In the nervous system, the selective expression of tyrosine hydroxylase (TH) leads to the development of catecholaminergic cells. TH is the rate- limiting enzyme in the synthesis of dopamine, norepinephrine and epinephrine, which are expressed in anatomically and functionally distinct locations within the central and peripheral nervous systems. This proposal seeks to define the regulatory elements that direct cell-specific expression and trans-synaptic induction of TH in cultured cells and transgenic mice. By mutational analyses of the 5' flanking region of the rat TH gene, two enhancer regions are required for full transcriptional activation in cultured cells: an AP1/E box region at -205 and an CRE site at -45. We propose to evaluate the role of these sites in transgenic mice using site- directed mutations. Since the CRE site has been implicated in mediating depolarization-induced transcription, the effect of CRE mutations will be assessed after trans-synaptic induction in vivo. Two paradigms will be investigated: tonic, activity-dependent expression in the dopaminergic periglomerular cells of the olfactory bulb and stress-dependent induction by cholinergic afferents in the adrenal and sympathetic ganglia. Using new CNS TH+ cell lines and deletion mapping in transgenic mice, we propose to identify sites responsible for TH expression in different TH+ cell types. Preliminary data from transgenic mice, suggest that multiple elements, both activators and repressors, may be involved. Fine mapping of the AP1/E box and the TH promoter regions is proposed in cultured cells. Using a panel of mutant AP1/E box sites assessed in the mapping experiments, proteins that bind transcriptionally active sites will be cloned from expression libraries and their relevance evaluated by several criteria.
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