C/EBP⍺ activates Irf8 expression in myeloid progenitors at the +56 kb

Development of type 1 conventional dendritic cells (cDC1) 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 novel paradigm for superenhancer function. We produced in vivo mutations of two C/EBP binding sites that comprise the Irf8 +56 kb enhancer that abrogate IRF8 expression in all myeloid progenitors and impair cDC1 development. These sites do not bind RUNX1 or RUNX3, and C/EBPα expression instead is regulated by their action at the Cebpa +37 kb enhancer, placing RUNX factors upstream of Cepba in regulating Irf8. Finally, we demonstrate that cis interactions between the +56 kb Irf8 enhancer and the previously reported +41 kb and +32 kb Irf8 enhancers are mandatory in the sequential progression of these stage specific constituent elements.