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

Abstract

The superoxide anion (O2−)-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 O2−-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 O2−-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 O2−-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-γ, transcription of gp91-phox was remarkably upregulated, and the cells were differentiated to macrophage-like cells that can produce O2−. Chromatin immunoprecipitation assay using the U937 cells during cultivation with IFN-γ 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 O2−-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 O2−-generating system in leukocytes.

DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation

“…4F). Because histone acetyltransferases (HATs) have previously been reported to acetylate NF-kB (23), we next determined whether blocking HATs with the HAT inhibitor (HATi) AA (44)(45)(46) could diminish K310 acetylation in SCRIPT-KD-DCs. As shown in Fig.…”

“…In addition to the broad specificity HATi AA (44)(45)(46), we also included the p300/ CBP-specific HATi's curcumin (47,48) and C646 (49) because p300/CBP has previously been implicated in NF-kB acetylation (23). As shown in Fig.…”

Protein kinase Cθ gene expression is oppositely regulated by GCN5 and EBF1 in immature B cells

“…RT-PCR was carried out essentially as described [11,12,16,17,[20][21][22]. PCRs were carried out using chicken PKCs primers listed in the previous report [12].…”

“…ChIP assay was performed using Chromatin Immunoprecipitation Assay Kit (Millipore, Billerica, MA, USA) as described [20][21][22][23]. Immunoprecipitation with anti-GCN5 antibody (Chemicon, Temecula, CA, USA), anti-acetylated histone antisera (Millipore) or anti-Flag M2 antibody (Sigma-Aldrich, St. Louis, MO, USA) was carried out.…”

“…Next, we performed ChIP assay using anti-GCN5 antibody and anti-acetylated histone antisera (Lys-9 of histone H3 (H3K9) and Lys-14 of histone H3 (H3K14)). These two Lys residues of histone H3 are typical acetylation sites catalyzed by GCN5 [10,[20][21][22][23]25]. Precipitated chromatins were amplified by PCR using ChIP primers for the 5 0 -flanking region of the chicken PKCh gene (Fig.…”