Regulation of the type I interferon response by chromatin organization (ATAC-seq of KO HoxB8-FL cells)

Antiviral type I interferons (IFN-I) including IFN-ß and multiple subtypes of IFN-a are encoded within a single locus. Whereas most cell types produce primarily IFN-ß, plasmacytoid dendritic cells (pDCs) respond to viruses by rapidly producing all IFN-I subtypes. We show that during pDC differentiation, the IFN-I locus translocates from the nuclear periphery and undergoes reorganization of topologically associated domains (TADs). Accordingly, IFN-I production by pDCs was strictly dependent on the TAD-organizing cohesin complex. Promoters of Ifna genes in pDCs were poised, which was imparted by the pDC-enriched transcription factor IRF8. Finally, we identified two distal regulatory regions that facilitated the expression of adjacent IFN-I genes. Thus, the IFN-I response is controlled by multilevel chromatin organization of the IFN-I locus, including its unique poised state in pDCs. Overall design: ATAC-seq was performed on purified pDCs (or bulk DCs for Irf8?) differentiated from HoxB8-FL progenitors transfected with sgRNAs targeting either Ifnar1, Irf7, Spib, Irf8 or a non-targeting sgRNA as a control (2 replicates per genotype).

抗病毒I型干扰素（IFN-I）包含干扰素β（IFN-β）与多种干扰素α（IFN-α）亚型，均由单个基因座编码。尽管多数细胞类型主要合成IFN-β，但浆细胞样树突状细胞（pDCs）可通过快速产生所有I型干扰素亚型来响应病毒感染。本研究揭示，在pDC分化过程中，I型干扰素基因座会从核周区域发生移位，并伴随拓扑关联结构域（TADs）的重塑。因此，pDC产生I型干扰素的过程严格依赖于调控TAD结构的黏连蛋白复合体。pDC内干扰素α基因的启动子处于预启动（poised）状态，这一表观状态由pDC富集的转录因子IRF8介导确立。本研究最终鉴定出两个远端调控区域，可促进邻近I型干扰素基因的表达。综上，I型干扰素应答由I型干扰素基因座的多层染色质结构调控，包括其在pDC中特有的预启动状态。
实验整体设计：从转染了靶向Ifnar1、Irf7、Spib、Irf8的单向导RNA（sgRNAs），或作为对照的非靶向sgRNA的HoxB8-FL祖细胞分化而来的纯化pDCs（针对Irf8靶点的实验采用批量DCs？）中提取样本，进行转座酶可及性测序（ATAC-seq），每组基因型设置2次生物学重复。