High IRF8 drives Mycl expression through interaction with PU.1. (A) H3K27ac ChIP-seq of the indicated populations for region surrounding the murine Mycl locus. (B) ChIP-seq of the indicated populations for IRF8, IRF4, BATF3, and PU.1 as labeled. (A and B) Putative enhancer regions are bound by a black box and defined by their distance from the transcription start site of Mycl. (C) Left: Flow cytometry analysis cDC1s (Ly6D⁻CD11c⁺MHCII⁺CD24⁺CD172⁻) derived from day 7 Flt3l cultured Lin⁻CD117^high BM progenitors infected with two retroviruses, one overexpressing BATF3 and human CD4 as a selection marker, and another overexpressing IRF8 and Thy1.1 as a selection marker. Right: Histograms of Mycl-GFP expression are derived from the indicated populations and gated into five subpopulations on the basis of Thy1.1-IRF8 expression levels. (D) Quantification of Mycl-GFP expression (MFI) in Irf8^+/+Mycl^GFP/+ cDC1s as a function of Thy1.1-IRF8 expression levels in BM progenitor populations infected with empty vectors, Thy1.1-IRF8 infected, or hCD4-BATF3 double-infected populations. Samples were normalized to maximum GFP signal detected among gates 1–5 for each independent experiment. (E) Representative histograms from which MFIs are derived in F as a function IRF8 expression levels defined by gates 1–5 in C. (F and G) As described in D, BM progenitors from Irf8^−/−Mycl^GFP/+ infected with Thy1.1-IRF8 or Thy1.1-IRF8-R294C with (G) or without (F) hCD4-BATF3. ***, P < 0.001. Data are representative of two independent experiments (n = 3–5). (H–J) Expression microarray analysis (n = 2) of cDCs generated by culturing whole BM from Irf4^−/−Irf8^−/− in Flt3l for 9 d, with retroviral overexpression of either Irf4 or Irf8 (H), or coexpression of Irf4 or Irf8 with Batf3 (I). (J)Mycl expression levels are quantified from duplicate experiments (H and I) relative to WT Flt3l-cultured cDC1 and cDC2. IP, immunoprecipitation.