Epigenetic modifications on the CpG island of Irf7 gene. (A) Flt3-pDCs were either untreated or treated with CpG-A (2 µg/ml) for 3 h, and nuclear versus cytoplasmic distribution of CXXC5 was analyzed by Western blotting. This experiment was repeated twice with similar results. (B) Cell lysates from Flt3L-differentiated WT and CXXC5^−/− (KO) pDCs were cross-linked and immunoprecipitated with anti-CXXC5. The enrichment of various regions of Irf7 promoter was measured by real-time PCR and quantified over respective inputs. This experiment was repeated three times, and data (n = 4, mean ± SD) were analyzed by Student’s t test (**, P < 0.01). TSS, transcription start site. (C and D) 500 ng genomic DNA from Flt3L-pDCs (C) and GM-CSF–cDCs (D) generated from WT and CXXC5^−/− mice were treated with sodium bisulfite, and the CGI region of Irf7 (500–1,000) was amplified by PCR and sequenced (n = 10). This experiment was conducted twice. (E) Flt3L-differentiated pDCs were cross-linked and sonicated, and chromatin fragments were immunoprecipitated with 5hmC antibodies. 5hmC levels at the promoter region of Irf7, Cxcl2, and Tnfα were quantified by real-time PCR (n = 3). This experiment was repeated once, and the data are presented as mean ± SD (***, P < 0.001). (F–H) Enrichment of Ezh2 and Sin3a and histone modifications in Irf7 CGI (500–1,000; F and G) and enrichment of pol II at the promoter region of Irf7, Tnfα, and Cxcl2 (H) in pDCs were quantified by real-time PCR (n = 3). These experiments were repeated twice with similar results (data were analyzed by Student’s t test and are presented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001).