Transcriptional Regulation of Neuronal Nicotinic Acetylcholine Receptor Genes

Abstract

To date, 11 members (␣2-␣9 and ␤2-␤4) of the neuronal nicotinic acetylcholine receptor gene family have been identified. These genes encode subunits that form distinct receptors with different pharmacological and physiological profiles in temporally and spatially restricted patterns within the nervous system. Distinct molecular mechanisms probably orchestrate the expression of various receptor subtypes, yet little is known of specific transcriptional regulatory elements and their associated factors that are responsible for this segregated pattern of expression. Here we report the identification of an element, in the 5-flanking region of the rat ␤4 subunit gene, containing a CA box that is necessary for ␤4 promoter activity in a transiently transfected cholinergic cell line, SN17. This element was shown to interact with a protein(s) in SN17 nuclear extracts that is antigenically related to the transcriptional activator Sp1. Furthermore, co-transfection experiments confirmed that Sp1 can transactivate a ␤4 promoter-reporter gene construct, indicating that Sp1 is necessary, at least in part, for transcriptional activation of the ␤4 subunit gene. Neuronal nicotinic acetylcholine (nACh)1 receptors belong to a large family of related neurotransmitter receptors that are expressed within the central and peripheral nervous systems (CNS and PNS). Little is known of their function within the CNS, although a recent report suggests that presynaptic nACh receptors may modify fast excitatory transmission in the CNS (1). Additionally, the loss of cholinergic neurons in pathological states involving alterations in cognition and memory (e.g. Alzheimer's disease) implicates nACh receptors in these processes (2). In support of this premise, transgenic mice lacking the nACh receptor ␤2 subunit performed poorly in passive avoidance testing, suggesting a defect in associative memory (3). Further underscoring the importance of nACh receptors within the CNS is a recent report describing the development of autosomal dominant, nocturnal, frontal lobe epilepsy in humans, resulting from a missense mutation in the ␣4 subunit gene (4). It is clear that a significant amount of work remains to be done in order to fully understand the function of nACh receptors, including the identification and characterization of individual subunit genes.Eleven members of the nACh receptor gene family have been identified to date, and they include ␣2-␣9 and ␤2-␤4 (5-20). These subunits have been demonstrated in either Xenopus oocytes or in vivo to form a variety of distinct heteromeric (␣2, ␣3, and ␣4 with either ␤2 or ␤4) and homomeric (␣7, ␣8, and ␣9) receptors with different pharmacological and physiological profiles (21-23). This functional heterogeneity likely results from incorporation of different ␣ and ␤ subunits into mature receptors (19,24). Further complicating the characterization of nACh receptors is the observation that individual nACh receptor subunit gene expression occurs in distinct yet overlapping temporally and spatially restricted patterns ...

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

“…This characteristic is also found in the rat ␤4 promoter (14) as well as in the promoters of the ␣2 (29), ␣3 (30, 31), ␣5 (7,17), and ␣7 (26, 32-34) subunits. From 5Ј-end deletion analysis (Fig.…”

“…In any case, the dominant role of the Sp1 element in the human promoter was further demonstrated by the fact that the double mutant of the Sp1 and NF-Y elements showed very low promoter activity in C2C12 and COS cells and, therefore, the activating effect of the NF-Y mutation was overcome by the detrimental effect of the Sp1 mutation. This predominant role of Sp1 has also been demonstrated for the rat promoter, since the alteration of the Sp1 element (CA box) produced a 90% decrease in promoter activity (14). In this case, additional factors appear to modulate promoter activity, such as the transcription factors Pur␣ (27), Sox10 (45,46), and the heterogeneous nuclear ribonucleoprotein K (28), the latter repressing Sp1 function totally.…”

“…In our laboratory we have previously isolated and characterized the bovine ␣7 (8, 26) and ␣5 (7) subunits promoters. 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).…”

“…Gene-Comparison of the ␤4 bovine promoter to its rat counterpart previously published (14,15) 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 ␤4 promoter.…”

“…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 (14,15), 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 ␣3 (16) and bovine (7) and human (17) ␣5 subunits, suggesting that this transcription factor plays a fundamental role in the expression of several nAChRs subunit genes.…”