Glucocorticoids (GCs) and epinephrine are important hormones released from the adrenal gland when the hypothalamic-pituitary-adrenal (HPA) axis is activated during stress. Glucocorticoids also play an important role in the expression of epinephrine since they control its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). Specifically, GCs activate PNMT transcription through the glucocorticoid receptor (GR), which binds to a recognitiOn site, a glucocorticoid response element (GRE), in the PNMT gene promoter/regulatory sequences. In addition to the GR, other transcriptional proteins, Egr-1, Sp1 and AP-2, may activate PNMT gene expression and it is likely that several may be required in concert for PNMT transcription. In the rat, these factors appear to be limited to the proximal 2 kb of PNMT promoter/regulatory sequences. The studies in this proposal will 1) identify transcriptional activators of PNMT through their canonical binding sequences in the first 2 kb of the rat PNMT promoter and verify their functionality using in vitro and in vivo methodologies, 2) identify whether the candidate factors are important during development, in tissue-specific expression or in response to specific stimuli, 3) determine whether interaction of the factors is required for PNMT transcription and 4) identify the mechanism by which the factors function. To establish the functionality of the factors, transient transfection assays coupled with pharmacologic paradigms, gel mobility shift assays with antibody and competitor DNA, in vitro and in vivo DNA footprinting and methylation interference assays, and antisense strategies will be utilized. To examine if the factors confer developmental, tissue or stimuli-specific expression, the expression of the mRNA and protein for the factor and PNMT will be correlated by RNase protection assay/in situ hybridization and immunohistochemistry/Wes-term analysis and/or enzyme activity as appropriate, using paradigms specific to those types of expression. Interactions between the candidate factors will be identified through their synergistic effects on reporter gene expression with PNMT promoter-reporter gene constructs and ablation/diminution of reporter gene activation with site-direCted mutt PNMT promoter-reporter gene constructs. Finally, the mechanisms by which the transcription factors function will be identified, including the activating receptor, signal transduction pathway, and gene and protein expression. These studies should provide critical information regarding the role of various transcription factors in adrenergic ontogeny and control of adrenergic function as well as further understanding of general mechanisms regulating gene expression. In mental illness, the HPA axis is an important component, and neurochemical deficits are likely not restricted to abnormal neural proteins. Mutations in DNA regulatory elements or the genes encoding the cognate transcriptional factors may be important in the etiology of psychiatric disease. It is therefore important to define these regulatory controls and their function, particularly, in the case of the stress hormone/neurotransmitter, epinephrine.
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