Abstract
The beta4 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 minus sign99 to minus sign64) 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 beta4 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 beta4 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 beta4 subunit gene in these three close species may be controlled by the general transcription factor Sp1. Nevertheless, other components could determine species-specific beta4 subunit expression.
Highlights
Cloning of nicotinic acetylcholine receptor1 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]
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, we suggest that both, Sp1 and NF-Y, are involved in the transcriptional regulation of the bovine 4 promoter
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]
Summary
Isolation and Analysis of the 5Ј-Flanking Sequence of the 4 Subunit—For the bovine promoter a cDNA probe corresponding to 218 bp at the beginning of the coding sequence and the contiguous 38 bp of 5Ј-untranslated region [10] was used to screen a genomic library. 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. Similar RNase protection experiments were carried out to find the 5Ј-end of human 4 mRNA In this case a 323-bp BamHI-SacI fragment whose 3Ј-end was at 30 bp from the initial ATG, was cloned into the pSPT19 vector to generate a 362-nucleotide probe which was labeled according to the instructions of the MaxiScript SP6 kit (Ambion). Deletion analysis of the most promoter-proximal region was performed by generating either appropriate restriction enzyme fragments or polymerase chain reaction fragments with suitable sense oligonucleotides and an antisense primer (5Ј-CTTTATGTTTTTGGCGTCTTCC-3Ј) that anneals to the pGL2-Basic vector, downstream of the site of transcription initiation. The equivalent anti-NF-Yb antibody (CBF-A, C-20) from Santa Cruz Biotechnology was used in later experiments with the human 4 promoter
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