ATAC-seq of dendritic cell progenitors. ATAC-seq of dendritic cell progenitors

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. Here we identified Irf8 enhancers and used CRISPR/Cas9 genome editing to assess their roles in Irf8 regulation. An enhancer 32 kilobases downstream of the Irf8 transcriptional start site (+32 kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41 kb Irf8 enhancer previously thought to be active only in plasmacytoid DCs (pDCs) was found to also be transiently accessible in cDC1 progenitors. Deletion of this enhancer reduced Irf8 expression in pDCs as expected, but also surprisingly prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41 kb Irf8 enhancer in DC progenitors is responsible for cDC1 fate specification. Overall design: ATAC-seq of three different dendritic cell progenitors (MDP, CDP, pre-cDC1) with three biological replicates of each progenitor

转录因子Irf8在普通树突状细胞祖细胞（common dendritic cell progenitor, CDP）中的诱导是经典1型树突状细胞（classical type 1 dendritic cell, cDC1）命运特化的必要条件，但调控该诱导过程的分子机制仍不明确。本研究首先鉴定了Irf8的增强子，并利用CRISPR/Cas9基因组编辑技术评估其在Irf8基因调控中的功能。研究发现，位于Irf8转录起始位点下游32 kb处的+32 kb Irf8增强子在成熟cDC1中具有活性，该增强子对于该细胞谱系的发育而非命运特化是必需的。与之相反，此前被认为仅在浆细胞样树突状细胞（plasmacytoid DCs, pDCs）中具有活性的+41 kb Irf8增强子，被发现也可在cDC1祖细胞中短暂开放。敲除该增强子不仅如预期般降低了pDC中的Irf8表达水平，还意外阻断了CDP中Irf8的诱导过程，并彻底取消了cDC1的命运特化。由此可见，树突状细胞祖细胞中+41 kb Irf8增强子的隐蔽激活是cDC1命运特化的核心驱动因素。实验设计概况：对三种不同的树突状细胞祖细胞——髓系树突状细胞祖细胞（MDP）、普通树突状细胞祖细胞（CDP）、前cDC1细胞（pre-cDC1）——进行转座酶可及性测序（ATAC-seq），每种祖细胞设置三个生物学重复。