Enhancer, Silencer, and Growth Factor Responsive Regulatory Sequences in the Promoter for the Mouse Choline Acetyltransferase Gene

Abstract

We describe here the isolation of the 5′ region of the mouse choline acetyltransferase (ChAT) gene and the functional characterization of regulatory regions that control its expression. ChAT catalyzes the synthesis of a neurotransmitter, acetylcholine, and is expressed specifically in cholinergic neurons. The 5′ flanking region of the mouse ChAT gene lacks a consensus TATA element and transcription initiates at multiple sites. The gene contains a strong enhancer in the promoter-proximal region, a weaker, nerve growth factor responsive enhancer located immediately upstream, and a silencer-like sequence yet further upstream. We identified the strong enhancer within the proximal promoter region -430 to -115 by its ability to activate a heterologous, minimal SV40 promoter. The ChAT enhancer functioned in both a position- and orientation-independent manner. The region from -825 to -430 contained weaker enhancer activity, but was modulated more strongly by exposure of transfected cells to NGF. The activity of the strong, -430 to -115 enhancer was not modulated by NGF. In our experiments, NGF coordinately increased expression of endogenous ChAT enzymatic activity and expression of reporter genes placed under the control of the ChAT promoter and enhancers. Activity of the ChAT proximal promoter is silenced in noncholinergic neuronal cell lines (B103 and F11) and in nonneuronal L6 myoblasts by an upstream region from -925 to -558. These results indicate that regulation of the ChAT gene is achieved by a combination of both positive and negative regulatory mechanisms whose interaction determines the expression of a cholinergic neuronal phenotype.