Optimization of the Irf8 +32 kb enhancer disrupts dendritic cell lineage segregation [scRNA-seq]

Autoactivation of lineage-determining transcription factors (TFs) mediates bistable expression to generate distinct cell phenotypes essential for complex body plans. Classical dendritic cells type 1 (cDC1) and type 2 (cDC2) provide non-redundant functions required for defense against distinct immune challenges. Interferon Regulatory Factor 8 (IRF8), the cDC1 lineage-determining TF, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32 kb enhancer, responsible for IRF8 autoactivation, has been tuned to possess low-affinity IRF8 binding sites. Incorporation of multiple high-affinity IRF8 binding sites into the Irf8 +32 kb enhancer induces erroneous IRF8 autoactivation in specified cDC2 progenitors, causing their redirection towards cDC1 and a novel hybrid DC subset with mixed lineage phenotypes. These developmental alterations critically impair both cDC1- and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of classical dendritic cells required for normal immune function.