The nervous system is composed of thousands of diverse cell types which exhibit functional specificity which is essential for appropriate cell-cell communication. The biochemical and biophysical properties of a cell are largely a function of the selection of genes expressed in that cell. The transcription of a gene into mRNA is regulated through the interaction of nuclear transcriptional modulatory proteins with genetic recognition elements within and surrounding that gene. The experiments in this proposal are designed to identify the factors which modulate the pattern of transcription of the rat dopamine beta-hydroxylase (DBH) gene. DBH catalyzes the conversion of dopamine to norepinephrine in the catecholamine biosynthetic pathway, and is expressed in specified brain nuclei, the sympathetic ganglia and the adrenal medulla. In the previous granting period we identified a regulatory element on the 5'-flanking region of the rat DBH gene which influences transcription in a cell type specific pattern. this 30 base regulatory element, which we have named DB1, functions as an enhancer of transcription in catecholaminergic cell lines, but not in fibroblast or epithelial cell lines. The experiments in this grant proposal will further characterize this DB1 DNA-protein interaction, and include (1) a determination of the precise binding site(s) of the DB1 binding factor on the DBH gene, (2) purification of the DB1 binding factor via DNA-affinity chromotography, (3) isolation of DB1 cDNA clone(s) followed by sequence analysis, (4) determination of the tissue distribution of the DB1 RNA transcript, and (5) characterization of the molecular composition of the DB1 binding complex. In addition to the studies which have defined the DB1 binding activity, we have identified a second binding site on the 5'-flanking region of the DBH gene corresponding to the recognition sequence of transcription factor AP-2. This binding activity was observed in extracts derived from neuroblastoma, but not epithelial cell lines. The experiments proposed will further characterize this AP-2 like binding activity, using biochemical and molecular genetic approaches similar to those described for the characterization of DB1. The functional significance of the AP-2 binding site will be studied by determining the ability of AP-2 to influence DBH gene transcription. One of the goals of the experiments in this proposal is to understand whether these transcriptional modulators of the DBH gene also function to influence transcription of other neuronal genes, especially those which are co- expressed in catecholaminergic cells. To address this question, we will determine the ability of the DB1 and AP-2 proteins to modulate the transcription of the tyrosine hydroxylase gene, which encodes the initial enzyme of the catecholamine biosynthetic pathway. Finally, we will characterize the functional importance of other, uncharacterized regions of the DBH gene, with regard to specificity of gene expression. The results of the experiments proposed in this application will provide an understanding of the genetic events which control the selection of a specific neurotransmitter phenotype.
