Ablation of cDC2 lineage specification by mutations within the −165 kb Zeb2 enhancer

Abstract

Abstract Efforts to determine the unique functions of the two types of classical dendritic cells, cDC1 and cDC2, are hindered by limited understanding of the divergence of the common dendritic cell progenitor (CDP). Some transcription factors act in commitment of already specified progenitors, such as Batf3 which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer, but how other factors control CDP divergence remains unknown. Here, we report the transcriptional mechanism of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis suggested that Nfil3 acts upstream of Id2, Batf3, and Zeb2 in cDC1 development but has not revealed its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and ChIP-seq analysis identified NFIL3 binding in the −165 kb Zeb2 enhancer at sites that also bind CCAAT-enhancer-binding proteins (C/EBPs). Mutation of these NFIL3/C/EBP sites by in vivo CRISPR/Cas9 targeting revealed functional redundancy, with C/EBPs and NFIL3 competing in binding these sites to support or repress Zeb2 expression, respectively. Mutation of these three NFIL3/C/EBP sites eliminated Zeb2 expression in myeloid, but not lymphoid progenitors, producing mice that completely lacked pre-cDC2 specification and mature cDC2 in vivo. These mice failed to make an appropriate TH2 response against H. polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths. Notably, Zeb2 expression is not required to maintain cDC2 identity, but acts only to block cDC1 specification. Thus, the competition between C/EBPs and NFIL3 binding at the −165 kb Zeb2 enhancer determines CDP divergence.