An Nfil3-Zeb2-Id2 pathway imposes Irf8 enhancer switching during cDC1 development (RNA-Seq)

Classical type 1 dendritic cells (cDC1s) are required for anti-viral and anti-tumor immunity, which has motivated a need to understand their development. The development of the cDC1 progentitor requires an E protein–dependent enhancer located 41 kilobases downstream of the transcription start site of the transcription factor IRF8 (+41 kb Irf8 enhancer) but its maturation instead requires the BATF3-dependent +32 kb Irf8 enhancer. To understand this switch, we performed single-cell RNA sequencing of the common dendritic cell progenitor (CDP) and identified a cluster of cells that expressed transcription factors that influence cDC1 development, such as Nfil3, Id2, and Zeb2. Genetic epistasis among these factors revealed that Nfil3 expression is required for the transition from Zeb2hi and Id2lo CDPs to Zeb2lo and Id2hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E protein activity to exclude plasmacytoid DC potential and explains the switch in Irf8 enhancer usage during cDC1 development. Overall design: To develop a comprehensive profile of heterogeneity within the CDP, we employed high-throughput single-cell RNA sequencing (scRNA-seq) of 10,000 CDPs.