ID2 secures cDC1 specification by antagonizing E proteins at a pleiotropic Zeb2 enhancer

Abstract The transcriptional regulator ID2 is required for type 1 classical dendritic cell (cDC1) specification, yet the mechanism has remained obscure. We previously identified the Zeb2 -165-kb enhancer as key to normal hematopoiesis, controlled by competing CEBP and NFIL3 inputs during myeloid dendritic cell divergence. Here, we uncover an unprecedented role for E proteins in myelopoiesis and demonstrate that ID2 promotes cDC1 development by antagonizing E protein activity at E-boxes within the Zeb2 enhancer. Deleting these E-boxes abolishes lymphoid B cell and plasmacytoid dendritic cell (pDC) development while skewing myelopoiesis toward cDC1s. Remarkably, E-box deletion rescues cDC1 development in Id2-deficient mice. These findings support a two-step model in which NFIL3 transiently represses Zeb2, followed by ID2-mediated inhibition of E proteins to stabilize cDC1 fate specification. Further, this work defines a paradigm of “site-specific pleiotropy,” wherein distinct transcription factor motifs–E-boxes and CEBP sites–within a single enhancer direct diverse cell fates.