Shared long-range regulatory elements coordinate expression of a gene cluster encoding nicotinic receptor heteromeric subtypes

The nicotinic acetylcholine receptor (nAChR) beta 4/alpha 3/alpha 5 gene cluster encodes several heteromeric transmitter receptor subtypes that are essential for cholinergic synaptic transmission in adrenal gland, autonomic ganglia, pineal gland, and several nuclei in the central nervous system. However, the transcriptional mechanisms coordinating expression of these subunit genes in different cell populations are unknown. Here, we used transgenic methods to investigate long-range transcriptional control of the cluster. A 132-kb P1-derived artificial chromosome (PAC) encoding the rat cluster recapitulated the neurally- and endocrine-restricted expression patterns of the endogenous beta 4/alpha 3/alpha 5 genes. Mutation of ETS factor binding sites in an enhancer, beta 43', embedded in the beta 4 3'-untranslated exon resulted in greatly diminished beta 4, alpha 3, and alpha 5 expression in adrenal gland and to a lesser extent in the superior cervical ganglion (SCG) but not in other tissues. Phylogenetic sequence analyses revealed several conserved noncoding regions (CNRs) upstream of beta 4 and alpha 5. Deletion of one of them (CNR4) located 20 kb upstream of beta 4 resulted in a dramatic decrease in beta 4 and alpha 3 expression in the pineal gland and SCG. CNR4 was sufficient to direct LacZ transgene expression to SCG neurons, which express the endogenous beta 4 alpha 5 subunits, and pineal cells, which express the endogenous beta 4 alpha 3 combination. Finally, CNR4 was able to direct transgene expression to major sites of expression of the endogenous cluster in the brain. Together, our findings support a model in which cell type-specific shared long-range regulatory elements are required for coordinate expression of clustered nAChR genes.