The long range goal of this project is to develop an understanding of the regulation of the cholinergic gene locus and its gene products, choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To achieve this goal three specific aims are proposed.
The first aim i s directed at elucidating the mechanism of cell specific expression of the cholinergic gene locus. This locus is unique in that it contains the genes for both ChAT and VAChT in the same transcriptional orientation. We will test the hypothesis that there are both upstream and downstream regulatory elements in the gene which direct cell specific expression of ChAT and/or VAChT. We will study the activity of these regulatory elements in the context of a """"""""double reporter gene"""""""" which measures both ChAT and VAChT transcription from the same construct. The second specific aim will test the hypothesis that the binding of the transcription factor REST to DNA is regulated by a neuron specific isoform of REST called REST4. REST4 is proposed to be a key regulator of the cholinergic gene locus as well as of a group of neuron specific genes which contain the neuron restrictive silencer element NRSE/RE1. In the third specific aim we propose to use a recently developed system for studying VAChT function. We have a mutant PC12 cell line which is devoid of both ChAT and VAChT, but contains synaptic vesicles, and can be reconstituted with ChAT and/or VAChT cDNAs. This provides a unique opportunity to study VAChT function in the absence of interfering background activity from endogenous VAChT. We will use this system to study the mechanism by which vesicle filling is regulated, to study trimeric G protein regulation of VAChT function, and to study a VAChT mutant which appears to act as a dominant negative by inhibiting endogenous VAChT activity.