The development and maintenance of a neurotransmitter system is one of the essential events in the generation of neural function and specificity. The events that influence the neurotransmitter phenotype of a specified cell are initiated during development and continue postnatally. Environmental stimuli and positional information stimulate intracellular signals that lead to neurotransmitter production. In nearly all situations studied the critical decisive regulatory event is the activation or repression of genes encoding the enzymes producing the neurotransmitter. This proposal aims to understand the genetic and biochemical regulatory elements of the DBH and TH genes that are critical for appropriate expression. In the previous granting period the applicants identified a homeodomain transcription factor termed, Arix which binds to the DBH gene and is expressed selectively in noradrenergic cells. Arix will regulate DBH gene transcription but it is dependent upon simultaneous activation of the cAMP second messenger pathway. Arix will also stimulate transcription of the DBH gene, independent of second messenger stimulation. In addition to the characterization of the positive genomic regulatory elements, they characterized a negative regulatory element of the DBH gene which functions to repress expression in the inappropriate cells. In this proposed granting period the goal is to define the molecular and biochemical mechanisms underlying these genetic regulatory events, and to begin testing the importance of these regulatory element in vivo. To achieve this goal the proteins that contribute to transcriptional regulation of TH and DBH will be identified and characterized. The mechanism by which these factors modify specific gene expression will be evaluated in cultured cells and in defined biochemical systems. In addition, the importance of one defined regulatory element of the DBH gene will be evaluated in vivo though construction of transgeneic animals. These experiments could be significant towards understanding the process by which a neuroendocrine cell carries out its specialized function. There are 4 specific aims: 1. Define the biochemical and molecular interactions between the newly identified homeodomain factor, Arix, and the cAMP system which leads to synergistic stimulation of DBH gene expression.; 2. Understand the basis of the difference between TH and DBH genes in requirements for Arix mediated transcriptional activation; 3. Identify and characterize the protein that interacts with the DBH silencer.; 4. Evaluate the importance of the CRE/Arix regulatory element in vivo by introduction of DNA containing mutation of this element into mice.
