-12Crossref PubMed Scopus (156) Google Scholar) as well as in mouse muscle C2C12 cells, that express the muscular-type nAChR (24Yu L. LaPolla R.J. Davidson N. Nucleic Acids Res. 1986; 14: 3539-3555Crossref PubMed Scopus (47) Google Scholar, 25Gardner P.D. Heinemann S. Patrick J. Brain Res. 1987; 427: 69-76PubMed Google Scholar). In both cell lines, the construct containing 74 bp of β4 promoter sequence (considering the initiation site labeled as +1 in Fig. 6 as reference for numbering) plus 97 bp of 5′-noncoding region (p74hβ4LUC) 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 p74hβ4LUC was 29 and 35% of the activity shown by pGL2Control in C2C12 and SHSY-5Y cells, respectively. When larger constructs were used (p255 and 960hβ4LUC) the relative luciferase activity decreased up to 60–70% of the activity observed with p74hβ4LUC. The activity of p74hβ4LUC in SHSY-5Y and C2C12 cells was about 90% reduced when 46 bp of the β4 promoter 5′-end were deleted further (p28hβ4LUC) and was barely detectable upon the additional deletion of 37 bp (p+9hβ4LUC). These results suggest that elements located between 74 and 28 bp of the transcription initiation site are essential for transcription. A search for transcription factors which could interact with elements at the proximal promoter region of the β4 subunit revealed the existence of two GC-boxes (labeled 1and 2 in Fig. 9 A) and an inverted CCAAT box with one mismatch in the core motif (labeled 3 in Fig. 9 A). 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 p74hβ4LUC (Fig. 9 B). Mutation of GC-box 1 had virtually no effect on functional activity relative to p74hβ4LUC. However, mutation of GC-box 2 resulted in 75, 84, and 83% decrease in transcript
Referência(s)