TNF REGULATES THE IN-VIVO OCCUPANCY OF BOTH DISTAL AND PROXIMAL REGULATORY REGIONS OF THE MCP-1 JE GENE/

In vivo genomic footprinting (IVGF) was used to examine regulatory sit e occupancy during the activation of the murine inflammatory response gene MCP-1/JE by TNF. In response to TNF, both promoter distal and pro ximal regulatory regions became occupied in vivo. EMSA analysis showed that while some of the factors involved in expression, including NF-k appa B, were translocated to the nucleus following TNF treatment, othe rs were already present and able to bind DNA in vitro. Protein kinase inhibitor studies showed that protein phosphorylation was required for TNF activation but not factor assembly. These studies provide evidenc e for a multistep model of TNF-mediated gene regulation involving chro matin accessibility, transcription factor complex assembly, and protei n phosphorylation.