Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks [Amplicon III]. Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks [Amplicon III]

Complex gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression within these networks is sufficiently perturbed it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map the connections between regulatory genes and their downstream targets. However, it has been challenging to identify the upstream regulators for genes of interest. Here we develop a system for systematic discovery of upstream regulators of critical immune factors – IL2RA, IL-2, and CTLA4 – in primary human T cells. Then, CRISPR perturbations coupled with RNA-Seq, and ATAC-Seq, allowed us to map the full network of these regulators’ target genes and enhancers. These regulators form highly interconnected networks with extensive feedback loops. Furthermore, this network is highly enriched for autoimmune-associated disease variants and genes. These results provide insight into how autoimmune associated disease genes are regulated in T cells as well as broader principles about the structure of human gene regulatory networks. Overall design: Amplicon sequencing to detect insertion/deletions at CRISPR guide RNA target sites.

复杂的基因调控网络（gene regulatory network）可确保关键基因以精准的水平表达。当这些网络内的基因表达发生显著扰动时，便可能引发疾病。为解析基因表达扰动如何在调控网络中扩散传导，我们首先需要绘制调控基因与其下游靶标之间的关联图谱。然而，针对目标基因鉴定其上游调控因子一直是一项颇具挑战性的工作。本研究开发了一套可在原代人类T细胞中系统性鉴定关键免疫因子——IL2RA、IL-2及CTLA4——上游调控因子的研究体系。随后，本研究结合CRISPR介导的基因扰动实验与RNA测序（RNA-Seq）、转座酶可及性测序（ATAC-Seq），完整绘制了这些调控因子的靶基因与增强子调控网络全貌。这些调控因子构成了具有大量反馈环路的高度互联调控网络。此外，该网络显著富集自身免疫相关疾病变异位点与疾病基因。本研究结果不仅解析了T细胞中自身免疫相关疾病基因的调控机制，同时也为人类基因调控网络整体结构的解析提供了新的理论依据。实验整体设计：采用扩增子测序技术，检测CRISPR向导RNA靶位点的插入/缺失突变。