Promoter Region of the Rat m4 Muscarinic Acetylcholine Receptor Gene Contains a Cell Type-specific Silencer Element

Abstract

We describe here the characterization of the rat m4 muscarinic acetylcholine receptor gene and the identification of its regulatory region. Two 5-noncoding exons are located approximately 5 kilobases upstream from the coding exon, and at least two alternatively spliced variants of m4 mRNA are expressed in the neuronal cell line PC12D. There are two transcription initiation sites. The promoter region is GC-rich, contains no TATA-box, but has two potential CAAT boxes and several putative binding sites for transcription factors Sp1 and AP-2. We assessed the m4 promoter activity functionally in transient expression assays using luciferase as a reporter. The proximal 435-base pair (bp) sequence of the 5-flanking region produced luciferase activity in both m4-expressing neuronal cell lines (PC12D and NG108 -15) and non-neuronal cell lines (L6 and 3Y1B). A longer fragment containing an additional 638-bp sequence produced luciferase activity only in m4-expressing neuronal cell lines. These data suggest that the proximal 435-bp sequence contains a constitutive promoter and that a 638-bp sequence farther upstream contains a cell type-specific silencer element. A consensus sequence for the neural-restrictive silencer element is found within this 638-bp segment. Muscarinic acetylcholine receptors (mAChR)1 are the members of the superfamily of G-protein-coupled receptors, which are characterized by the presence of seven putative transmembrane domains. mAChRs are widely expressed in the central and the peripheral nervous system. In the brain, mAChRs are thought to play critical roles in higher functions, including attention, regulation of movement, learning, and memory (1, 2). Five subtypes of mAChR (m1-m5) have been identified by molecular cloning (3). Each subtype of mAChRs shows a distinct and complicated distribution in peripheral tissues and brain, suggesting subtype-specific functions (4 -7). Although the coding region of each mAChR subtype has been cloned and sequenced, the precise structures of their 5Ј-noncoding regions and regulatory regions have not been reported. Cloning and analysis of the genetic regulatory elements of these genes should yield important insights into the mechanisms that underlie the tissue-and site-specific expression of each subtype of mAChR.In this study, we focused on defining the sequences that regulate m4 mAChR gene expression. In mammals, the m4 mAChR gene is expressed predominantly in the central nervous system, although its expression has been detected in some peripheral tissues such as rabbit lung (but not human or rat lung) (8, 9). In rat brain, m4 mRNA is present in the cerebral cortex, striatum, olfactory bulb, and pyramidal cell layer of the hippocampus (4, 7, 10, 11). m4 mAChR has been suggested to function not only postsynaptically but also presynaptically in some regions (12, 13). As a first step toward elucidating the regulation mechanism of m4 mAChR gene expression, we have characterized the rat m4 mAChR gene and identified its regulatory region. EXPERIMENTAL PROCEDURESCell Cul...

Neural Specific Expression of the m4 Muscarinic Acetylcholine Receptor Gene Is Mediated by a RE1/NRSE-type Silencing Element

“…Recently another group have published results of experiments characterizing the rat m4 promoter (27). The conclusions drawn by Mieda et al (27) are very similar to our original work (1) except in the position of the transcription initiation site, which they claim is located 336 bp downstream from the initiation point defined previously by us.…”

“…The conclusions drawn by Mieda et al (27) are very similar to our original work (1) except in the position of the transcription initiation site, which they claim is located 336 bp downstream from the initiation point defined previously by us. We have compared the expression levels obtained in NG108 and PC12 cell lines with our reporter constructs to the levels reported by Mieda et al (27) and found them to be practically identical, despite the fact that their constructs include a further 270 bp of exon 1 as defined in Ref. 1.…”

Neuronal Expression of Zinc Finger Transcription Factor REST/NRSF/XBR Gene

“…The highest levels of rREST mRNA were detected in the neurons of hippocampus and the nuclei of pons/medulla and midbrain. Previous studies have shown that neuronal genes that contain NRSE-like sequences (Schoenherr et al, 1996) may convey NRSF/REST/ XBR-mediated repression (Kraner et al, 1992;Mori et al, 1992;Pathak et al, 1994;Thiel et al, 1994;Lo ¨nnerberg et al, 1996;Mieda et al, 1996;Wood et al, 1996). Analyses of the data available in the literature about the expression profiles of REST/ NRSF/XBR target genes revealed that expression of some of these genes (M4 muscarinic receptor, NMDAR1, and GABA-A receptor) is limited to the brain regions in which rREST mRNA is not expressed or expressed at low levels.…”

Molecular properties of muscarinic acetylcholine receptors

“…Mieda, 116,117) together with Saffen, studied the regulation of M 4 gene expression and identified regulatory functions of the 5′-flanking region in the M 4 receptor gene: the proximal 435-bp sequence contains a constitutive promoter and the 635-bp sequence a cell-type-specific silencer element. Furthermore, the neuron-restrictive silencer/repressor element (NRSE/RE1) upstream of the M 4 gene was shown to be necessary and sufficient to repress M 4 promoter activity in non-neuronal cells.…”