gp91-phox Gene Expression System in Leukocytes Via Controlling GCN5 Regulates the Superoxide-Generating

The superoxide anion (O 2 2)-generating system is an important mechanism of innate immune response against microbial infection in phagocytes and is involved in signal transduction mediated by various physiological and pathological signals in phagocytes and other cells, including B lymphocytes. The O 2 2-generating system is composed of five specific proteins: p22-phox, gp91-phox, p40-phox, p47-phox, p67-phox, and a small G protein, Rac. Little is known regarding epigenetic regulation of the genes constituting the O 2 2-generating system. In this study, by analyzing the GCN5 (one of most important histone acetyltransferases)-deficient DT40 cell line, we show that GCN5 deficiency causes loss of the O 2 2-generating activity. Interestingly, transcription of the gp91-phox gene was drastically downregulated (to ∼4%) in GCN5-deficient cells. To further study the involvement of GCN5 in transcriptional regulation of gp91-phox, we used in vitro differentiation system of U937 cells. When human monoblastic U937 cells were cultured in the presence of IFN-g, transcription of gp91-phox was remarkably upregulated, and the cells were differentiated to macrophage-like cells that can produce O 2 2. Chromatin immunoprecipitation assay using the U937 cells during cultivation with IFN-g revealed not only that association of GCN5 with the gp91-phox gene promoter was significantly accelerated , but also that GCN5 preferentially elevated acetylation levels of H2BK16 and H3K9 surrounding the promoter. These results suggested that GCN5 regulates the O 2 2-generating system in leukocytes via controlling the gp91-phox gene expression as a supervisor. Our findings obtained in this study should be useful in understanding the molecular mechanisms involved in epigenetic regulation of the O 2 2-generating system in leukocytes. G eneration of superoxide anion (O 2 2) is an important function in phagocytes (1) and B lymphocytes (2). On stimulation, the O 2 2-generating system in leukocytes carries an electron from NADPH to molecular oxygen and generates O 2 2 , which is released either outside the cells or inside phagosomes. Five specific proteins are essential for the O 2 2-generating system: namely, large (gp91-phox) and small (p22-phox) subunits of cytochrome b558 in membranes (3–6) and cy-tosolic p40-phox, p47-phox, and p67-phox proteins (7–10). The cytochrome is dormant in resting leukocytes but becomes activated during phagocytosis to generate O 2 2 , a precursor of micro-bicidal oxidants. Activation of the cytochrome requires stimulus-induced membrane translocation of these cytosolic proteins and small G protein Rac (11, 12). The importance of the system is emphasized by a genetic …