Localization of Sequences Determining Cell Type Specificity and NGF Responsiveness in the Promoter Region of the Rat Choline Acetyltransferase Gene

Abstract

A genomic clone containing 7 kb of 5' flanking sequences from the rat choline acetyltransferase (ChAT) gene was isolated and shown to contain a TATA box-like sequence and several consensus binding sites for the transcription factor AP1. Two constructs containing 450 and 1450 base pairs (bp), respectively, of 5' flanking sequences promoted expression of a fused chloramphenicol acetyltransfersase (CAT) gene when transfected into fibroblast FR3T3, Sertoli TM4, phaeochromocytoma PC12 and cholinergic neuronal SN6 cell lines. In contrast, a longer construct containing 3850 bp of 5' flanking sequence allowed CAT activity only in the cholinergic cell line SN6. CAT activity with this construct was suppressed in the three other cell lines, indicating that the distal region of the ChAT promoter contains a cell type-specific silencer-like element that restricts ChAT gene expression to cholinergic cells. Treatment of PC12 cells with nerve growth factor (NGF) increased the promoter activity of the -450 and -1450 constructs approximately four-fold and allowed promoter activity from the -3850 construct, indicating that elements involved in NGF responsiveness of the ChAT promoter are contained in the first 450 bp of upstream sequence. These results support a model in which gene transcription controlled by cell-type specific regulatory elements contribute to the establishment, maintenance and plasticity of the cholinergic transmitter phenotype in the nervous system.