Transcription Factors NF-Y and Sp1 Are Important Determinants of the Promoter Activity of the Bovine and Human Neuronal Nicotinic Receptor β4 Subunit Genes

Abstract

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. 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 ciselements governing basal transcriptional activity. Although their sequences differ from the one of the rat ␤4 promoter (14...

Transcriptional Repression by a Conserved Intronic Sequence in the Nicotinic Receptor α3 Subunit Gene

“…Several laboratories are actively pursuing this hypothesis, and substantial work has revealed a number of shared regulatory features between the two genes. First, by using in vitro approaches, it has been shown that the ␣3 and ␤4 promoters can directly interact with and be trans-activated by the widely expressed transcription factors Sp1 and Sp3 (56,59,62,65,70). This observation extends to the third gene in the cluster, the ␣5 subunit gene, which also appears to be regulated by Sp1 (73).…”

“…In this regard, it is important to note that a growing body of evidence suggests that the ␣3 and ␤4 subunit genes may be coordinately expressed, whereas the ␣5 gene may be independently regulated (47, 49 -51). A number of laboratories, including our own, have identified several cis-acting transcriptional regulatory elements (52-66) and trans-acting regulatory factors (59,65,(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77) that are involved in the expression of the clustered nACh receptor subunit genes. The majority of the regulatory elements thus far described are involved in activating transcription and are located upstream of the nACh receptor genes that they regulate.…”

Transcriptional Regulation by Activation and Repression Elements Located at the 5′-Noncoding Region of the Human α9 Nicotinic Receptor Subunit Gene

“…For this purpose we chose the bovine ␣7 and the human ␤4 promoters, which had been characterized previously by us (19,20) and have different transcriptional requirements. The Sox boxes were located between the luciferase reporter gene and the mentioned promoters, and the activity of the resultant constructs in C2C12 cells was compared with that shown by the corresponding parent constructs.…”

Shared Long-Range Regulatory Elements Coordinate Expression of a Gene Cluster Encoding Nicotinic Receptor Heteromeric Subtypes

“…The ␤4, ␣3, and ␣5 promoters are all able to direct expression of reporters in neural cell lines, and each can be transactivated by the ubiquitously expressed family of Sp transcription factors (5,6,9,10,54,61) and the neurally restricted…”