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• W2034703630 abstract "PHOX2A is a paired-like homeodomain transcription factor that participates in specifying the autonomic nervous system. It is also involved in the transcriptional control of the noradrenergic neurotransmitter phenotype as it regulates the gene expression of tyrosine hydroxylase and dopamine-β-hydroxylase. The results of this study show that the human orthologue of PHOX2A is also capable of regulating the transcription of the human α3 nicotinic acetylcholine receptor gene, which encodes the ligand-binding subunit of the ganglionic type nicotinic receptor. In particular, we demonstrated by chromatin immunoprecipitation and DNA pulldown assays that PHOX2A assembles on the SacI-NcoI region of α3 promoter and, by co-transfection experiments, that it exerts its transcriptional effects by acting through the 60-bp minimal promoter. PHOX2A does not seem to bind to DNA directly, and its DNA binding domain seems to be partially dispensable for the regulation of α3 gene transcription. However, as suggested by the findings of our co-immunoprecipitation assays, it may establish direct or indirect protein-protein interactions with Sp1, thus regulating the expression of α3 through a DNA-independent mechanism. As the α3 subunit is expressed in every terminally differentiated ganglionic cell, this is the first example of a “pan-autonomic” gene whose expression is regulated by PHOX2 proteins. PHOX2A is a paired-like homeodomain transcription factor that participates in specifying the autonomic nervous system. It is also involved in the transcriptional control of the noradrenergic neurotransmitter phenotype as it regulates the gene expression of tyrosine hydroxylase and dopamine-β-hydroxylase. The results of this study show that the human orthologue of PHOX2A is also capable of regulating the transcription of the human α3 nicotinic acetylcholine receptor gene, which encodes the ligand-binding subunit of the ganglionic type nicotinic receptor. In particular, we demonstrated by chromatin immunoprecipitation and DNA pulldown assays that PHOX2A assembles on the SacI-NcoI region of α3 promoter and, by co-transfection experiments, that it exerts its transcriptional effects by acting through the 60-bp minimal promoter. PHOX2A does not seem to bind to DNA directly, and its DNA binding domain seems to be partially dispensable for the regulation of α3 gene transcription. However, as suggested by the findings of our co-immunoprecipitation assays, it may establish direct or indirect protein-protein interactions with Sp1, thus regulating the expression of α3 through a DNA-independent mechanism. As the α3 subunit is expressed in every terminally differentiated ganglionic cell, this is the first example of a “pan-autonomic” gene whose expression is regulated by PHOX2 proteins. The high degree of cellular heterogeneity of the mammalian nervous system is because of distinct specification and differentiation processes that mainly rely on transcriptional control mechanisms mediated by transcription factors having discrete temporal patterns of region-specific and cell type-specific expression. PHOX2A and PHOX2B are paired-like homeodomain proteins that have been shown to play a pivotal role in the development of the three peripheral divisions of the autonomic nervous system (1Howard M.J. Dev. Biol. 2005; 277: 271-286Crossref PubMed Scopus (124) Google Scholar). They are also expressed in all of the noradrenergic neurons of the brainstem, in some cranial sensory ganglia that participate in autonomic reflexes, and in a subset of cranial motor neurons (2Brunet J.F. Pattyn A. Curr. Opin. Genet. Dev. 2002; 12: 435-440Crossref PubMed Scopus (189) Google Scholar). None of the components of the autonomic nervous system develop properly in Phox2b knock-out mice (3Pattyn A. Morin X. Cremer H. Goridis C. Brunet J. Nature. 1999; 399: 366-370Crossref PubMed Scopus (669) Google Scholar), whereas Phox2a null mutants show an apparently less severe phenotype that only involves the agenesis of the Locus coeruleus and atrophy of the cranial sensory ganglia (4Morin X. Cremer H. Hirsch M. Kapur R.P. Goridis C. Brunet J. Neuron. 1997; 18: 411-423Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar); nevertheless, they do not feed and die on the day of birth. The different phenotypes of Phox2 knock-out mutants along with their asynchronous onset of expression during development underscore that the two factors are not functionally equivalent. This has been more directly demonstrated by reciprocal gene replacement experiments (5Coppola E. Pattyn A. Guthrie C.S. Goridis C. Studer M. EMBO J. 2005; 24: 4392-4403Crossref PubMed Scopus (68) Google Scholar) that led to the conclusion that biochemical differences may be responsible for the specific function of each paralogue. It is worth noting that PHOX2 proteins are also involved in the transcriptional control of the neurotransmitter phenotype (6Goridis C. Rohrer H. Nat. Rev. Neurosci. 2002; 3: 531-541Crossref PubMed Scopus (285) Google Scholar); they play a fundamental role in the terminal differentiation of the orthosympathetic system as they regulate the gene expression of tyrosine hydroxylase and dopamine-β-hydroxylase (DBH), 4The abbreviations used are: DBH, dopamine-β-hydroxylase; EMSA, electromobility gel shift assay; ChIP, chromatin immunoprecipitation; DAPI, 4,6-diamino-2-phenylindole; nAChR, nicotinic acetylcholine receptor; TK, thymidine kinase. 4The abbreviations used are: DBH, dopamine-β-hydroxylase; EMSA, electromobility gel shift assay; ChIP, chromatin immunoprecipitation; DAPI, 4,6-diamino-2-phenylindole; nAChR, nicotinic acetylcholine receptor; TK, thymidine kinase. two limiting enzymes in catecholamine synthesis. In particular, it has been shown that PHOX2A can synergize with dHAND in the regulation of the DBH promoter (7Xu H. Firulli A.B. Zhang X. Howard M.J. Dev. Biol. 2003; 262: 183-193Crossref PubMed Scopus (53) Google Scholar, 8Rychlik J.L. Gerbasi V. Lewis E.J. J. Biol. Chem. 2003; 278: 49652-49660Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Because of their function in the development of the entire autonomic nervous system, it might be expected that PHOX2 proteins also control the expression of specific genes of the parasympathetic and enteric branches or pan-autonomic genes common to all three autonomic divisions. As no information is yet available concerning this fundamental issue, we tested this hypothesis using the human α3 nicotinic receptor subunit gene as a possible target of PHOX2A regulation. All post-ganglionic neurons respond to preganglionic stimulation by a fast excitatory post-synaptic potential, which triggers the initiation of the post-synaptic spike. The fast excitatory post-synaptic potential is because of the activation of neuronal nicotinic acetylcholine receptors (nAChRs) whose pharmacological blockade abolishes all autonomic nervous system activity (9Taylor P. Brunton L.L. Lazo J.S. Parker K.L. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. 11th Ed. McGraw-Hill Inc., New York2006: 217-236Google Scholar). Neuronal nAChRs form a family of acetylcholine-gated cation channels that are expressed in the autonomic and sensory ganglia, the adrenal medulla, and distinct areas of the central nervous system and have a quaternary structure consisting of five transmembrane subunits assembled around a central channel. Twelve distinct neuronal nicotinic subunits have been cloned so far and classified into two subfamilies of nine α subunits (α2–α10) and three β subunits (β2–β4) (10Gotti C. Clementi F. Prog. Neurobiol. 2004; 6: 363-396Crossref Scopus (784) Google Scholar). The α3 subunit appears precociously during neural crest cell differentiation (11Howard M.J. Gershon M.D. Margiotta J.F. Dev. Biol. 1995; 170: 479-495Crossref PubMed Scopus (20) Google Scholar), and its expression becomes very abundant in all of the ganglionic neurons of the autonomic nervous system, where it assembles with β4 or β2 (and, in some receptor molecules, also with α5) to form the ganglionic type nAChR. Many knock-out mice lacking the α3 subunit succumb early after birth, and most of the remaining die during the next 6–8 weeks because of the severe abnormalities of autonomic functions (12Wang N. Orr-Urtreger A. Korczyn A.D. Prog. Neurobiol. 2002; 68: 341-360Crossref PubMed Scopus (66) Google Scholar). Thus, the α3 subunit is a very typical pan-autonomic gene responsible for relevant functions of terminally differentiated ganglionic neurons. The aim of this study was to investigate whether PHOX2A regulates the expression of the human α3 nAChR subunit gene, and the possible molecular mechanisms underlying this process. Cell Lines and Cultures—The SH-SY5Y and IMR32 human neuroblastoma cell lines and the DAOY human medulloblastoma cell line were grown in RPMI 1640 medium (CAMBREX Bio Science, Inc., Rockland, ME), 10% fetal calf serum (Euroclone Life Science Division, Milan, Italy), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine (Sigma). The HeLa cell line was grown in Dulbecco’s modified Eagle’s medium (Sigma), 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mml-glutamine, and 10 mm sodium pyruvate (Sigma). Plasmid Construction—The 4.3 KN, the 1 KN, and the BglII-NcoI human α3 promoter reporter plasmids have been described previously by Fornasari et al. (13Fornasari D. Battaglioli E. Flora A. Terzano S. Clementi F. Mol. Pharmacol. 1997; 51: 250-261Crossref PubMed Scopus (46) Google Scholar). The BsiWI-AccIII (60) α3 minimal promoter construct as well as the plasmids, BglII-NcoI mutA, BglII-NcoI mutB, and BglII-NcoI mutAB bearing mutations either in the A or B or both Sp1-binding sites have been described by Terzano et al. (14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The SacI-NcoI construct was obtained by digesting 0.35 BN plasmid (13Fornasari D. Battaglioli E. Flora A. Terzano S. Clementi F. Mol. Pharmacol. 1997; 51: 250-261Crossref PubMed Scopus (46) Google Scholar) with SacI, followed by re-ligation. The α5 –240/+180 construct has been described by Flora et al. (15Flora A. Schulz R. Benfante R. Battaglioli E. Terzano S. Clementi F. Fornasari D. J. Neurochem. 2000; 75: 18-27Crossref PubMed Scopus (41) Google Scholar). The human PHOX2A cDNA was cloned by means of reverse transcription-PCR, using RNA purified from SH-SY5Y cells; polymerase Superscript II (Invitrogen) was used for reverse transcription, and Pfx proofreading polymerase (Invitrogen) was used for amplification. The oligonucleotides were designed on the basis of the sequence deposited at the NCBI with accession number NM_005169; the upper primer corresponded to nucleotides 1–25 (5′-CTT GCG TTG CAC CCG GGC TGA GTG C-3′); the lower primer was complementary to nucleotides 997–1027 (5′-GAT TGG TCT TCA GGG CGG GGC CGG GCT TC-3′). The PCR product was subcloned in the pGEM-T Easy vector (Promega), and completely sequenced on both strands. The cDNA was excised by means of EcoRI digestion and cloned into pcDNA3.0 vector (Invitrogen), and the construct was named PHOX2A/pcDNA3. Recombinant PCR was used to introduce point mutations into the homeodomain of PHOX2A (16Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2080) Google Scholar). The oligonucleotides used to generate the mutations were Ph2aR53W-UP, 5′-CCA GAA CCG CTG GGC CAA GTT CC-3′; Ph2aR53W-LOW, 5′-GGA ACT TGG CCC AGC GGT TCT GG-3′; Ph2aN51H-UP, 5′-TTC CAG CAC CGC CGG GCC AAG-3′; Ph2aN51H-LOW, 5′-CCC GGC GGT GCT GGA ACC AG-3′; the external primers were N-ATG-UP, 5′-GGG CCG ATG GAC TAC TCC TAC C-3′ and Ph2aCT-LOW, 5′-TTG CGG CCG CCT AGA AGA GAT TGG TCT TCA GGG C-3′ (the changed codons are in boldface and underlined). The resulting DNA was subcloned into the pGEM-T easy vector (Promega) and sequenced on both strands. The cDNA was excised by means of EcoRI digestion and cloned into pcDNA3.0 vector (Invitrogen), and the constructs were named PHOX2A-R53W/pcDNA3 and PHOX2A-N51H/pcDNA3. The 4xTK-luc construct was obtained by cloning into the pGL3-basic vector a synthetic double-stranded oligonucleotide (see Fig. 6A for the sequence) containing four copies (three in the right orientation and one in the opposite orientation) of domain II of the DBH promoter, bearing a well described PHOX2A-binding site (17Kim H.S. Seo H. Yang C. Brunet J.F. Kim K.S. J. Neurosci. 1998; 18: 8247-8260Crossref PubMed Google Scholar) upstream of the TK promoter. Transfections and Luciferase Assays—The transfection and co-transfection experiments were performed by means of lipofection, as described by Flora et al. (18Flora A. Lucchetti H. Benfante R. Goridis C. Clementi F. Fornasari D. J. Neurosci. 2001; 21: 7037-7045Crossref PubMed Google Scholar), using 4 × 104 HeLa cells, 5 × 104 DAOY cells, or 2 × 105 IMR32 cells. The luciferase assays were carried out using the dual luciferase reporter assay system (Promega, Madison, WI) as described previously (18Flora A. Lucchetti H. Benfante R. Goridis C. Clementi F. Fornasari D. J. Neurosci. 2001; 21: 7037-7045Crossref PubMed Google Scholar, 19Battaglioli E. Gotti C. Terzano S. Flora A. Clementi F. Fornasari D. J. Neurochem. 1998; 71: 1261-1270Crossref PubMed Scopus (44) Google Scholar). All of the transfections were performed in duplicate, and each construct was tested in at least three independent experiments using different batches of the plasmid preparation. Preparation of Synthetic Peptide and Antibody Production—A peptide corresponding to the human PHOX2A sequence, CKPGPALKTNLF, located in the C terminus of the protein was synthesized as described in Cargnin et al. (20Cargnin F. Flora A. Di Lascio S. Battaglioli E. Longhi R. Clementi F. Fornasari D. J. Biol. Chem. 2005; 280: 37439-37448Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The affinity-purified polyclonal antibodies from chicken egg yolk were produced by Davids Biotechnologie (Ragensburg, Germany). In Vitro Protein Expression—The in vitro expression of PHOX2A-wild type and PHOX2A-R53W was obtained by means of a commercial rabbit reticulocyte lysate system (TnT quick-coupled transcription/translation system, Promega, Madison, WI) as described previously (20Cargnin F. Flora A. Di Lascio S. Battaglioli E. Longhi R. Clementi F. Fornasari D. J. Biol. Chem. 2005; 280: 37439-37448Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Protein Preparation and Western Blot Analyses—Ten micrograms of nuclear extract, prepared as described in Terzano et al. (14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), or 2 μl of in vitro expressed proteins were separated by means of 10% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell). The membranes were preincubated for 1 h with blocking buffer (5% nonfat dry milk, 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Tween 20), after which the primary antibodies were added at an appropriate dilution and incubated for 2 h before the secondary antibodies conjugated with horseradish peroxidase (Davids Biotechnologie) were added and incubated for 1 h. After appropriate washes, the bands were revealed using Super Signal West Dura (Pierce). Standard molecular weights (New England Biolabs, Inc., Beverly, MA) were loaded in parallel, and the relative mass of PHOX2A was calculated by means of computer-assisted analysis. Competition experiments were carried out using PHOX2A peptide in a 10:1 mass ratio with the corresponding antibodies. Immunofluorescence—DAOY cells plated on 1.7 × 1.7-cm2 glass coverslips were grown to 50% confluency and transfected with PHOX2A/pcDNA3, PHOX2A-R53W/pcDNA3, and PHOX2A-N51H/pcDNA3 plasmids or mock-transfected with the empty vector. Immunofluorescence was performed as described in Cargnin et al. (20Cargnin F. Flora A. Di Lascio S. Battaglioli E. Longhi R. Clementi F. Fornasari D. J. Biol. Chem. 2005; 280: 37439-37448Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The primary antibody was used at a dilution of 1:400 and revealed by means of an anti-chicken Texas Red-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). Competition assays using the specific peptide were performed as described above. Electrophoretic Mobility Shift Assays (EMSAs)—The EMSAs were performed as described by Terzano et al. (14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and Cargnin et al. (20Cargnin F. Flora A. Di Lascio S. Battaglioli E. Longhi R. Clementi F. Fornasari D. J. Biol. Chem. 2005; 280: 37439-37448Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The oligonucleotide PRS1 has been described by Kim et al. (17Kim H.S. Seo H. Yang C. Brunet J.F. Kim K.S. J. Neurosci. 1998; 18: 8247-8260Crossref PubMed Google Scholar). All of the oligonucleotides were purchased from Invitrogen. Chromatin Immunoprecipitation Assays (ChIP)—Chromatin immunoprecipitation was carried out as described previously (20Cargnin F. Flora A. Di Lascio S. Battaglioli E. Longhi R. Clementi F. Fornasari D. J. Biol. Chem. 2005; 280: 37439-37448Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Chromatin was incubated overnight at 4 °C with 5 μgof each antibody (anti-PHOX2A (Davids Biotechnologie, Germany), anti-human Sp1, and anti-acetylated histone H4 (Upstate, Charlottesville, VA), and chicken preimmune IgY (Davids Biotechnologie)), and the immunocomplexes were collected on monoclonal anti-chicken IgY-agarose beads or protein G/agarose bead slurry (Invitrogen), preadsorbed with 20 μg/μl tRNA and 10 μg/μl salmon sperm DNA (Sigma). After washings and elution, the cross-linking was reversed by heating to 65 °C overnight, and the samples were purified on columns (High Pure PCR product purification kit, Roche Diagnostics). For the PCR detection of the immunoprecipitated chromatin, 5% of the purified DNA was used as a template with the following primers: ChIP[α3prom]UP, 5′-CTC CTT CCT GGT GGT GGT GAC-3′, and ChIP[α3prom]LOW, 5′-GGG CTC CTC TCC GCT TGC-3′ to amplify the –386/–127 region of the nAChR α3 subunit promoter (numbering according to Ref. 14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar); and ChIP[α5prom]UP, 5′-CTC TGC TCC AGG GTC GCA C -3′, and ChIP[α5prom]LOW, 5′-GAG TGT GAG TCG TGA GAC AAA ACG-3′, to amplify the α5 promoter. The DNA samples were heated to 95 °C for 2 min, followed by 47 cycles of heating at 95 °C for 30 s, annealing at 64 (α3 promoter) or 61 °C (α5 promoter) for 30 s, and extension at 72 °C for 30 s. Transient chromatin immunoprecipitation assays were carried out as described in Wells and Farnham (21Wells J. Farnham P.J. Methods (San Diego). 2002; 26: 48-56Crossref PubMed Scopus (207) Google Scholar). Briefly, IMR32 cells were transfected with 5 μg of different α3 promoter plasmids (BglII-NcoI, BglII-NcoI mutA, BglII-NcoI mutB, or BglII-NcoI mutAB constructs). After 24 h, chromatin was prepared for immunoprecipitation as described above. For the PCR detection of the immunoprecipitated chromatin, specific primers designed on plasmid backbone sequences were used as follows: GL2 primer, 5′-CTT TAT GTT TTT GGC GTC TTC C-3′, and RV3 primer, 5′-CTA GCA AAA TAG GCT GTC CC-3′. The DNA samples were heated to 95 °C for 3 min, followed by 38 cycles of heating at 95 °C for 45 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. DNA Pulldown Assays—Five picomoles of the SacI-NcoI or –240/+180 DNA fragments of the α3 and α5 regulatory regions were biotinylated by filling in and incubated with streptavidin-coated magnetic beads (Roche Diagnostics); 250 μg of IMR32 nuclear extracts were added and incubated on ice for 40 min in binding buffer (4% Ficoll, 20 mm Hepes, pH 7.9, 1 mm MgCl2, 0.5 mm dithiothreitol), 100 mm final salt concentration (NaCl + KCl), and 25 μg of poly[d(I-C)] as the competitor. After magnetic separation, the beads were extensively washed with a binding buffer containing 25% glycerol instead of Ficoll and resuspended in protein loading buffer. The samples were analyzed by Western blotting as described previously. In Vitro DNase I Footprinting Assays—The 376-bp probe, containing the SacI-NcoI region of the α3 promoter, was obtained as described in Terzano et al. (14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). To generate the probe corresponding to the multimerized domain II of the DBH promoter, we subcloned the double-stranded oligonucleotide shown in Fig. 6A into the KpnI-BglII sites of pSP73 vector (Promega). The plasmid was then digested with HindIII to label the bottom strand. In each footprinting reaction, 2 fmol of probes (corresponding to 20,000–30,000 cpm) were incubated with 50 μgof nuclear extract, prepared as described previously (14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), and the reactions were performed using a previously described procedure (14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). DNase I (DNase I-RNase free; Roche Diagnostics) was used at concentrations of 0.05 units/μg DNA without extract and 1.0 unit/μg DNA in the presence of extract, and the samples were processed as described previously (14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Co-immunoprecipitation Assays—IMR32 cells were grown to 70% confluency and harvested after 24 h, when the nuclear extract was prepared for immunoprecipitation. The nuclei were obtained as described previously (14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Nuclear proteins were extracted in lysis buffer containing non-ionic detergent (0.5% Triton X-100, 50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.5 mm EDTA, 10 mm imidazole, 10% glycerol, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, Sigma protease inhibitors mixture) and precleared using protein G/agarose bead slurry (Invitrogen) and rabbit preimmune IgG or chicken preimmune IgY (Santa Cruz Biotechnology). The precleared extracts were incubated overnight at 4 °C with 5 μg of each primary antibody (polyclonal chicken anti-PHOX2A antibody (Davids Biotechnologie), polyclonal rabbit anti-Sp1 antibody (Santa Cruz Biotechnology), and preimmune rabbit IgG or preimmune chicken IgY (Santa Cruz Biotechnology)), and the immunocomplexes were captured by protein G/agarose bead slurry (Invitrogen). Because of the poor binding of chicken antibodies to protein G, a bridging antibody (rabbit anti-chicken IgG, Upstate) was added to enhance immunocomplexes capture. The beads were collected by centrifugation and gently washed and resuspended in sample loading buffer. The immunocomplexes were dissociated from the beads by boiling the samples, then separated by SDS-PAGE, and transferred on nitrocellulose membrane. Western blotting was performed as described previously using the primary anti-PHOX2A, anti-Sp1, and anti-CREB-1 antibodies (Santa Cruz Biotechnology). Transcriptional Effects of PHOX2A on the Activity of the α3 Promoter—To evaluate whether PHOX2A plays a role in α3 expression, we performed co-transfection experiments in which a vector expressing PHOX2A was introduced into DAOY and HeLa cells (not shown), together with different reporter constructs bearing the luciferase reporter gene under the control of different parts of the α35′-regulatory region or different promoters used as positive and negative controls (Fig. 1A). The 4.3 KN construct containing the whole intergenic region between β4 and α3 (13Fornasari D. Battaglioli E. Flora A. Terzano S. Clementi F. Mol. Pharmacol. 1997; 51: 250-261Crossref PubMed Scopus (46) Google Scholar, 14Terzano S. Flora A. Clementi F. Fornasari D. J. Biol. Chem. 2000; 275: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 22Raimondi E. Rubboli F. Moralli D. Chini B. Fornasari D. Tarroni P. De Carli L. Clementi F. Genomics. 1992; 12: 849-850Crossref PubMed Scopus (37) Google Scholar) was stimulated by ∼3-fold (Fig. 1B), to the same extent as the 4xTK plasmid (Fig. 1B), and in a range of transactivation that was comparable with that observed using the native DBH promoter (23Swanson D.J. Adachi M. Lewis E.J. J. Biol. Chem. 2000; 275: 2911-2923Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Progressive deletions at the 5′ end of the α3 regulatory region caused fluctuations in the response to PHOX2A (Fig. 1B, see constructs 1 KN and SacI-NcoI) but did not prevent the transcription factor from exerting its regulatory functions. Furthermore and to our surprise, the BsiWI-AccIII construct containing the 60-bp α3 minimal promoter was the most responsive to PHOX2A (Fig. 1B). As expected, the SV40 promoter was not transactivated by PHOX2A, thus indicating the specific nature of the transcriptional effects on the α3 promoter (Fig. 1B), and the α5 promoter construct (corresponding to the –1011/+180 plasmid described in Ref. 15Flora A. Schulz R. Benfante R. Battaglioli E. Terzano S. Clementi F. Fornasari D. J. Neurochem. 2000; 75: 18-27Crossref PubMed Scopus (41) Google Scholar) showed a negligible and not statistically significant transcriptional response to PHOX2A (Fig. 1B). This lack of effect paralleled our previous finding that PHOX2B is unable to transactivate this nAChR subunit gene (18Flora A. Lucchetti H. Benfante R. Goridis C. Clementi F. Fornasari D. J. Neurosci. 2001; 21: 7037-7045Crossref PubMed Google Scholar). As these data suggested that PHOX2A could regulate α3 transcription by acting through its minimal gene promoter, we decided to carry out biochemical experiments to ascertain whether PHOX2A could actually assemble on the α3 promoter. Characterization of a Polyclonal Antibody Directed against the Human PHOX2A Protein—To follow the expression of the human PHOX2A protein, we produced an anti-peptide polyclonal antibody in chicken eggs and then verified its specificity by means of various criteria and techniques. We first made a Western blot analysis using nuclear extracts prepared from IMR32 or HeLa cells in parallel with the PHOX2A protein obtained by means of in vitro transcription/translation. The in vitro expressed PHOX2A protein was recognized by the antibody as a doublet with apparent molecular masses of 38.1 and 35.7 kDa, whereas no signal was detectable using the unprogrammed lysate (Fig. 2A, lanes 1 and 3). The same doublet was also detected in the IMR32 and SH-SY5Y nuclear extracts (Fig. 2, A, lane 2, and B, lanes 1 and 2), whereas no band was observed in the HeLa cells, as expected (Fig. 2, A, lane 4, and B, lane 3). To corroborate these data, we carried out competition experiments involving the addition of an excess of the PHOX2A peptide used for immunization. As shown in Fig. 2A, the specific PHOX2A bands disappeared from the lanes containing the IMR32 nuclear extracts or the in vitro expressed PHOX2A protein (lanes 5 and 6). The antibody was also tested against nuclear extracts prepared from HeLa cells transfected with an expression vector bearing mouse (Fig. 2B, lane 5) or human PHOX2A cDNA (Fig. 2B, lane 6); in both cases, the antibody recognized two bands with apparent molecular masses of 35.7 and 38.1 kDa, whereas no bands were detectable in the mock-transfected HeLa cells (Fig. 2B, lane 4). The difference in the apparent molecular weights of the two forms of PHOX2A is compatible with the alternative use of two in-frame methionine residues surrounded by perfect Kozak sequences (24Kozak M. Annu. Rev. Cell Biol. 1992; 8: 197-225Crossref PubMed Scopus (415) Google Scholar), which are conserved in both human and mouse orthologues (18Flora A. Lucchetti H. Benfante R. Goridis C. Clementi F. Fornasari D. J. Neurosci. 2001; 21: 7037-7045Crossref PubMed Google Scholar, 25Valarche I. Tissier-Seta J.P. Hirsch M.R. Martinez S. Goridis C. Brunet J.F. Development (Camb.). 1993; 119: 881-896Crossref PubMed Google Scholar). Immunofluorescence was used to confirm that the examined antibody recognized PHOX2A in its native state. DAOY cells (which do not endogenously express the antigen) were transfected with PHOX2A/pcDNA3 and probed with the antibody. Fig. 2C shows the nuclei of transfected DAOY cells stained with DAPI (panel c) or revealed by the anti-PHOX2A antibody (panel d). The antibody labeled the nuclei of a few cells, as may be expected in a transient transfection experiment, but did not produce any signal when mock-transfected cells were probed (Fig. 2C, panels a and b). Similarly, no signal was detectable in the PHOX2A/pcDNA3-transfected cells if the antibody was previously incubated with an excess of the PHOX2A peptide (Fig. 2C, panels e and f). It is well known that DBH is a PHOX2A target gene, and so we used the PRS1 oligonucleotide to verify whether the examined antibody was capable of supershifting complexes containing PHOX2A. PRS1 corresponds to a region of 22 nucleotides within domain IV of the DBH promoter (26Yang C. Kim H. Seo H. Kim C. Brunet J. Kim K. J. Neurochem. 1998; 71: 1813-1826Crossref PubMed Scopus (118) Google Scholar), contains two ATTA core motifs for homeodomain proteins, and has been shown to bind PHOX2A (17Kim H.S. Seo H. Yang C. Brunet J.F. Kim K.S. J. Neurosci. 1998; 18: 824" @default.
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