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

Abstract

The genes encoding the nicotinic acetylcholine receptor ␣3, ␣5, and ␤4 subunits are genomically clustered. These genes are co-expressed in a variety of cells in the peripheral and central nervous systems. Their gene products assemble in a number of stoichiometries to generate several nicotinic receptor subtypes that have distinct pharmacological and physiological properties. Signaling through these receptors is critical for a variety of fundamental biological processes. Despite their importance, the transcriptional mechanisms underlying their coordinated expression remain to be completely elucidated. By using a bioinformatics approach, we identified a highly conserved intronic sequence within the fifth intron of the ␣3 subunit gene. Reporter gene analysis demonstrated that this sequence, termed "␣3 intron 5," inhibits the transcriptional activities of the ␣3 and ␤4 subunit gene promoters. This repressive activity is position-and orientation-independent. Importantly, repression occurs in a cell type-specific manner, being present in cells that do not express the receptor genes or expresses them at very low levels. Electrophoretic mobility shift assays demonstrate that nuclear proteins specifically interact with ␣3 intron 5 at two distinct sites. We propose that this intronic repressor element is important for the restricted expression patterns of the nicotinic receptor ␣3 and ␤4 subunit genes.Neuronal differentiation is a consequence of extrinsic and intrinsic regulatory cascades that ultimately act to regulate, both positively and negatively, gene expression. This process yields mature neurons that express a limited set of genes encoding proteins that perform specific functions (1-9). Part of this repertoire of genes is that of encoding proteins required for neuronal signaling, including neurotransmitter biosynthetic enzymes and their cognate neurotransmitter receptors. Acetylcholine is an excitatory neurotransmitter that interacts with both ionotropic and G-protein-coupled receptors. Signaling through ionotropic nicotinic acetylcholine (nACh) 2 receptors is involved in a variety of behaviors ranging from muscle contraction to memory formation (10 -13). In addition, acetylcholine and its receptors play important roles in neural development (14 -17). Not surprisingly, compromised signaling through nACh receptors is implicated in a number of neurological disorders (18 -35). Thus, understanding the molecular details underlying nACh receptor expression will shed light on various aspects of neural development and function as well as contribute to our understanding of several neuropathological conditions.In the nervous system, nACh receptors are encoded by a conserved family of genes consisting of at least 12 members, ␣2-␣10 and ␤2-␤4 (10, 11, 36 -39). A significant effort has been put forth to elucidate transcriptional mechanisms controlling expression of the nACh receptor genes (40, 41). We and others have focused on the regulation of a genomic cluster of receptor genes, those encoding the ␣3, ␣5, and ␤4 subunits (4...

Alternative CHRNB4 3′-UTRs Mediate the Allelic Effects of SNP rs1948 on Gene Expression

“…Several studies, looking at the transcriptional level, have revealed a cell-type specific enhancer positioned in the rat β4 3′-UTR [37], [39], [42], [44], [48], [50]. By contrast, we found decreased luciferase expression in all our test plasmids when compared to the empty pGL3 vector, along with no differences among cell lines tested.…”

“…Although the transcriptional regulation of the CHRNA5/A3/B4 cluster of genes has been extensively studied in rats by several groups [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], little is known about the impact of human non-coding SNPs in this cluster of genes on the expression of these subunits. Only few studies describing the functional features of the α3 nAChR [51], [52], [53], [54] and α5 nAChR [55] subunit promoters have been done using human sequences, but none of them have looked at rs1948, located in the 3′-untranslated region (3′-UTR) of CHRNB4 .…”

Aversion to Nicotine Is Regulated by the Balanced Activity of β4 and α5 Nicotinic Receptor Subunits in the Medial Habenula

“…Subsequently, the virus was resuspended in 50-100 µl 1x PBS, aliquoted and stored at -70 °C. The titer of concentrated lentivirus was determined by transducing 1  10 Yellow boxes: exons, green box: eGFP cassette, white box: polyadenylation signal (pA), black arrows: direction of transcription, red crosses: truncated transcription, cis-regulatory elements marked in red: conserved non-coding region (CNR4), 43' enhancer (Xu et al, 2006), E1/E2: SP1 and SP3 binding sites (Bigger et al, 1997) and 3-i: transcriptional silencer (Medel and Gardner, 2007).…”

“…The upstream sequences of Chrna5, encoding exon 1 splice variants (Flora et al, 2000), are missing in the BAC transgene ( Figure 3A). To promote correct expression of Chrnb4, the BAC included the intergenic and 5' flanking regions encompassing the cis-regulatory elements that coordinate co-transcriptional control of the genes in the cluster (Bigger et al, 1997;Medel and Gardner, 2007;Xu et al, 2006). As a result of these modifications in the BAC transgene, these mice (referred to as Tabac mice for Transgenic a3b4a5 cluster) express high levels of 4 but not 5 ( Figure 3B), and expression of 3 is replaced by expression of an eGFP reporter cassette to monitor the sites expressing the transgene ( Figures 3C-H).…”