C/EBPα activates <i>Irf8</i> expression in myeloid progenitors at the +56-kb enhancer to initiate cDC1 development

Development of type 1 conventional dendritic cells (cDC1s) underlies the capacity to generate antiviral and antitumor immune responses. Here, we identify the basis for cDC1 development from its earliest progenitors, determining the hierarchy of several required transcription factors and uncovering a series of mandatory cis interactions between constituent enhancers within the <i>Irf8</i> superenhancer. We produced in vivo mutations of two C/EBPα binding sites that comprise the <i>Irf8</i> +56-kilobase (kb) enhancer that markedly reduced IRF8 expression in all myeloid progenitors and impaired cDC1 development. These sites did not bind RUNX1 or RUNX3, and C/EBPα expression was instead regulated by their action at the <i>Cebpa</i> +37-kb enhancer, placing RUNX factors upstream of <i>Cebpa</i> in regulating <i>Irf8</i>. Last, we demonstrate that cis interactions between the +56-kb <i>Irf8</i> enhancer and the previously reported +41- and +32-kb <i>Irf8</i> enhancers are mandatory in the sequential progression of these stage-specific constituent elements.