Centralized control of dynamic gene regulatory circuits governs human T cell rest and activation [H3K27ac_CUTnRUN]
The ability of individual cells to maintain a distinct identity and respond to transient environmental signals requires tightly controlled regulation of gene networks. However, how discrete sets of nuclear regulators coordinate dynamic circuits that respond to extracellular cues remains poorly defined. This is prominent in CD4+ T cells, where cells must respond to diverse immunogenic signals to orchestrate effective adaptive immune responses and maintain immune homeostasis. Here, we performed CRISPR screens in multiple primary human CD4+ T cell contexts to identify nuclear regulators that control expression of IL2RA, a canonical marker of T cell activation that is differentially regulated between pro-inflammatory effector T cells and anti-inflammatory regulatory T cells. Strikingly, we discovered that the majority of identified regulators function within discrete cell type and stimulation timepoints, and a subset even had opposite effects across conditions. Using single-cell transcriptomics and surface proteomics after pooled perturbation of context specific screen hits, we characterized many factors as regulators of overall rest or activation and constructed state-specific regulatory networks. KLF2, MYB, and SOCS3 were distinguished as interconnected repressors of activation in resting T cells while GATA3 and MYC are required for proper stimulation response. Upstream of these circuits, MED12 – a component of the Mediator complex – serves as a dynamic orchestrator of trans regulators across conditions, governing both cell type and stimulation specific gene expression. Chromatin analysis revealed how MED12 coordinates key regulators as T cells transition to and from rest and activation. We demonstrated that CRISPR ablation of MED12 blunted this transition and protected T cells from activation-induced cell death. Overall design: H3K27ac CUT&RUN for CRISPR KO primary human CD4+ Teffs and CD4+ Tregs. Samples include both resting and stimulated Teffs and Tregs.  AAVS1 KOs samples serve as wild-type controls for the dataset.