Central control of dynamic gene circuits governs T cell rest and activation

The ability of cells to maintain distinct identities and respond to transient environmental signals requires tightly controlled regulation of gene networks^1-3. 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^4-8. 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^9-11. 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.