Catecholamine (CA) neurotransmitters play a profound role in diverse brain functions. Tyrosine hydroxylase (TH) catalyzes the conversion of tyrosine to L-dopa, the first and rate-limiting step in CA biosynthesis, and the dopamine beta-hydroxylase (DBH) converts dopamine to noradrenaline. Expression of these genes is regulated in a distinct spatiotemproal fashion. The long-term goal of this project is to understand the mechanisms controlling the expression of the CA neuron-specific genes at the molecular level using the DBH and TH genes. Our previous experiments identified and characterized several regulatory regions or sequence elements critical for transcriptional control of these genes, including a very recent identification of the potential importance of sequences within the first intron of the rat TH gene. The following experiments are proposed to isolate and characterize transcription factors that regulate CA neuron-specific gene expression in the brain.
Aim 1 : The putative cell-specific cis-elements and enhancer that may reside in the first intron and distal upstream area of the TH gene will be delineated. Deletional and site-directed mutational analyses, footprinting, and gel shift assays will be used to further define potential regulatory sequence motifs.
Aim 2 : Transcription factors binding to the cell-specific proximal promoter (domain II) and silencer elements will be cloned using the yeast genetic selection system and lambda gt11 screening method. Using cell culture experiments, we identified critical sequence elements in the upstream region of the human DBH gene. This sequence information will be used to isolate the cDNAs encoding the cognate protein factors. The functional role of the isolated factors will be studied by molecular techniques such as sequence analysis, DNA-binding assay, co-transfection assay, and in situ hybridization.
Aim 3 : The role of homeodomain protein factors, i.e., Phox2a and Phox2b, for the phenotypic expression of CA neurons will be investigated. The potential role of these protein factors on TH and DBH gene expression will be investigated using DNA binding and transient co-transfection assays. The role of Phox2a in phenotypic determination of CA neurons in vivo will be addressed by generating and characterizing transgenic mice that ectopically express Phox2a in dopaminergic neurons or in the cerebellar Purkinje cells. Dysregulation of CA neurotransmitters has been implicated in several psychiatric and neurological disorders. The proposed studies will advance basic understanding of CA neuron-specific gene regulation at the molecular level and thereby provide insight into candidate mechanisms involved in the pathophysiology of those human afflictions in which CA metabolism may by dysregulated.
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