The ability of cells to maintain distinct identities and respond to transient environmental signals requires tightly controlled regulation of gene networks^1,2,3. These dynamic regulatory circuits that respond to extracellular cues in primary human cells remain poorly defined. The need for context-dependent regulation is prominent in T cells, where distinct lineages must respond to diverse signals to mount effective immune responses and maintain homeostasis^4,5,6,7,8. Here we performed CRISPR screens in multiple primary human CD4⁺ T cell contexts to identify regulators that control expression of IL-2Rα, a canonical marker of T cell activation transiently expressed by pro-inflammatory effector T cells and constitutively expressed by anti-inflammatory regulatory T cells where it is required for fitness^9,10,11. Approximately 90% of identified regulators of IL-2Rα had effects that varied across cell types and/or stimulation states, including a subset that even had opposite effects across conditions. Using single-cell transcriptomics after pooled perturbation of context-specific screen hits, we characterized specific factors as regulators of overall rest or activation and constructed state-specific regulatory networks. MED12 — a component of the Mediator complex — serves as a dynamic orchestrator of key regulators, controlling expression of distinct sets of regulators in different T cell contexts. Immunoprecipitation–mass spectrometry revealed that MED12 interacts with the histone methylating COMPASS complex. MED12 was required for histone methylation and expression of genes encoding key context-specific regulators, including the rest maintenance factor KLF2 and the versatile regulator MYC. CRISPR ablation of MED12 blunted the cell-state transitions between rest and activation and protected from activation-induced cell death. Overall, this work leverages CRISPR screens performed across conditions to define dynamic gene circuits required to establish resting and activated T cell states.

细胞保持独特身份和响应瞬态环境信号的能力需要严格控制基因网络的调节^1,2,3。这些对原代人类细胞中的细胞外线索做出反应的动态调节回路仍然不明确。在 T 细胞中，对环境依赖性调节的需求非常突出，其中不同的谱系必须对不同的信号做出反应，才能产生有效的免疫反应并维持体内平衡^4,5,6,7,8。在这里，我们在多个原代人 CD4⁺ T 细胞环境中进行了 CRISPR 筛选，以确定控制 IL-2Rα 表达的调节因子，IL-2Rα 是促炎效应 T 细胞瞬时表达的 T 细胞活化的经典标志物，由抗炎调节性 T 细胞组成型表达，这是健身所必需的^9,10,11.大约 90% 的已鉴定 IL-2Rα 调节因子在不同细胞类型和/或刺激状态下具有不同的作用，包括一个子集甚至在不同条件下具有相反的作用。在对特定环境的筛选命中进行合并扰动后，使用单细胞转录组学，我们将特定因素表征为整体静止或激活的调节因子，并构建了状态特异性调节网络。MED12 是 Mediator 复合体的一个组分，充当关键调节因子的动态协调器，控制不同 T 细胞环境中不同调节因子的表达。免疫沉淀-质谱分析显示 MED12 与组蛋白甲基化 COMPASS 复合物相互作用。MED12 是组蛋白甲基化和编码关键环境特异性调节因子（包括其余维持因子 KLF2 和多功能调节因子 MYC）的基因表达所必需的。 MED12 的 CRISPR 消融减弱了静息和激活之间的细胞状态转换，并防止激活诱导的细胞死亡。总体而言，这项工作利用跨条件进行的 CRISPR 筛选来定义建立静息和活化 T 细胞状态所需的动态基因回路。