The divergence of the common dendritic cell progenitor^1,2,3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages^4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors—such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer^4,6—but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis⁷ suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the –165 kb Zeb2 enhancer⁸ at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPβ. In vivo mutational analysis using CRISPR–Cas9 targeting showed that these NFIL3–C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3–C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (T_H2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting T_H2 responses to helminths^9,10,11. Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the –165 kb Zeb2 enhancer.

^{人们对共同树突细胞祖细胞1,2,3} (CDP)分化为传统 1 型和 2 型树突细胞（分别为 cDC1 和 cDC2）谱系^4,5的了解甚少。一些转录因子在已经指定的祖细胞的承诺中发挥作用，例如 BATF3，它稳定+32 kb Irf8增强子^4,6处的Irf8自激活，但控制 CDP 初始分歧的机制仍然未知。在这里，我们报告了 CDP 分歧的转录基础，并描述了 pre-cDC2 规范的首要要求。遗传上位分析⁷表明Nfil3在cDC1发育中作用于Id2、Batf3和Zeb2的上游，但没有揭示其机制或靶标。对新生成的 NFIL3 报告小鼠的分析显示，在 cDC1 规范期间，NFIL3 表达极其短暂。CUT&RUN 和染色质免疫沉淀随后进行测序，鉴定出内源性 NFIL3 结合在 –165 kb Zeb2增强子⁸的三个位点上，这些位点也结合了 CCAAT 增强子结合蛋白 C/EBPα 和 C/EBPβ。使用 CRISPR-Cas9 靶向的体内突变分析表明，这些 NFIL3-C/EBP 位点在功能上是冗余的，其中 C/EBP 支持这些位点的Zeb2 表达，而 NFIL3 抑制 Zeb2 的表达。所有三个 NFIL3-C/EBP 位点的三重突变消除了骨髓中Zeb2 的表达，但不消除淋巴祖细胞中的表达，导致前 cDC2 规范完全丧失和体内成熟的 cDC2 发育。这些小鼠没有产生针对Heligmosomoides polygyrus感染的辅助性T 2 ( _TH 2)细胞反应，这与cDC2支持对蠕虫的_{TH 2 反应一致}^9,10,11。因此，CDP 分化为 cDC1 或 cDC2 是通过 NFIL3 和 C/EBP 在 –165 kb Zeb2增强子处的竞争来控制的。