Elements between the protein-coding regions of the adjacent ?4 and ?3 acetylcholine receptor genes direct neuron-specific expression in the central nervous system (original) (raw)
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Journal of Neurobiology, 1997
elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three regions, we investigated the activity of 02732 //47 in clustered neuronal nicotinic acetylcholine receptor vivo. Transgenic mice were generated, which carry the (nAchR) subunit genes ordered b4, a3, and a5 over-lacZ gene fused downstream of 02732 //47. Expreslap extensively in the peripheral nervous system sion of the lacZ transgene is restricted to neurons of (PNS) but only partially in the central nervous system the CNS; no expression was detected in the PNS or (CNS). We have begun to investigate cell type-spein nonneural tissues. LacZ -positive cells were detected cific cis elements regulating these genes by analyzing virtually exclusively in a subset of CNS nuclei that in both cell culture and transgenic mice, a 2.8-kb fragtranscribe the endogenous a3 gene. Some overlap was ment (02732//47) containing the a3 promoter reseen with the b4 gene, but nearly none with the a5 gion, the b4/a3 intergenic region, and a portion of the gene. Our results demonstrate that cis elements posi-b4 3-untranslated exon. The 02732 //47 fragment is tioned between the a3 and b4 coding regions are impreferentially active in PC12 cells relative to nonneuportant for establishing part of the restricted CNS ral cell lines. Deletion analysis revealed a cell typepatterns of b4, a3, and a5 gene transcription. ᭧ 1997 specific positive transcriptional element positioned in John Wiley & Sons, Inc. J Neurobiol 32: 311-324, 1997 the b4 3-untranslated exon. The positive element is Keywords: neuronal nicotinic receptors; cis-acting elelikely to be an enhancer and not a second a3 proments; neuron-specific regulation; transgenic mice; moter, because no a3 exons are present in this region. gene regulation; ligand-gated ion channel gene Having shown in cell culture that cell-type specific cis
European Journal of Pharmacology, 2000
Receptors assembled from the products of a neuronal b4a 3a 5 NAChR gene cluster depend on these genes being coordinately regulated in particular populations of neurons. Little is known, however, about the transcriptional mechanisms that are likely to underlie their co-expression in correct neuronal cell types. We have identified several regulatory elements and transcription factors that influence transcription of the a 3 and b4 genes. The promoters of these genes appear to contain a common cis element that binds Sp1 transcription factors. They can be activated by the POU-domain factor SCIP and activation does not require SCIP binding sites. Between these two promoters is a cell type specific enhancer called b43 X . This enhancer has little activity in non-neuronal cells and is preferentially active in particular populations of central neurons. The clustered genes are potential targets of ETS factors as the ETS domain factor, Pet-1 can activate b43 X -dependent transcription. The neuron-selective properties of b43 X and its location suggest that it is a component of the cis regulatory information required to control expression of the b4 and a 3 genes in specific populations of neurons. q
European Journal of Neuroscience, 1998
Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes compose a family of genes. The major isoform of nAChR in the brain is made up of the α4 and β2 subunits and possesses a high affinity for nicotine. To investigate the mechanisms of the regulation of the nAChR α4 gene expression in mouse, its genomic DNA was cloned and characterized. The transcription initiation site was mapped by primer extension and RNase protection experiments and localized at about 254 bp upstream of the translation initiation site. The 5Ј flanking region of this gene did not have typical TATA box but GC-rich sequences were found around the initiation site. Methylation analysis of this region revealed that genomic DNAs from liver and muscle are partially methylated, whereas little methylation was observed in genomic DNA from brain. To characterize the cis-acting elements driving cellspecific expression of the α4 subunit gene, we produced lines of transgenic mice which carry a series of fragments of the α4 gene fused with bacterial lacZ as a reporter gene. An 11.5-kb DNA fragment containing 9 kb of the region upstream of the transcription initiation site and the first intron was found to confer an expression pattern which coincides rather well with the endogenous gene expression pattern at early embryonic stages, suggesting that the elements necessary for the onset of α4 gene expression are located in this region. A DNA fragment containing the 1.8-kb upstream sequence and the first intron drove expression of lacZ in a limited subset of α4 expressing cells, whereas the 1.8-kb upstream sequence alone did not elicit any significant expression. These results show that both upstream and intronic sequences are important for cell-specific expression of the nAChR α4 gene.
Cell specificity of a rat neuronal nicotinic acetylcholine receptor α7 subunit gene promoter
Neuroscience Letters, 2006
Neuronal nAChRs are pentameric transmembrane proteins which function as ligand-gated ion channels and are composed of multiple ␣ and  subunits. Nine neuronal nAChR ␣ subunit genes (␣2-␣10) and three nAChR  subunit genes (2-4) have been identified. nAChR subtypes are heteromers, composed of various combinations of nAChR subunits or homomers composed of ␣7, ␣8, or ␣9 subunits. nAChR subtypes are widely expressed in the nervous system, yet each subunit has a distinct and unique pattern of expression. This report focuses on the expression of the nAChR ␣7 gene since homomeric nAChRs can be formed from this one subunit, simplifying a study of the expression of a specific nAChR subtype. ␣7 nAChRs are involved in several important biological activities in addition to synaptic transmission including mediating neurite outgrowth, neuronal development and cell death, and in presynaptic control of neurotransmitter release. Transcriptional regulation of ␣7 gene expression may be important to control the location and timing of these events. We previously isolated a rat ␣7 nAChR promoter and studied expression in PC12 cells. In this study we examined the expression of the ␣7 promoter in PC12, HEK293, L6, SN17 and Neuro-2A cells in order to define elements necessary for cell-specific expression. Elements promoting expression of ␣7 in muscle and fibroblasts were identified. We also demonstrated that several other nAChR genes are also expressed in SN 17 and Neuro-2A cells, supporting use of these cell lines as models to study transcriptional control of nAChR genes.
The Minimal Promoter of the Human α3 Nicotinic Receptor Subunit Gene
Journal of Biological Chemistry, 2000
The minimal promoter of the human ␣ 3 nicotinic receptor subunit gene has been mapped to a region of 60 base pairs and found to contain two Sp1 sites, one of which is essential for promoter activity. DNase footprinting has revealed the presence of another region of interaction with nuclear factors (named F2) immediately downstream of the Sp1 sites. This region has been found to be functional since it is capable of stimulating the minimal promoter. The F2 protection is completely and specifically competed by an AP2 consensus oligonucleotide that has been proved to bind AP2␣ exclusively. However, the AP2␣ recombinant protein was unable to bind the F2 region directly, thus suggesting that AP2␣ may participate in F2 protection by protein-protein interactions with other nuclear factors. The minimal promoter has been shown to be stimulated by two additional regions, one located downstream of F2 and the other upstream of the minimal promoter itself. In neuronal cells, the combined stimulatory activities of these three regions have synergistic effects, whereas in nonneuronal cells, there is a negative interference between the upstream and downstream regions. These opposite transcriptional effects may account for at least part of the neuro-specific expression profile of the ␣ 3 gene. The identification of the genetic mechanisms underlying the expression of neural genes is rapidly becoming an invaluable approach for developing our understanding of the generation and maintenance of distinct neural phenotypes. Neuronal cholinergic-nicotinic receptors consist of subunits whose anatomical distribution and developmental regulation represent an attractive model for addressing such crucial questions (1-3). These molecules form a family of acetylcholine-gated cation channels, expressed in the autonomic and sensory ganglia, the adrenal medulla, and in distinct areas of the central nervous system, with a quaternary structure consisting of five transmembrane subunits assembled around a central channel. Eleven distinct neuronal nicotinic subunits have been cloned so far and classified into two subfamilies of eight ␣ (␣ 2-␣ 9) and three  subunits ( 2- 4) (1, 4). In heterologous expression
Journal of Biological Chemistry, 2000
The minimal promoter of the human ␣ 3 nicotinic receptor subunit gene has been mapped to a region of 60 base pairs and found to contain two Sp1 sites, one of which is essential for promoter activity. DNase footprinting has revealed the presence of another region of interaction with nuclear factors (named F2) immediately downstream of the Sp1 sites. This region has been found to be functional since it is capable of stimulating the minimal promoter. The F2 protection is completely and specifically competed by an AP2 consensus oligonucleotide that has been proved to bind AP2␣ exclusively. However, the AP2␣ recombinant protein was unable to bind the F2 region directly, thus suggesting that AP2␣ may participate in F2 protection by protein-protein interactions with other nuclear factors. The minimal promoter has been shown to be stimulated by two additional regions, one located downstream of F2 and the other upstream of the minimal promoter itself. In neuronal cells, the combined stimulatory activities of these three regions have synergistic effects, whereas in nonneuronal cells, there is a negative interference between the upstream and downstream regions. These opposite transcriptional effects may account for at least part of the neuro-specific expression profile of the ␣ 3 gene. The identification of the genetic mechanisms underlying the expression of neural genes is rapidly becoming an invaluable approach for developing our understanding of the generation and maintenance of distinct neural phenotypes. Neuronal cholinergic-nicotinic receptors consist of subunits whose anatomical distribution and developmental regulation represent an attractive model for addressing such crucial questions (1-3). These molecules form a family of acetylcholine-gated cation channels, expressed in the autonomic and sensory ganglia, the adrenal medulla, and in distinct areas of the central nervous system, with a quaternary structure consisting of five transmembrane subunits assembled around a central channel. Eleven distinct neuronal nicotinic subunits have been cloned so far and classified into two subfamilies of eight ␣ (␣ 2-␣ 9) and three  subunits ( 2- 4) (1, 4). In heterologous expression
European Journal of Neuroscience, 1997
Within the chick central nervous system, expression of the 3 nicotinic acetylcholine receptor gene is restricted to a subset of retinal neurons, the majority of which are ganglion cells. Transient transfection in retinal neurons and in neural and non-neural cells from other regions of the chick embryo allowed the identification of the cis-regulatory domain of the 3 gene. Within this domain, a 75-base pair fragment located immediately upstream of the transcription start site suffices to reproduce the neuron-specific expression pattern of 3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the 3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of 3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic helix-loop-helix transcription factors indicate that the cis-regulatory domain of 3 has the remarkable property of discriminating accurately between related members of the basic helix-loop-helix protein family. The sequence located immediately 3 of the E-box participates in this selection, and the E-box acts in concert with the nearby CAAT box.
Characterization of a Rat Neuronal Nicotinic Acetylcholine Receptor α7 Promoter
Journal of Biological Chemistry, 2001
Neuronal nicotinic acetylcholine receptors (nAChRs) containing the ␣7 subunit are expressed in the central nervous system, autonomic nervous system, retina, adrenal medulla, and PC12 cells. ␣7 nAChRs have been implicated in several important biological activities apart from synaptic transmission such as mediating neurite growth and presynaptic control of neurotransmitter release. A 178-base pair promoter was sufficient to drive high level expression of the ␣7 gene in PC12 cells. The ␣7 promoter was also cell-specific, expressing in PC12 cells but not in L6 rat muscle cells. Within our minimal rat ␣7 nAChR promoter we identified two sequences important for basal level expression. Mutation of a GC-rich sequence at ؊172 relative to the translational start site led to an increase in activity of the promoter, indicating the presence of a negative regulatory element. Upstream stimulatory factor-1 acted to regulate ␣7 expression positively by binding to an E-box at ؊116. A site directly adjacent to the upstream stimulatory factor-1 binding site was shown to bind Egr-1. Sp1 and Sp3 binding also occurred downstream from or overlapping the Egr-1 binding site in the rat ␣7 promoter. Several transcription factors interact in close proximity to control expression of the rat ␣7 nicotinic receptor gene. Neuronal nicotinic acetylcholine receptors (nAChRs) 1 mediate synaptic transmission in many parts of the vertebrate central nervous system, as well as in autonomic ganglia, retina, and adrenal medulla. Neuronal nAChRs are pentameric structures (1, 2) that function as ligand-gated ion channels and are composed of multiple ␣ and  subunits. Nine neuronal nAChR ␣ subunit genes (␣2-␣10) and three nAChR  subunit genes (2-4) have been identified (3-16). One specific subtype of neuronal nAChR is sensitive to ␣-bungarotoxin and composed of ␣7 subunits. ␣7 nAChRs possess several characteristics that set them apart from most