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. 2015 Sep;116(9):2008-17.
doi: 10.1002/jcb.25155.

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

Josias B Frazão  1 , Alison Thain  2 , Zhiqing Zhu  2 , Marcos Luengo  3 , Antonio Condino-Neto  1 , Peter E Newburger  2
Affiliations

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

Josias B Frazão et al. J Cell Biochem. 2015 Sep.

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-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.

Keywords: CYBB; ENHANCER; GENE EXPRESSION; NF-κB; PHAGOCYTE OXIDASE.

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Figures

Fig. 1
Fig. 1
Location of the putative NF-κB binding site upstream from the CYBB gene. The schematic diagram indicates the positions of DNase-I hypersensitive sites (HS) I, II, III, and IV;(25) the NF-κB sitewithin HS-II; and the transcription start site.
Fig. 2
Fig. 2
Gel shiftanalysis of the putative CYBB NF-κB site. Panel A: Binding of recombinant human p50.32P-5′ end-labeled NFκB.cybb oligonucleotide (0.5 ng,0.035 pmol) was incubated with rh-p50 (0.012 gel shift units) and analyzed by electrophoresis on a native polyacrylamide gel. Lane 1: free probe. Lanes 2–5: probe plus rh-p50. Lanes 3 and 4 contain a 100-fold excess of unlabeled specific competitors: NF-κB consensus sequence (NFκB.cons) and CYBB NF-κBsite (NFκB.cybb), respectively. Lane 5 contains a 100-fold excess of non-specific competitor DNA containing an AP-1 binding site. Panel B: Binding of HL-60 nuclear extract. End labeled NFκB.cybb probe was incubated with nuclear extract from HL-60 cells with or withoutTNF-α treatment, as indicated in the top margin, and then analyzed by electrophoresis on a native polyacrylamide gel. Incubations also included, as indicated above the image. Lanes4and 5: excess unlabeled oligonucleotide representing specific (“S”: NFκB.cons) or non-specific (“NS”: AP-1) competitors. Lane5: recombinant human I-κBα. Lanes 7–9: antibody to human p50 or p65, or preimmune IgG, as indicated. Labels in the right margin indicate bandsA, B, and C as discussed in the text. The images are each representative of three independent experiments.
Fig. 3
Fig. 3
ChIP-PCR analysis of the distant upstream NF-κB site. Protein-DNA complexes immunoprecipitated by anti-p50 and anti-p65 from HL-60 cells wereanalyzed by PCR amplification of the CYBB NF-κB binding site, CYBB exon 9 (negative control), and the IκBα NF-κB binding site (positive control) with nested primers. PCR templates were derived, as indicated in the top margin, from total cellular DNA, ChIP with anti-NF-κB antibodies or control immunoglobulin. Template-free PCR controls are shown for the outer and nested set of primers. The image is representative of three independent experiments.
Fig. 4
Fig. 4
Chromosome conformation capture (3C) analysis of interactions between the NF-κB site and the CYBB genomic region in NB4 cells. Bars and error brackets indicate mean and SD for interaction frequencies of the anchor primer (so indicated) with primers at the indicated genomic positions, with the most distal primer interaction defined as frequency = 1 (bars and error brackets represent mean ± SD of triplicate PCR amplifications for two biological replicates, indicated by blue and red bars). The positions of the putative NF-κB siteand CYBB geneelements mapped on a UCSC genome browser image (http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg18&position=chrX%3A37500000–37560000) showing (bottom to top) × chromosome genomic scale, DNase I hypersensitive sites (University of Washington DNasel Hypersensitivity by Digital DNasel data, including myeloid cell lines K562 and NB4, and GM12878, a lymphoblastoid B cell line that weakly expresses CYBB), and enhancer- and promoter-associated histone mark H3K4Me1 (mono-methylation of lysine 4 of the H3 histone protein; ENCODE data from eight cell lines, including GM12878 in salmon color).
Fig. 5
Fig. 5
Effect of NF-κB inhibitors on CYBB expression in response to LPS. Left Panel: Northern blot analysis of RNA from human peripheral blood neutrophils (upper lanes) and monocytes (lower lanes), treated with LPS, salicylate, or indomethacin as indicated in the overlying grid. RNA was probed with radiolabeled cDNA complementary to the CYBB gene or a normalization probe complementary to 18s RNA, as indicated in the left margin. The images are representative of three independent experiments. Right Panel: Quantification of data from a representative northern blot experiment. The bars indicate the fold change of CYBB transcript levels (normalized to levels of 18s rRNA) in neutrophils (upper graph) and monocytes (lower graph) treated with LPS, salicylate, or indomethacin as indicated in the overlying grid.
Fig. 6
Fig. 6
RT-PCR analysis of CYBB expression in U937 cells transfected with I-κB “super-repressor.” Total RNA from U937 cells was analyzed by RT-PCR as described in Materials and Methods. The panels show gel electrophoresis bands of amplicons from the CYBB and ACTB (β-actin) genes, as indicated. Lanes 1 and 2: native U937 cells. Lanes 3 and 4: U937 cells transfected with empty vector. Lanes 5 and 6: U937 cells transfected with IκBα “super-repressor” (serine to alanine mutations at serines 32 and 36). Lanes 2, 4, and 6: cells induced to monocytic differentiation with dibutyryl cAMP.
Fig. 7
Fig. 7
Expression of luciferase reportersdriven by the CYBB proximal promoter with the native or scrambled NF-κB binding sequence. U937 cells transfected, as indicated, with either pCMV-Bsd-Luc-NFκB-1,500 carrying the putative CYBB upstream NF-κB sequence and CYBB 15,00 bp proximal promoter driving the luciferase gene, or pCMV-Bsd-Luc-scramble-1,500, containing a scrambled sequence of the NF-κB site. In addition, cells were cotransfected, as indicated, with either pCMV4-3HA-κBa expressing the IκBα “super-repressor” or with the empty vector pCMV4-3HA. These combinations of transfectants were incubated, as indicated, with no cytokine or with IFN-γ for 24 h before transfection or with IFN-γ for 24 h and a further 48 h after transfection. The bars and error brackets represent means and SEMs of six independent experiments; asterisks indicate significance at P < 0.05 (one-tailed Mann-Whitney test) for comparison of data from the groups plotted at each end of the vertical line.
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