The long term objectives of this research are to understand the role of the catecholamine biosynthetic enzyme, tyrosine hydroxylase, in the etiology and treatment of severe psychiatric and nourological disorders. In this application we propose to determine the mechanisms which-regulate expression of the TH gene. Specifically, we will define cis-acting sequences involved in the hormonal and neural-specific regulation of tyrosine hydroxylase. Transient expression assays will be used together with deletion mutations of the promoter region to delineate sequences involved in transcriptional regulation. The important DNA sequence will be further defined by point mutagenesis experiments. Gel retardation and DNase I footprinting experiments will be used to identify binding of trans-acting factors. We will demonstrate their physical association with specific sequences defined by the deletion and point mutagenesis experiments. Information gained from these experiments will be used to design retroviral/tyrosine hydroxylase minigenes. Cell lines containing the mutant tyrosine hydroxylase genes will be tested for their ability to be regulated by normal physiological stimuli. Results from these experiments are necessary prerequisites for future in vivo studies investigating tissue and hormonal specificity in tranagenic animals. Additionally, these studies will further our understanding of the structural requirements for tyrosine hydroxylase gene regulation and expression. Understanding such structural requirements will help us understand the mechanisms which contribute to neuronal phonotypic diversity and development and perhaps, atypical catecholamine expression.
