IN-VIVO FOOTPRINTING OF THE MOUSE INDUCIBLE NITRIC-OXIDE SYNTHASE GENE - INDUCIBLE PROTEIN OCCUPATION OF NUMEROUS SITES INCLUDING OCT AND NF-IL6

A wide variety of cells usefully but sometimes destructively produce n itric oxide via inducible nitric oxide synthase (iNOS). Data obtained by gel shift analysis and reporter assays have linked murine iNOS gene induction by cytokines and bacterial products with the binding of a n umber of proteins to a proximal promoter, as well as to a distal enhan cer of the iNOS gene. Nevertheless, these techniques do not necessaril y reflect protein occupation of sites in vivo. To address this, we hav e used dimethyl sulphate in vivo footprinting to determine binding eve nts in the two murine iNOS transcription control regions, using a clas sical lipopolysaccharide induction of RAW 264.7 macrophages. Protein-D NA interactions are absent before activation. Exposure to lipopolysacc haride induces protection at a NF-kappa B site and hypersensitivity at a shared gamma-activated site/interferon-stimulated response element within the enhancer. Protections are seen at a NF-IL6, and an Oct site within the promoter. We also observe modulations in guanine methylati on at two regions which do not correspond to any known putative bindin g elements. Furthermore, we confirm the probable involvement of interf eron regulatory factor-1 (binding to its -901 to -913 site) and the bi nding of NF-kappa B to its proximal site. Our data demonstrate an abun dance of hitherto-unrecognised protein-DNA binding events upon simple lipopolysaccharide activation of the iNOS gene and suggests a role for protein-protein interactions in its transcriptional induction.