The monocyte chemoattractant protein-1 (MCP-1) functions to amplify inflammatory responses and is associated with the modulation of tumor immunity, atherosclerosis, rheumatoid arthritis, fibrotic lung disease, and allergic responses. MCP-1 is strongly induced by TNFalpha, a pleiotropic cytokine that plays an important role in tumor surveillance, inflammation, cachexia, septic shock,and HIV activation.Together, MCP-1 and TNF amplify inflammatory responses and have the potential to exacerbate disease. To be able to design novel therapies targeting TNF induction of MCP-1, the molecular mechanism of MCP-1 regulation must be elucidated. In this application the TNF induction of MCP-1, encoded by JE gene in the mouse, will be studied as a model system to determine how chemokines are regulated by primary inflammatory mediators. Using deletion analysis of the promotor, EMSAs, and in vivo genomic footprinting (IVGF), they have defined two complex regulatory regions that are responsible for MCP-1/JE expression. The distal region, located 2.5 kb upstream from the transcription start site is responsible for TNF induction and is composed of three elements: two B sites and an element termed Site A. The proximal region contains three sites as well, including an SPI binding element. Our results showed that with the exception of the most distant element, Site A, both regulatory regions were unoccupied prior to TNF treatment. This was true for the proximal elements, even though these factors were present in three nucleus prior to TNF treatment and could bind DNA in vitro. Upon TNF treatment both regions became occupied. They hypothesize that TNF initiated the assembly of factors by either inducing the activation of a factor, such as NK-kB, altering chromatin organization, or both. Because Site A was occupied in vivo prior to TNF treatment and was required for maximal TNF induction, they hypothesize that this element may serve a unique function, perhaps as a control element for factor assembly or as boundary/insulator element. Following TNF induction, IVGF detected the formation pronounced hypersensitive site between the kB elements. They propose that this site may be indicative of complex protein interactions in the distal region. Experiments proposed in this application will test these hypothesis:
Aim 1 will determine the cis-acting elements that are required for assembly of factors in vivo;
Aim 2 will determine the function of Site A and clone the factors that bind its DNA;
and Aim 3 will investigate protein-protein interactions in the distal region.
