In vivo Footprinting of the Mouse Inducible Nitric Oxide Synthase Gene: Inducible Protein Occupation of Numerous Sites Including Oct and NF-IL6

Abstract

A wide variety of cells usefully but sometimes destructively produce nitric 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 number of proteins to a proximal promoter, as well as to a distal enhancer of the iNOS gene. Nevertheless, these techniques do not necessarily reflect protein occupation of sites in vivo. To address this, we have used dimethyl sulphate in vivo footprinting to determine binding events in the two murine iNOS transcription control regions, using a classical lipopolysaccharide induction of RAW 264.7 macrophages. Protein-DNA interactions are absent before activation. Exposure to lipopolysaccharide induces protection at a NF-kappaB 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 methylation at two regions which do not correspond to any known putative binding elements. Furthermore, we confirm the probable involvement of interferon regulatory factor-1 (binding to its -901 to -913 site) and the binding of NF-kappaB to its proximal site. Our data demonstrate an abundance 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.