The neuropoietic cytokines, ciliary neurotrophic factor (CNTF) and leukemia inhibitory (LIF), are neurotrophic factors that promote neuronal survival in many different neuronal populations. While scientists are beginning to understand the initial steps which lead to CNTF and LIF's actions, the intermediate steps which alter patterns of neurons gene expression leading to long term changes in neuronal function are still unknown. CNTF and LIF induce expression of the gene that encodes the neuropeptide, vasoactive intestinal peptide (VIP), through a 180bp cytokine response element (CyRE) in the VIP promoter. The VIP CyRE is a necessary and sufficient to mediate a dramatic and specific transcriptional response to these neuropoietic cytokines in a cell specific and cytokine-specific manner. In neuroblastoma cells, CNTF and LIF induce the transcription factors Stat and AP-1 to bind to specific sites within the VIP CyRE. These important, but ubiquitous, proteins can not by themselves explain the specificity exhibited by the VIP CyRE. The objective of this proposal is to identify and characterize other functional components that mediate cytokine-induced transcription through the VIP CyRE. The central hypothesis is that cytokine-mediated regulation of VIP gene expression is dependent on previously uncharacterized transcription factors that act together with known signaling pathways to generate CNTF- and LIF- specific gene induction. The PI has identified a previously uncharacterized region of the VIP CyRE, located at its 3' end, that contribute significantly to its cytokine-mediated induction. In this application, the PI proposes to investigate the contribution of proteins which bind to this 3' region of (I) characterizing unidentified nuclear proteins and their binding sites within this 3' region, (ii) characterizing a functionally important transcription factor that binds to a partially characterized novel site in this 3' region, and (iii) cloning this functionally important transcription factor. At the completion of this research, the PI expects to have characterized the 3' region of the VIP CyRE and discovered which are the important sequences in this region that contribute to cytokine- mediated induction of the CyRE. The PI also expects to have characterized and cloned a novel transcription factor that contributes significantly to the cytokine mediated induction of VIP gene expression. Understanding the mechanisms through which these cytokines regulate neuronal gene expression will assist the PI in establishing the molecular basis of enhanced neuronal survival and may enable the manipulation of these signaling pathways and transcription factors to therapeutic benefit.
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