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Regulation of CYBB Gene Expression in Human Phagocytes by a Distant Upstream NF‐κB Binding Site
Author(s) -
Frazão Josias B.,
Thain Alison,
Zhu Zhiqing,
Luengo Marcos,
CondinoNeto Antonio,
Newburger Peter E.
Publication year - 2015
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.25155
Subject(s) - microbiology and biotechnology , biology , chromatin immunoprecipitation , binding site , hypersensitive site , electrophoretic mobility shift assay , promoter , gene expression , gene , genetics
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‐κB 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‐κB 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‐κB binding to the site in intact HL‐60 cells. Chromosome conformation capture (3C) assays demonstrated physical interaction between the NF‐κB binding site and the CYBB promoter region. Inhibition of NF‐κB activity by salicylate reduced CYBB expression in peripheral blood neutrophils and differentiated U937 monocytic leukemia cells. U937 cells transfected with a mutant inhibitor of κB “super‐repressor” showed markedly diminished CYBB expression. Luciferase reporter analysis of the NF‐κB site linked to the CYBB 5′ flanking promoter region revealed enhanced expression, augmented by treatment with interferon‐γ. These studies indicate a role for this distant, 15 kb upstream, binding site in NF‐κB regulation of the CYBB gene, an essential component of phagocyte‐mediated host defense. J. Cell. Biochem. 116: 2008–2017, 2015. © 2015 Wiley Periodicals, Inc.