Signaling through nicotinic acetylcholine (nACh) receptors underlies a diverse array of behaviors. In order for appropriate signaling to occur via nACh receptors, it is necessary for the genes encoding the receptor subunits to be expressed in a highly regulated temporal and spatial manner. Here we report a transgenic mouse approach to characterize the transcriptional regulation of the gene encoding the nACh receptor β4 subunit. nACh receptors containing this subunit play critical roles in both the central and peripheral nervous systems. We demonstrate that a 2.3-kilobase pair fragment of the β4 5′-flanking region is capable of directing reporter gene expression in transgenic animals. Importantly, the transcriptional activity of the promoter region is cell-type-specific and developmentally regulated and overlaps to a great extent with endogenous β4 mRNA expression. These data indicate that the 2.3-kilobase pair fragment contains transcriptional regulatory elements critical for appropriate β4 subunit gene expression. Keywords nicotinic receptor; gene expression; transcriptionSignaling through neuronal nicotinic acetylcholine (nACh) receptors underlies several fundamental biological processes both during development and in the adult (Albuquerque et al., 2009). In the central nervous system, presynaptic nACh receptors modulate release of most classical neurotransmitters including norepinephrine, ACh, glutamate and GABA (Albuquerque et al., 2009;Dani and Bertrand, 2007;Engelman and MacDermott, 2004). Postsynaptic nACh receptors are intimately involved in fast ACh-mediated synaptic transmission in addition to activity-dependent gene expression, which is critical for synaptic Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 March 31. Dani and Bertrand, 2007;Hu et al., 2002;Ji et al., 2001). Within the peripheral nervous system, nACh receptors mediate fast excitatory transmission in most, if not all, autonomic ganglia and are involved in modulating visceral and somatic sensory transmission (Genzen et al., 2001;Hu and Li, 1997;Steen and Reeh, 1993;Sucher et al., 1990;Wang et al., 2002). More recently, numerous studies have revealed the expression of nACh receptors on non-neuronal cells and evidence is accumulating indicating that the receptors play crucial roles in signal transduction underlying many physiological processes outside the nervous system (Gahring and Rogers, 2006;Spindel, 2003;Wessler and Kirkpatrick, 2008). The importance of nACh receptor-mediated signaling is reflected ...
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...
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