Identification and characterization of TF1(phox), a DNA-binding protein that increases expression of gp91(phox) in PLB985 myeloid leukemia cells.
The CYBB gene encodes gp91(phox), the heavy chain of the phagocyte-specific NADPH oxidase. CYBB is transcriptionally inactive until the promyelocyte stage of myelopoiesis, and in mature phagocytes, expression of gp91(phox) is further increased by interferon-gamma (IFN-gamma) and other inflammatory mediators. The CYBB promoter region contains several lineage-specific cis-elements involved in the IFN-gamma response. We screened a leukocyte cDNA expression library for proteins able to bind to one of these cis-elements (-214 to -262 base pairs) and identified TF1(phox), a protein with sequence-specific binding to the CYBB promoter. Electrophoretic mobility shift assay with nuclear proteins from a variety of cell lines demonstrated binding of a protein to the CYBB promoter that was cross-immunoreactive with TF1(phox). DNA binding of this protein was increased by IFN-gamma treatment in the myeloid cell line PLB985, but not in the non-myeloid cell line HeLa. Overexpression of recombinant TF1(phox) in PLB985 cells increased endogenous gp91(phox) message abundance, but did not lead to cellular differentiation. Overexpression of TF1(phox) in myeloid leukemia cell lines increased reporter gene expression from artificial promoter constructs containing CYBB promoter sequence. These data suggested that TF1(phox) increased expression of gp91(phox).
Eklund
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Lurleen B. Wallace Tumor Institute, Department of Hematology and Oncology, and the Comprehensive Cancer Center, University of Alabama, Birmingham School of Medicine, Birmingham, Alabama 35294, USA.
Kakar
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D001483:Base Sequence; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007951:Leukemia, Myeloid / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D007371:Interferon-gamma; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases