Expression of the Rat m4 Muscarinic Acetylcholine Receptor Gene Is Regulated by the Neuron-restrictive Silencer Element/Repressor Element 1

Abstract

Neuronal cell-specific expression of the rat m4 muscarinic acetylcholine receptor (mAChR) is regulated by a silencer element. A likely mediator of this silencing is the neuron-restrictive silencer element/repressor element 1 (NRSE/RE1), which is present 837 base pairs (bp) upstream from the transcription initiation site of the m4 mAChR gene (Wood, I. C., Roopra, A., Harrington, C., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940; Mieda, M., Haga, T., and Saffen, D. W. (1996) J. Biol. Chem. 271, 5177-5182). In the present study, we examined whether this putative NRSE/RE1 functions as a silencer. Transient expression assays using m4 mAChR promoter/luciferase expression vectors showed that the m4 NRSE/RE1 is necessary and sufficient to repress m4 promoter activity in non-neuronal L6 cells. m4 promoter activity was only partially repressed, however, in neuronal NG108-15 cells exogenously expressing the neuronal-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST). By contrast, the promoter activity of the type II sodium channel (NaII) gene was nearly completely repressed in NRSF/REST-expressing NG108-15 cells. Experiments with expression vectors containing chimeric promoters revealed that the NRSE/RE1 elements derived from both the m4 and NaII genes are independently sufficient to silence NaII gene promoter activity, but only partially repress m4 mAChR gene promoter activity in NRSF/REST-expressing NG108-15 cells. Thus, the repression activity of NRSF/REST depends upon the species of promoter to which it is linked. Gel-shift assays showed that the NRSF/REST is the only protein that binds to a 92-bp segment from the m4 mAChR promoter containing NRSE/RE1. This and the fact that m4 promoter activity was completely repressed in L6 cells suggest that the proteins that bind to the m4 constitutive promoter may be different from those in NG108-15 cells. Deletion analysis of the m4 constitutive promoter revealed that a 90-bp segment immediately upstream from the transcription initiation site contains significant promoter activity. Gel-shift assays revealed that several proteins in nuclear extracts prepared from L6 and NG108-15 cells bind to this 90-bp segment and that some of these proteins are L6 or NG108-15 cell-specific. These data support the idea that the repression activity of NRSF/REST depends upon the species of promoter to which it is linked and upon the proteins that bind to those promoters.