Transcriptional Regulation by Activation and Repression Elements Located at the 5-Noncoding Region of the Human 9 Nicotinic Receptor Subunit Gene* Received for publication, July 2, 2003, and in revised form, July 14, 2003 Published, JBC Papers in Press, July 14, 2003, DOI 10.1074/jbc.M307043200 Luis M. Valor‡, Mar Castillo§, Jose ´ A. Ortiz¶, and Manuel Criado From the Department of Biochemistry and Molecular Biology and the Instituto de Neurociencias, Universidad Miguel Herna ´ndez-Consejo Superior de Investigaciones Cientı´ficas, 03550 San Juan, Alicante, Spain The 9 subunit is a component of the neuronal nico- tinic acetylcholine receptor gene superfamily that is ex- pressed in very restricted locations. The promoter of the human gene has been analyzed in the human neuroblas- toma SH-SY5Y, where 9 subunit expression was de- tected, and in C2C12 cells that do not express 9. A proximal promoter region (from 322 to 113) showed maximal transcriptional activity in SH-SY5Y cells, whereas its activity in C1C12 cells was much lower. Two elements unusually located at the 5-noncoding region exhibited opposite roles. A negative element located be- tween 15 and 48 appears to be cell-specific because it was effective in C2C12 but not in SH-SY5Y cells, where it was counterbalanced by the presence of the promoter region 5 to the initiation site. An activating element located between 66 and 79 and formed by two adja- cent Sox boxes increased the activity of the 9 promoter about 4-fold and was even able to activate other promot- ers. This element interacts with Sox proteins, probably through a cooperative mechanism in which the two Sox boxes are necessary. We propose that the Sox complex provides an initial scaffold that facilitates the recruiting of the transcriptional machinery responsible for 9 sub- unit expression. Neuronal nicotinic acetylcholine receptors (nAChRs) 1 are members of a supergene family of ion channels gated by neu- rotransmitters (1). They are pentameric oligomers composed of related subunits, which are commonly classified as agonist- binding (designated 2–10) and structural (2–4) subunits. Unlike some nAChR subunits that have a relatively broad expression, the 9 subunit has been found only in very re- stricted areas such as the pituitary pars tuberalis, the olfactory epithelium, and the cochlea (2, 3). This limited expression could be the consequence of tight mechanisms of transcriptional reg- ulation, which might be of great interest in understanding how the regional and developmental expression of neuronal nAChRs is controlled at the transcriptional level (see Ref. 4 for a review). For this reason, here we have analyzed the human 9 promoter, finding that two cis-elements unusually located at the 5'-noncoding region of 9 transcripts control in opposite ways the basal transcriptional activity of the 9 subunit gene. EXPERIMENTAL PROCEDURES Isolation and Analysis of the 5'-Flanking Sequence of the 9 Sub- unit—The human 9 coding sequence was obtained by PCR from a human pituitary cDNA library (Clontech, Heidelberg, Germany) by using the information contained in the GenBank TM sequence AJ243342. A fragment from the 5'-end was used to screen a human genomic library constructed in EMBL-3 SP6/T7 (Clontech) and tested as described previously (5). A bacteriophage clone was purified and characterized. It contained 4,600 bp of 5'-flanking region and at least the first exon. 5'-RACE Analysis of 5' mRNA Ends—The 5'-end of 9 mRNA was mapped by 5'-RACE, as primer extension and RNase protection meth- ods did not yield satisfactory results. For this purpose the Marathon Ready cDNA system from Clontech was applied to the previously men- tioned cDNA library from human pituitary as indicated by the manu- facturer. Two antisense oligonucleotides at the first and second exons (see Fig. 1) were used in parallel assays of DNA amplification. Their sequences were: from position +201 to +180, 5'-CTCAGTCTGGAAG- CAGCAAAG-3'; +602 to +578, 5'-CGTAATCTGCAGG-GTCACAT- TCAGG-3'. The resulting products were cloned and sequenced. RT-PCR Analysis—Poly (A) + RNA was directly selected from SH- SY5Y cell lysates by oligo(dT)-Dynabeads (Dynal, Oslo, Norway) accord- ing to the manufacturer’s instructions. 9 subunit transcripts, as well as 7 and 4 transcripts as positive controls, were detected by an RT-PCR assay (Access RT-PCR System from Promega, Madison, WI). Briefly, samples of mRNA (200 ng) or in vitro synthesized cRNA (30 ng) in a final volume of 20 l were reverse transcribed with 2 units of avian myeloblastosis virus (AMV) reverse transcriptase (45 min at 48 °C), and PCR was further performed for 30 cycles (30 s at 94 °C, 45 s at 60 °C, and 1 min at 68 °C) with 2 units of Tfl DNA polymerase. The amplified DNA fragments extended over at least 1 intron or more, to rule out the possibility of amplifying a potential contamination of genomic DNA. Anyway, RT-PCR performed in the absence of AMV reverse tran- scriptase or RNA did not yield any DNA fragment. A similar analysis was performed with mRNA from C2C12 cells and 35 PCR cycles, and 7 but not 9 transcripts were detected. Plasmid Constructions—All 9 promoter-luciferase gene fusions were made in the pGL2-Basic vector (Promega), introducing in its polylinker upstream of the luciferase gene the suitable 9 promoter fragments. These fragments were generated with restriction enzymes and cloned directly into pGL2-Basic or subcloned first in pBluescript and then transferred to pGL2-Basic. Deletion analysis of the most promoter-proximal region was performed by generating either appro- priate restriction enzyme fragments or PCR fragments with suitable sense oligonucleotides and an antisense primer (5'-CTTTATGTTTTT- * This work was supported by grants from the Ministries of Educa- tion (PM98-0104) and Science and Technology (BMC2002-00972) of Spain and from the Generalitat Valenciana (CTIDIB/2002/138). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” 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) AJ576315. ‡ Recipient of predoctoral fellowships from the Generalitat Valenci- ana and Consejo Superior de Investigaciones Cientı ´ficas-Bancaja. § Recipient of a fellowship from the Ministry of Science and Technol- ogy (MCyT) of Spain. ¶ Supported by a grant from the MCyT of Spain (‘‘Ramo ´n y Cajal’’ program).  To whom correspondence should be addressed. Tel.: 34-965919479; Fax: 34-965919484; E-mail: Manuel.Criado@umh.es. 1 The abbreviations used are: nAChR, nicotinic acetylcholine recep- tor; HMG, high mobility group; GST, glutathione S-transferase; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription PCR. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 39, Issue of September 26, pp. 37249 –37255, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. 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