The murine monocyte chemoattractant protein-1 (MCP-1) is an inducible gene product that functions to recruit monocytes and T cells to sites of inflammation. MCP-1 has been shown to play an important role in normal wound healing, allergy, atherosclerosis, and tumor immunity. Tumor necrosis factor (TNF) and platelet derived growth factor (PDGF) both regulate the expression of MCP-1. MCP-1 gene expression is controlled by two complex regulatory regions that are separated by 2.3 kb of DNA. The proximal region requires Sp1 for its activity and is sufficient for PDGF induction while TNF induction requires both the proximal and distal regions. The distal region requires NF-kB p65 for its activity. In vivo, the proximal region is unoccupied by transcription factors prior to stimulation, even though these factors (e.g., Sp1) are present and active in the nucleus. Moreover, cell lines deficient in Sp1 and NF-kb apart. These data suggest that transcription factor assembly is coordinate. Furthermore, inhibitors of histone deacetylation were shown to stimulate MCP-1 expression, suggesting a role for nucleosome modification in this system. From these observations, they hypothesize that MCP-1 expression requires long-distance protein-protein interactions and that changes in chromatin structure are required for transcription factor access and activity. To determine if this hypothesis is correct, experiments are proposed to: 1) determine if an how chromatin structure is altered following TNF or PDGF treatment; 2) determine the properties of p65 and Sp1 that might be responsible for coordinate assembly or changes in chromatin structure; 3) determine the role of coactivators on activation of this system; and 4) examine interactions between NF-kB, Sp1, and coactivators on naked and chromatin MCP-1 DNA in vitro. Results from this study will describe the molecular mechanism by which MCP-1 is regulated by TNF and PDGF, as well as provide knowledge about how complex enhancers communicate over long distances to regulate gene expression. It is hoped that this knowledge will aid in the development of novel therapies to regulate macrophage and lymphocyte infiltration in cancer, as well as in many other diseases and pathologies.
