Cis-regulation between Irf8 enhancers during cDC1 development (ATAC-Seq). Cis-regulation between Irf8 enhancers during cDC1 development (ATAC-Seq)

Type 1 classical dendritic cells (cDC1s) development requires the transcription factor IRF8, and IRF8 mutations cause severe combined immunodeficiency, including disseminated bacille Calmette-Guérin (BCG) disease. IRF8 regulated enhancers are required for Irf8 expression in early DC progenitors and mature cDC1s. The E-protein-dependent +41 kb enhancer gains accessibility and activates transcription in common DC progenitors (CDPs), and its depletion abrogated pre-cDC1s specification. The Batf3-dependent +32 kb enhancer gains accessibility and activates transcription in pre-cDC1 progenitors, and its depletion impaired cDC1 maturation. The +32 kb Irf8 enhancer locus bears an enhancer transcript, and the production of enhancer RNA (eRNA) Gm39266 is dependent on +41 kb Irf8 enhancer. It was unclear whether the +32 kb and +41 kb enhancers act independently or cooperate to regulate Irf8 expression. We dissected the mechanisms using +32/+41 compound heterozygous mice, and found that while the +41 kb enhancer suffices for pre-cDC1 progenitor specification, the +32 kb and +41 kb enhancers must reside on one allele to support cDC1 maturation. The +41 kb enhancer cis-regulates chromatin accessibility and BATF3 binding at +32 kb enhancer, independent of eRNA Gm39266 transcripts or transcription across +32 kb Irf8 enhancer. Overall design: Examination of chromatin accessibility in the pre-cDC1 progenitors from Irf8 +32+/- and +32/+41 compound heterozygous mice by ATAC-seq.

1型经典树突状细胞（cDC1s）的发育依赖于转录因子IRF8，而IRF8突变可导致重症联合免疫缺陷，包括播散性卡介苗（bacille Calmette-Guérin, BCG）病。受IRF8调控的增强子是早期树突状细胞祖细胞以及成熟cDC1s中Irf8基因表达所必需的。依赖于E蛋白的+41kb增强子在常规树突状细胞祖细胞（common DC progenitors, CDPs）中获得染色质开放性并激活转录，其敲除会阻断前cDC1s的特化过程。依赖于Batf3的+32kb增强子在前cDC1祖细胞中获得染色质开放性并激活转录，其敲除会损害cDC1s的成熟过程。+32kb Irf8增强子位点存在增强子转录本，而增强子RNA（enhancer RNA, eRNA）Gm39266的生成依赖于+41kb Irf8增强子。目前尚不明确+32kb与+41kb增强子是独立发挥调控作用，还是协同调控Irf8基因的表达。本研究通过构建+32/+41复合杂合小鼠解析了其调控机制，研究发现：尽管+41kb增强子足以介导前cDC1祖细胞的特化，但+32kb与+41kb增强子必须位于同一等位基因上，才能支持cDC1s的成熟过程。+41kb增强子可顺式调控+32kb增强子区域的染色质开放性与BATF3结合，且这一调控过程不依赖于eRNA Gm39266转录本或+32kb Irf8增强子区域的跨区域转录。总体实验设计：通过转座酶可及性测序（assay for transposase-accessible chromatin using sequencing, ATAC-seq）分析Irf8 +32+/-及+32/+41复合杂合小鼠前cDC1祖细胞的染色质开放性。