PU.1 is dominant and HAF-1 supplementary for activation of the gp91(phox) promoter in human monocytic PLB-985 cells.
Gp91(phox) is a key component of the phagocyte NADPH oxidase. Mutations of its promoter found in patients with chronic granulomatous disease cause deficient binding of PU.1 and HAF-1. Because the two factors bind to the same site (Pu box) of the promoter, we attempted to clarify their relative in vivo contributions to activation of the gp91(phox) promoter in monocytically differentiated PLB-985 cells using a dual luciferase reporter assay and a gel shift competition assay. We found that the activity of a series of single-point-mutated promoters increases or decreases according to an increase or decrease, respectively, in the affinity of the promoters to PU.1 but not to HAF-1. Two of 7 mutants showing weak binding affinity to PU.1 exhibited moderate promoter activity and normal binding affinity for HAF-1. These results suggest PU.1 is the dominant activator and HAF-1 is supplementary. The increased promoter activity of single-, double-, and triple-point-mutated constructs with sequences closer to that of the Ets-binding element correlates with their binding affinity to PU.1 but not to HAF-1, supporting that PU.1 is a more efficient activator than HAF-1. In contrast to co-expressed wild-type PU.1, dominant-negative PU.1 significantly inhibited the activity of a PU.1-optimised gp91(phox) promoter construct. Therefore, we conclude that PU.1 and HAF-1 binding to the Pu box is dominant and supplementary, respectively, for activation of the gp91(phox) promoter in human monocytic cells.
Islam
Md Rafiqul
MR
Department of Host-Defense Biochemistry, Institute of Tropical Medicine, Nagasaki University, Sakamoto, 852-8523, Japan.
Fan
Chun
C
Fujii
Yoshito
Y
Hao
Li Jun
LJ
Suzuki
Shoichi
S
Kumatori
Atsushi
A
Yang
Dan
D
Rusvai
Erzsebet
E
Suzuki
Norio
N
Kikuchi
Hiroshi
H
Nakamura
Michio
M
D001667:Binding, Competitive; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D005799:Genes, Dominant; D006609:High Mobility Group Proteins; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics*; D008957:Models, Genetic; D009000:Monocytes / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D000099232:Proto-Oncogene Protein Spi-1
D006609:High Mobility Group Proteins; D008156:Luciferases; D008562:Membrane Glycoproteins; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C102078:HAF-1 protein, human; C056558:CYBB protein, human