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

Abstract

The nicotinic acetylcholine receptor (nAChR) ␤4/␣3/␣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 ␤4/␣3/␣5 genes. Mutation of ETS factor binding sites in an enhancer, ␤43, embedded in the ␤4 3-untranslated exon resulted in greatly diminished ␤4, ␣3, and ␣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 ␤4 and ␣5. Deletion of one of them (CNR4) located 20 kb upstream of ␤4 resulted in a dramatic decrease in ␤4 and ␣3 expression in the pineal gland and SCG. CNR4 was sufficient to direct LacZ transgene expression to SCG neurons, which express the endogenous ␤4␣3␣5 subunits, and pineal cells, which express the endogenous ␤4␣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.Recent evidence from whole-genome studies has led to the conclusion that gene clustering is common in the eukaryotic genome (8, 26). Two well-studied clusters are the ␤-globin and Hox loci, in which clustering is thought to facilitate interactions between gene-proximal elements in or near individual promoters and shared long-range regulatory elements. These interactions are necessary for correct cell type-and stage-specific switching of ␤-globin gene expression during erythropoiesis and coordinate expression of Hox genes in order for them to fulfill their roles in limb development and in patterning the main body axis (34, 52). Many gene clusters are expressed either exclusively or predominantly in the nervous system, but the mechanisms involved are not understood (19,51,67). The prevalence of eukaryotic gene clusters suggests that the identification of the transcriptional mechanisms governing their expression will be important for understanding the differentiation of specific cell types and perhaps the molecular basis of disease (29).A phylogenetically conserved cluster of nicotinic acetylcholine receptor (nAChR) subunit genes, ␤4/␣3/␣5, encodes heteromeric neurotransmitter-gated cation channels that are critically important for fast cholinergic synaptic transmission (7,11,13). Several observations indicate that the clustered genes are coordinately regulated. First, a...

The CHRNA5/A3/B4 Gene Cluster Variability as an Important Determinant of Early Alcohol and Tobacco Initiation in Young Adults

“…The importance of this tightly linked region in the potential regulation of the CHRNA5/A3/B4 gene cluster stems from transcriptional analysis of nicotinic receptors in rodents, where McDonough and Deneris (44) discovered a novel enhancer positioned in the 3′-untranslated exon of the CHRNB4 gene. The location of this enhancer within the CHRNB4 gene may be under selective pressure for maintaining tight linkage of the clustered neuronal CHRNA5/A3/B4 genes (19,44). To date, there is no functional evidence for a human CHRNB4 3′UTR-enhancer, but this region appears to be conserved between rodents and humans (Dr. Deneris, personal communication).…”

“…The phylogenetically conserved cluster of nAChRs subunit genes, the α5 α3 and β4 gene-cluster (A5A3B4), encodes heteromeric channels important in fast cholinergic synaptic transmission. The three subunits are co-expressed in autonomic ganglia and several structures of the brain (19).…”

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

“…Third, in a recent elegant study by the Deneris laboratory (66), an enhancer, referred to as ␤43Ј, located in the 3Ј-untranslated region of the ␤4 subunit gene, was shown to play a critical role in vivo in the expression of the clustered genes in the adrenal gland. In addition, this group demonstrated that a conserved noncoding region, CNR4, located ϳ20 kb pairs upstream of the ␤4 coding sequence, plays a key role in directing expression of the clustered genes in the pineal gland and superior cervical ganglion (66). CNR4 may also be important for cell typespecific expression of the clustered genes in the central nervous system (66).…”

“…Second, the ␣3 and ␤4 promoters can be trans-activated by the cell type-specific regulatory factors, Sox10 and SCIP/Tst-1/Oct-6 (53,74). Third, in a recent elegant study by the Deneris laboratory (66), an enhancer, referred to as ␤43Ј, located in the 3Ј-untranslated region of the ␤4 subunit gene, was shown to play a critical role in vivo in the expression of the clustered genes in the adrenal gland. In addition, this group demonstrated that a conserved noncoding region, CNR4, located ϳ20 kb pairs upstream of the ␤4 coding sequence, plays a key role in directing expression of the clustered genes in the pineal gland and superior cervical ganglion (66).…”

“…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)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(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.…”