Abstract
Expression of neurotransmitter receptors encoded by the nicotinic acetylcholine receptor (nAchR) subunit gene cluster depends on coexpression of the beta4, alpha3, and alpha5 subunits in certain kinds of neurons. One way in which coexpression might be achieved is through the regulation of promoters in the cluster by neuron-selective enhancers. The beta43' enhancer is located between the beta4 and alpha3 promoters and it directs cell type-specific expression in cell lines. It is not known, however, whether beta43' is active in neurons. Therefore, we assayed beta43' in dissociated rat sympathetic ganglia cultures, which contain nAchR-positive neurons as well as nAchR-negative non-neuronal cells. Reporters controlled by the alpha3 promoter and beta43' were expressed in a neuron-selective manner; greater than 90% and up to 100% of luciferase expression was detected in neurons. Neuron selectivity was maintained when beta43' was placed next to ubiquitously active viral promoters. In contrast, replacing beta43' with the SV40 enhancer eliminated neuron selectivity. The enhancer is composed of at least two separate but functionally interdependent elements, each of which interacts with a different type of ETS domain factor. These findings support a model in which beta43' controls neuronal expression of one or more genes in the cluster through interactions with a combination of ETS factors.
Highlights
Likely to provide additional insight into the mechanisms underlying neuronal differentiation and development of neuronal diversity
Expression of neurotransmitter receptors encoded by the nicotinic acetylcholine receptor subunit gene cluster depends on coexpression of the 4, ␣3, and ␣5 subunits in certain kinds of neurons
Using adrenal chromaffin cell-derived PC12 cells, which express the clustered genes, we identified an enhancer positioned within the 4 3Ј-untranslated exon
Summary
Luciferase Reporters—Reporters for cell line transfections were prepared using pGL2 series vectors and those used for sympathetic ganglia culture transfections were prepared using pGL3 series vectors (Promega, Madison, WI). Enhancer sequences were placed upstream of either the SV40 or ␣3 promoters using synthetic oligonucleotides and convenient restriction sites or by polymerase chain reaction. The same enhancer fragment was cloned upstream of SV40P or AdMLP. The AdMLP construct contains sequences Ϫ55/ϩ10 of the adenovirus 2 major late promoter cloned upstream of the luciferase reporter. To prepare the SV40E/␣3P construct, an ␣3 promoter restriction fragment was cloned into pGL3c in place of the SV40p. Luciferase reporters containing four GAL4binding sites upstream of the ␣3 promoter (4XG-␣3P) or AdMLP (4XGAdMLP) were prepared from pGL2– 4XG [20] using convenient restriction sites. 43Ј reporters prepared using synthetic oligonucleotides were confirmed by dideoxy sequencing using Sequenase reagents according to the manufacturer’s protocol (Amersham Pharmacia Biotech). The CGS-ETS-2 expression construct, encoding ETS-2 amino acid residues 281– 445, was made by subcloning an XbaI/SmaI fragment of the ETS-2 cDNA into a cytomegalovirus promoter-containing plasmid [18] using standard methods
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