Extended Data Fig. 8: C/EBP factors bind to the –165 kb Zeb2 enhancer to support cDC2 and monocyte development.
From: Ablation of cDC2 development by triple mutations within the Zeb2 enhancer
a, Rank of the C/EBPα and C/EBPβ CUT&RUN peaks at the –165 kb Zeb2 enhancer. b, Representative flow plots showing GFP-reporter activity in monocytes transduced with empty retrovirus (minimal CMV promoter only) or retroviruses expressing the –165 kb Zeb2 enhancer or mutants of the indicated NFIL3/C/EBP binding sites. Data shown are one of three similar experiments. A bar graph on the right shows mean values of MFI ± s.d. c, Representative flow plots showing cDCs differentiated from WT CDPs retrovirally expressing C/EBPγ or C/EBPδ. Data shown are one of two similar experiments. d, Representative flow plots showing monocytes differentiated from WT CDPs retrovirally expressing C/EBPα or C/EBPβ (LAP isoform) (pre-gate: CD317– B220– cells). cDCs expressing empty-retroviral vectors are shown as controls (dashed black lines) for Ly-6C and CD115 expression in MHCII– CD11c– cells. Data shown are one of two similar experiments. e, Frequency of cDC1s, cDC2s and monocytes in all infected cells (left panel) or infected cDCs (right panel) differentiated from WT or Δ1+2+3 CDPs retrovirally expressing C/EBPα or C/EBPβ (LAP isoform). Data are pooled from four independent experiments. f, Single-cell RNA transcriptome analysis of CDPs showing Cebpa, Cebpb and Cebpe expression visualized with Loupe Browser. g, h, Frequency of cDC2 and representative flow plots showing in vivo developmental potential of Cebpbfl/fl CD117hi BM progenitors transduced with empty retrovirus or retrovirus expressing Cre recombinase. Recipient spleens were analyzed for the presence of retroviral transduced and donor-derived cDCs. Data are pooled from two independent experiments (n = 4 for each retrovirus). i, Gene expression microarray analysis of hematopoietic stem and progenitor cells from WT or Cebpa knockout mice. The cells were left un-treated or treated with Flt3L for 4 h. Shown are averages of quadruplicate gene expression values of the indicated genes. Mean ± s.d.; h: unpaired, two-tailed Mann−Whitney test.
