The long range goal of our research is to gain a better understanding of the regulation of membrane excitability. Our studies continue to focus on regulation of the voltage-dependent sodium channel, which is responsible for the generation of action potentials in most excitable tissues. Experiments in the previous funding period revealed that expression of a major sodium channel type in the central nervous system, type II, is likely due to a novel transcriptional repression mechanism that prevents expression of this channel in non-neural tissues. We showed that the molecular basis of this mechanism was the DNA-binding protein that we termed REST. Further, because a large number of other "neural-specific" genes contain REST-binding sites, we proposed that REST played an important role in determining the neural phenotype. The new specific aiins will test our hypothesis that REST is indeed a critical factor for acquisition of neural fate. We will perform gain-of-function and loss-of function studies of REST in cell lines and in transgenic mice to determine whether REST controls expression of target genes in the chromosome (aim l). We will also examine, as a measure of the extent of influence of REST, the ability of ectopic expression of REST to block the induction of sodium channel excitability by Nerve Growth Factor. Preliminary data suggests that REST expression is repressed at a developmental time when neural progenitor cells differentiate to acqulre neural fates. The elucidation of upstream factors that repress REST expression during neurogenesis is attacked in specific aim 2. The upstream regulatory region of REST will be isolated and tested in transient expression assays for the presence of DNA elements that regulate its cell type specific expression. The ability of the cloned REST sequences expressed in transgenic mice to recapitulate the temporal and spatial pattern of expression of the endogenous REST gene will be confirmed and subsequent experiments will identify the precise DNA elements required for the absence of REST expression in neuronal cells. The proposed studies will contribute importantly to the revelation of mechanisms controlling the appearance and maintenance of membrane excitability and of factors critical for regulation of mammalian neurogenesis.