Extended Data Fig. 2: AICEs in mouse and human IRF8 enhancers show low binding affinities to the BATF3-JUNB-IRF8 complexes.

(a) Competitive EMSA showing binding of ³²P-dCTP-labeled Ctla4 probe with the indicated unlabeled competitors at increasing amounts (0, 25, 100, 400, and 1600⁻fold excess). The NEs used were combined from 293FT cells transfected with Junb and Batf3 and 293FT cells transfected with Irf8. Bands corresponding JUNB-BATF3-IRF8 complexes and free probes are indicated by the upper and lower arrows, respectively. Data shown are representative of two similar experiments. (b) EMSA performed with ³²P-dCTP-labeled probes on various NEs: NEs from 293FT cells transfected with Junb and Batf3 (B), combined NEs from 293FT cells transfected with Junb and Batf3 and 293FT cells transfected with IRF8 (B + I), and NEs from MutuDCs. Bands corresponding JUNB-BATF3-IRF8 and JUNB-BATF3 complexes are indicated by the upper and lower arrows, respectively. Data shown are representative of three similar experiments. (c) Alignment of a 161 bp region encompassing the four AICEs in the mouse Irf8 + 32 kb enhancer (chr8:120,768,539-120,768,699, mm10) across several mammalian species, colored by conservation (Jalview). The six most probable AICEs (defined using FIMO and the AICE PWM from Fig. 1b) are indicated. (d) Retroviral reporter analysis of the mouse Irf8 + 32 kb enhancer and the human IRF8 + 48 kb enhancer in cDC1s and cDC2s obtained from KitL/Flt3L cultured mouse BM cells. Shown are enhancer-driven GFP expression in transduced cDC1s (pre-gate: B220^- CD11c⁺ MHCII⁺ CD24⁺ Sirpa⁻ Thy1.1⁺ cells) and transduced cDC2s (pre-gate: B220⁻ CD11c⁺ MHCII⁺ Sirpa⁺ Thy1.1⁺ cells). Numbers are GFP geometric MFI. Data shown are representative of two similar experiments. (e) Competitive EMSA performed with ³²P-dCTP-labeled Ctla4 probe in the presence of 200-fold excess of various unlabeled competitors. Competitors derived from the human +48 kb enhancer are denoted as ‘Hu’. Bands corresponding JUNB-BATF3-IRF8 complexes and free probes are indicated by the upper and lower arrows, respectively. Data shown are representative of two similar experiments.