The interferon family of cytokines regulates several physiologic responses, such as antiviral, antitumor, and immune functions. They are in clinical use for the therapy of a number of cancers, viral diseases and neurodegenerative disorders. By interacting with other cytokines IFNs form a large network of intercellular signaling molecules that control neoplastic cell growth and host defenses against pathogens. Previous studies from our laboratory identified a novel response element called, the gamma-IFN activated transcriptional element (GATE), in the promoter of the IRFg/p48/ISGF3gamma gene. During the last funding period, we have characterized two GATE-binding factors and identified one of them as transcription factor CCAAT/Enhancer Binding protein-beta (C/EBP-beta). This factor regulates a number of disparate process including cell differentiation, energy metabolism, immune response, tumor growth and apoptosis. The deletion of C/EBP-beta gene in mice causes immunodeficiency, loss of macrophage dependent innate antitumor and antibacterial defenses. Since IRF9 was the only C/EBP-beta dependent IFN-gamma regulated gene known thus far we hypothesized that there might be other undefined genes under the control of IFN-gamma/C/EBP-beta pathway. We have now generated macrophage cell lines from wildtype and C/EBP-beta null mice and identified several genes. One of them is the death associated protein kinasel (DAPK1), a crucial regulator of apoptosis, cell cycle, and metastasis. This gene is frequently inactivated and/or deleted in several human cancers.
In specific aim 1, we propose to investigate the role of C/EBP-beta in regulating the DAPK gene, using promoter mutational analysis, in vivo foot printing, chromatin histone immunoprecipitation (CHIP) assays to demonstrate the physiologic relevance of C/EBP-beta to DAPK gene regulation.
In specific aim 2 we will investigate the role of MAPKinases in regulating IFN-induced DAPK-transcription. We also present preliminary evidence for the involvement of a novel protein, TRAP220, a component of the human transcriptional mediator machinery as a regulator of IFN-driven transcriptional response through C/EBP-beta. We will investigate the physiologic relevance of TRAP220 in regulating IFN-gamma driven transcription of DAPK using RNAi and ChIP assays. Lastly, we will investigate the disruption of MAPK/CEBP/TRAP220 interactions in human cancer cells that do not express endogenous DAPK1. This study will integrate signaling pathways and transcriptional control mechanisms that control cellular death responses through C/EBP-beta.
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