Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells [ATAC-seq]

Cis-regulatory elements (CRE) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of adjacent T cell costimulatory genes CD28, CTLA4, and ICOS encoding regulators of cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells – both Conventional and Regulatory subsets – uncovered gene-, cell subset- and stimulation-specific CREs. Integrating these data with CRISPR knockout (KO) screens and ATAC-seq characterization identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. Lastly, we discovered and extensively validated a critical CTCF boundary that governs this locus, serving to reinforce CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis. Primary human Treg cells were isolated, subjected to AAVS1 control or FOXP3 knockout, and harvested for ATAC-seq.

顺式调控元件（Cis-regulatory elements, CRE）可与反式调控因子（trans regulators）相互作用，协同调控基因表达，但多基因位点内的转录调控协同机制尚未通过实验手段阐明。本研究旨在解析调控相邻T细胞共刺激基因CD28、CTLA4及ICOS动态表达的顺式调控元件，上述基因编码细胞介导免疫的调控因子。在原代人T细胞（涵盖常规T细胞与调节性T细胞亚群）中开展的平铺式CRISPR干扰（CRISPR interference, CRISPRi）筛选，鉴定出了基因特异性、细胞亚群特异性及刺激条件特异性的顺式调控元件。将上述筛选数据与CRISPR敲除（CRISPR knockout, KO）筛选结果及ATAC-seq染色质可及性表征数据整合后，本研究明确了可在特定CRISPRi响应元件处调控染色质状态、进而控制共刺激基因表达的反式调控因子。最后，本研究发现并经全面验证了一个关键的CTCF边界元件，该元件可调控该基因位点：一方面强化顺式调控元件与CTLA4的相互作用，另一方面亦可阻断CD28的异常激活。本研究直接在原代人T细胞亚群中系统性绘制了顺式调控元件及其关联的反式调控因子，克服了长期以来的实验局限，从而破译了这一对免疫稳态至关重要的复杂多基因位点内的情境依赖性基因调控程序。研究人员分离了原代人调节性T细胞（regulatory T cell, Treg），分别以AAVS1位点对照或FOXP3敲除处理，并收取细胞用于ATAC-seq测序分析。