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. 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.

单个细胞维持独特身份并响应瞬时环境信号的能力，离不开对基因网络的精准调控。然而，离散的核调节因子如何协同构建响应胞外信号的动态通路，目前仍未得到清晰阐明。这一问题在CD4阳性T细胞（CD4+ T cell）中尤为突出：这类细胞必须响应多样化的免疫原性信号，以协调有效的适应性免疫应答并维持免疫稳态。
本研究在多种原代人CD4阳性T细胞培养环境中开展CRISPR筛选（CRISPR screen），旨在鉴定调控IL2RA表达的核调节因子。IL2RA是T细胞活化的经典标志物，其表达调控模式在促炎性效应T细胞与抗炎性调节性T细胞之间存在显著差异。
令人意外的是，本研究发现绝大多数鉴定得到的调节因子仅在特定细胞类型和刺激时间点发挥功能，甚至有部分调节因子在不同实验条件下的作用完全相反。通过对筛选获得的环境特异性靶点进行批量扰动后的单细胞转录组学（single-cell transcriptomics）与表面蛋白质组学（surface proteomics）分析，我们将诸多因子归类为静息状态或活化状态的调控因子，并构建了状态特异性的调控网络。
其中，KLF2、MYB与SOCS3被鉴定为静息T细胞中相互关联的活化抑制因子，而GATA3与MYC则是T细胞对刺激产生恰当应答所必需的核心因子。在这些调控通路上游，中介复合物（Mediator complex）组分MED12可作为跨条件的转录调节动态协调因子，同时调控细胞类型特异性与刺激特异性的基因表达。染色质分析进一步揭示了MED12如何在T细胞往返于静息与活化状态的过程中协同关键调控因子。本研究证实，MED12的CRISPR敲除会阻滞这一状态转换，并保护T细胞免于活化诱导的细胞死亡。
本数据集包含针对原代人CD4阳性效应T细胞（Teffs）与CD4阳性调节性T细胞（Tregs）的CRISPR敲除样本的H3K27ac CUT&RUN检测数据，样本涵盖静息与活化状态的Teffs和Tregs。其中AAVS1敲除样本作为该数据集的野生型对照。