Regulation of CYBB Gene Expression in Human Phagocytes by a Distant Upstream NF-kappaB Binding Site

Abstract

The human CYBB gene encodes the gp91-phox component of the phagocyte oxidase enzyme complex, which is responsible for generating superoxide and other downstream reactive oxygen species essential to microbial killing. In the present study, we have identified by sequence analysis a putative NF-kappaB binding site in a DNase I hypersensitive site, termed HS-II, located in the distant 5' flanking region of the CYBB gene. Electrophoretic mobility assays showed binding of the sequence element by recombinant NF-kappaB protein p50 and by proteins in nuclear extract from the HL-60 myeloid leukemia cell line corresponding to p50 and to p50/p65 heterodimers. Chromatin immunoprecipitation demonstrated NF-kappaB binding to the site in intact HL-60 cells. Chromosome conformation capture (3C) assays demonstrated physical interaction between the NF-kappaB binding site and the CYBB promoter region. Inhibition of NF-kappaB activity by salicylate reduced CYBB expression in peripheral blood neutrophils and differentiated U937 monocytic leukemia cells. U937 cells transfected with a mutant inhibitor of kappaB "super-repressor" showed markedly diminished CYBB expression. Luciferase reporter analysis of the NF-kappaB site linked to the CYBB 5' flanking promoter region revealed enhanced expression, augmented by treatment with interferon-gamma. These studies indicate a role for this distant, 15 kb upstream, binding site in NF-kappaB regulation of the CYBB gene, an essential component of phagocyte-mediated host defense.