Project description: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 gene regulators coordinate circuits that respond to extracellular cues remains poorly defined. The need for context-dependent regulation is prominent in human CD4+ T cells, where distinct cell lineages must respond to diverse signals to orchestrate effective adaptive immune responses and maintain homeostasis. Here, we performed CRISPR screens in multiple primary human CD4+ T cell contexts to identify regulators that control expression of IL2RA, which is a canonical marker of T cell activation in pro-inflammatory effector T cells (Teffs) and constitutively expressed in anti-inflammatory regulatory T cells (Tregs) where it is required for fitness. Strikingly, the majority of identified regulators are required in discrete cell type and stimulation timepoints, and a subset even had opposite functional effects in different 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. Upstream of these networks, MED12 – a component of the Mediator complex – serves as a dynamic orchestrator of regulators across conditions, governing both cell type- and stimulation-specific gene expression. MED12 interacts with the histone modifying complex COMPASS, affecting chromatin state and expression of genes encoding key context- and lineage-specific regulators; including rest maintenance factor KLF2 and activation promoting genes. We demonstrated that CRISPR ablation of MED12 blunted this cell state transition and protected T cells from activation-induced cell death. Overall, this work leverages CRISPR screens performed across primary cell conditions to identify context-specific regulators and dynamic gene circuits required to establish resting and activated T cell states.

Project description:The RNA polymerase II transcriptional Mediator subunit MED12 is broadly implicated in vertebrate brain development, and genetic variation in human MED12 is associated with X-linked intellectual disability and neuropsychiatric disorders. Although prior studies have begun to elaborate the functional contribution of MED12 within key neurodevelopmental pathways, a more complete description of MED12 function in the developing nervous system, including the specific biological networks and cellular processes under its regulatory influence, remains to be established. Herein, we sought to clarify the global contribution of MED12 to neural stem cell (NSC) biology through unbiased transcriptome profiling of mouse embryonic stem (ES) cell-derived NSCs following RNAi-mediated MED12 depletion. A total of 240 genes (177 up, 73 down) were differentially expressed in MED12-knockdown versus control mouse NS-5 (mNS-5) NSCs. Gene set enrichment analysis revealed MED12 to be prominently linked with “cell-to-cell interaction” and “cell cycle” networks, and subsequent functional studies confirmed these associations. Targeted depletion of MED12 led to enhanced NSC adhesion and upregulation of cell adhesion genes, including Syndecan 2 (SDC2). Concomitant depletion of both SDC2 and MED12 reversed enhanced cell adhesion triggered by MED12 knockdown alone, confirming that MED12 negatively regulates NSC cell adhesion by suppressing the expression of cell adhesion molecules. MED12-mediated suppression of NSC adhesion is a dynamically regulated process in vitro, enforced in self-renewing NSCs and alleviated during the course of neuronal differentiation. Accordingly, MED12 depletion enhanced adhesion and prolonged survival of mNS-5 NSCs induced to differentiate on gelatin, effects that were bypassed completely by growth on laminin. On the other hand, MED12 depletion in mNS-5 NSCs led to reduced expression of G1/S phase cell cycle regulators and a concordant G1/S phase cell cycle block without evidence of apoptosis, resulting in a severe proliferation defect. These findings suggest that MED12 contributes to the maintenance of NSC identity through a functionally bipartite role in suppression and activation of gene expression programs dedicated to cell adhesion and G1/S phase cell cycle progression, respectively. MED12 may thus contribute to the regulatory apparatus that controls the balance between NSC self-renewal and differentiation, with important implications for MED12-linked neurodevelopmental disorders.

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Centralized control of dynamic gene regulatory circuits governs human T cell rest and activation [Perturb-CITE-seq]

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