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-spe cific NADPH oxidase. CYBB is transcriptionally inactive until the prom yelocyte stage of myelopoiesis, and in mature phagocytes, expression o f gp91(phox) is further increased by interferon-gamma (IFN-gamma) and other inflammatory mediators. The CYBB promoter region contains severa l lineage-specific cis-elements involved in the IFN-gamma response. We screened a leukocyte cDNA expression library for proteins able to bin d to one of these cis-elements (-214 to -262 base pairs) and identifie d TF1(phox), a protein with sequence-specific binding to the CYBB prom oter. 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(ph ox) message abundance, but did not lead to cellular differentiation. O verexpression of TF1(phox) in myeloid leukemia cell lines increased re porter gene expression from artificial promoter constructs containing CYBB promoter sequence, These data suggested that TF1(phox) increased expression of gp91(phox).