The transcriptional regulator ID2 is required for specification of type 1 classical dendritic cells (cDC1), but the precise mechanism by which ID2 enforces cDC1 fate has been incompletely defined. Previous work identified a distal regulatory element, the Zeb2 −165-kb enhancer, as essential for normal hematopoiesis and capable of integrating competing transcription factor inputs during myeloid dendritic cell divergence. The present study interrogates how transcription factors acting at this single enhancer instruct divergent lineage outcomes and how ID2 interfaces with those inputs to secure cDC1 specification.
Using genome-wide binding and chromatin assays, the authors document that both CEBP family transcription factors and E proteins occupy the Zeb2 −165-kb enhancer. The enhancer contains discrete motif classes—CEBP recognition sites and E-box motifs—each serving as binding platforms for distinct regulatory factors. Figures provided in the article demonstrate binding of CEBPs and E proteins at this locus, establishing the enhancer as a hub for integrating multiple transcriptional signals.
Targeted deletion of the E-box motifs within the −165-kb enhancer produced a striking loss of B cell and plasmacytoid dendritic cell (pDC) development. These data indicate that E-box–dependent recruitment of E proteins to the enhancer is required for the normal formation of these lymphoid and dendritic lineages, linking E-box occupancy to lineage-specific gene regulatory outcomes.
In addition to effects on B cells and pDCs, deletion of the E-boxes altered innate lymphoid cell (ILC) development and skewed myelopoiesis toward the cDC1 lineage. The experiments reported show that removal of E-box motifs not only impairs certain lymphoid fates but also redistributes progenitor output within the myeloid compartment in favor of cDC1s.
Complementary perturbations of the enhancer reveal that both CEBP sites and E-boxes within the −165-kb element contribute to differentiation of classical dendritic cell subsets. The data indicate that discrete motif classes inside a single enhancer can exert separable influences on cell-fate decisions, with CEBP inputs and E-box/E protein inputs directing different aspects of cDC subset specification.
Normal Zeb2 expression requires intact CEBP sites and E-boxes in the −165-kb enhancer. The authors show that perturbation of these motifs impacts Zeb2 transcriptional output, linking motif-specific transcription factor occupancy to quantitative control of Zeb2 levels and thereby to downstream lineage choices.
The study reports that NFIL3 transiently represses Zeb2 as an early step in cDC1 fate initiation. NFIL3 input at the enhancer appears to provide a temporal repression of Zeb2, priming progenitors for subsequent stabilization of cDC1 identity by other factors.
A central finding is that ID2 promotes cDC1 development by antagonizing E protein activity at E-box motifs within the Zeb2 −165-kb enhancer. Loss of ID2 impairs cDC1 specification; however, deletion of the E-box motifs that recruit E proteins paradoxically rescues cDC1 development in Id2-deficient mice. This genetic epistasis supports a model in which ID2 acts, at least in part, by blocking E protein function at the enhancer to repress Zeb2 and permit cDC1 fate stabilization.
The authors document reciprocal regulatory interactions in which ZEB2 promotes B cell and pDC development by repressing ID2. This reciprocal antagonism contributes to lineage bifurcation, such that enhancer-mediated control of Zeb2 and ID2 levels forms a regulatory circuit directing alternative hematopoietic outcomes.
Collectively, the results support a two-step model: first, NFIL3 transiently represses Zeb2; second, ID2 inhibits E proteins to stabilize cDC1 fate. More broadly, the work defines a paradigm of “site-specific pleiotropy,” whereby distinct transcription factor motifs—E-boxes and CEBP sites—within a single enhancer encode instructions for multiple, divergent cell fates. This highlights how modular motif architecture within an enhancer can specify context-dependent regulatory outcomes across hematopoietic lineages.
The authors report that datasets generated in the study are deposited in Gene Expression Omnibus (GEO). Specifically cited accessions include lin− KIThi-int BM scRNA-seq (GSE336861), lin− KIThi-int FLT3+ BM CUT&RUN (GSE326868) and cMoP CUTAC (GSE326954). The study also reanalyzed several publicly available datasets (ATAC-seq, H3K27ac ChIP-seq, CUT&RUN and ChIP-seq datasets) with their relevant GEO or ENCSR accessions provided in the manuscript.
Figures in the article illustrate: binding of CEBP and E proteins at the enhancer; lineage consequences of E-box deletion for B cells and pDCs; effects on ILC development; contributions of CEBP sites and E-boxes to cDC subset differentiation; requirement of both motif classes for normal Zeb2 expression; NFIL3-mediated repression of Zeb2 in initiating cDC1 specification; ID2-dependent repression of Zeb2 to promote cDC1s; and reciprocal repression of ID2 by ZEB2 in promoting B cell and pDC fates. GEO accession numbers and reanalyzed datasets are listed in the article for users seeking primary data.
Note: This summary and rewrite reflect only the content provided in the source preview and figure legends. Details of experimental methods, quantitative results, statistical analyses and full author discussions are available in the full article and the deposited datasets referenced by accession number.