PU.1 as an essential activator for the expression of gp91^phoxgene in human peripheral neutrophils, monocytes, and B lymphocytes

Abstract

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91 phox ) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91 phox gene revealed a single-base mutation (C 3 T) at position ؊53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position ؊53 of the gp91 phox promoter, and the mutation at position ؊53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position ؊50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position ؊56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions ؊53 and ؊50 significantly reduced the gp91 phox promoter activity; however, the mutation at position ؊56 did not affect the promoter activity. In transient cotransfection study, PU

Cooperation of STAT-1 and IRF-1 in Interferon-γ-induced Transcription of the gp91 Gene

“…Electrophoretic Mobility Shift Assays (EMSAs)-Preparation of the double-stranded oligonucleotide probes and EMSAs were performed as described previously (18). In competition assays, a 100-molar excess of unlabeled competitor oligonucleotides was added prior to the addition of the probe to the mixture, which was then preincubated on ice for 15 min.…”

“…Preparation of Nuclear Extracts-Nuclear extracts were prepared from U937 cells treated or untreated with IFN-␥ as described previously (18) with some modifications. Approximately 1 ϫ 10 7 cells were swollen in 400 l of ice-cold buffer A (10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM dithiothreitol/Complete TM Protease Inhibitor Mixture (Roche Molecular Biochemicals)/Phosphatase Inhibitor Mixture I and II (Sigma)).…”

“…After a 15-min incubation on ice, the cells were incubated at 37°C under 5% CO 2 , 95% air for 5 h in RPMI 1640 containing 10% fetal calf serum with or without 100 units/ml IFN-␥. Reporter activities were measured as described previously (18).…”

“…Interferon regulatory factor (IRF)-2 also activates the gp91 phox promoter through two interferonstimulated response elements (ISRE) in the absence of CDP binding (15,17). PU.1 works as a pivotal transcription factor of the gp91 phox gene by binding a PU.1/hematopoietic-associated factor (HAF)-1-binding element centered at bp Ϫ53 in human neutrophils, monocytes, and B-lymphocytes (18). HAF-1 is a multiprotein complex, and its components are still not defined, but PU.1, IRF-1, ICSBP, and Elf-1 are picked out as candidates (19,20).…”

YY1 Binds Five cis-Elements and Trans-activates the Myeloid Cell-restricted gp91 Promoter

“…Fractions exhibiting abundant BID DNA binding activity (0.2-0.3 M KCl) were pooled and dialyzed to 0.1 M KCl. For mini-nuclear extracts, 10 7 HEL cells were transiently transfected with 20 g of CB6 ϩ or CB6-YY1 expression plasmids as described above, incubated for 12 h, and mini-nuclear extracts prepared as described (20).…”