Promoter region of the rat m4 muscarinic acetylcholine receptor gene contains a cell type-specific silencer element.

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.