Histone deacetylation of CIITA promoters by DcR3 occurs via activation of JNK and ERK. (A) Expression of RFX factors and CIITA in MDMs. cDNAs were prepared from MDMs treated with either mock (medium only), hIgG1, or DcR3 for 6 days and the expression of mRNAs corresponding to HLA-DR, RFX5, RFXAP, and the 3 CIITA isoforms was examined by RT-PCR analysis. (B) Effects of trichostatin A (TSA) on the expression of HLA-DR. MDMs were pretreated with TSA for 30 minutes, followed by DcR3 treatment for 6 days. Expression of HLA-DR was examined by flow cytometry with allophycocyanin-conjugated anti–HLA-DR mAb. The open histogram represents isotype control, and the shaded histogram corresponds to specific staining. (C) Effect of DcR3 on the acetylation of H3 associated with CIITA promoters. ChIP was performed with antibodies specific for acetylated histone H3 (AcH3). The immunoprecipitates were analyzed by PCR to determine the presence of the regulatory region of the CIITA genes. The proximal promoter of the GAPDH was amplified as an internal control. (D) Kinetics of MAPK activation after DcR3 treatment. Cell lysates were collected at the indicated time points after DcR3 treatment to examine the activation status of ERK, JNK, and p38 using antibodies specific for the phosphorylated form of each MAPK. (E) Effects of MAPK inhibitors on the expression of HLA-DR. Macrophages were pretreated with SP600125, SB203580, and PD98059 for 30 minutes, followed by DcR3 treatment for 6 days. Expression of HLA-DR in MDMs was determined by flow cytometry with allophycocyanin-conjugated anti–HLA-DR mAb. Open histograms represent isotype control and shaded histograms correspond to specific staining. (F) Effects of MAPK inhibitors on the acetylation of H3 associated with CIITA promoters. MDMs were prepared as in panel E, and chromatin was immunoprecipitated with antibodies specific for AcH3; this was followed by PCR to determine the presence of the CIITA regulatory regions. The proximal promoter of the GAPDH was amplified as an internal control.