Dynamic Foxp3-chromatin interaction controls tunable Treg cell function [PSI-ChIP]

Nuclear factor Foxp3 determines regulatory T (Treg) cell fate and function via mechanisms that remain unclear. Here we investigate the nature of Foxp3-mediated gene regulation in suppressing autoimmunity and antitumor immune response. Contrasting with previous models, we find that Foxp3-chromatin binding is regulated by Treg activation states, tumor microenvironment, and antigen and cytokine stimulations. Proteomics studies uncovered dynamic proteins within the Foxp3 proximity upon TCR or IL-2 receptor signaling in vitro, reflecting intricate interactions among Foxp3, signal transducers, and chromatin. Pharmacological inhibition and genetic knockdown experiments indicate that NFAT and AP-1 protein Batf are required for enhanced Foxp3-chromatin binding in activated Treg cells and tumor-infiltrating Treg cells to modulate target gene expression. Furthermore, mutations at Foxp3 DNA-binding domain destabilize Foxp3-chromatin association. These representative settings delineate context-dependent Foxp3-chromatin interaction, suggesting that Foxp3 associates with chromatin by hijacking DNA-binding proteins resulting from Treg activation or differentiation, which is stabilized by direct Foxp3-DNA binding, to dynamically regulate Treg cell function according to immunological contexts. Overall design: We developed a comparative proteomics method by projecting the spatial information (PSI) of nuclear proteins with peroxidase–mediated proximity ligation, thus revealing the protein components of the cis-regulatory elements bound by regulatory T cell lineage–specifying factor Foxp3. By incorporating this method with ChIP sequencing, we performed high efficient immunoprecipitation and ChIP sequencing.

核转录因子Foxp3（Nuclear factor Foxp3）可通过尚未阐明的分子机制决定调节性T（Treg）细胞的命运与功能。本研究旨在探究Foxp3介导的基因调控在抑制自身免疫与抗肿瘤免疫应答中的本质。与既往研究模型相悖，我们发现Foxp3-染色质结合行为受Treg细胞活化状态、肿瘤微环境、抗原及细胞因子刺激的调控。蛋白质组学研究揭示了体外T细胞受体（TCR, T cell receptor）或白细胞介素-2（IL-2, interleukin-2）受体信号刺激下，Foxp3邻近区域的动态蛋白谱，反映出Foxp3、信号转导分子与染色质之间的复杂相互作用。药物抑制与基因敲低实验表明，活化Treg细胞及肿瘤浸润Treg细胞中，活化T细胞核因子（NFAT, nuclear factor of activated T cells）与激活蛋白1（AP-1, activator protein 1）家族蛋白Batf对于增强Foxp3-染色质结合以调控靶基因表达是必需的。此外，Foxp3 DNA结合结构域的突变会削弱Foxp3与染色质的结合稳定性。上述一系列代表性实验阐明了依赖于免疫环境的Foxp3-染色质相互作用模式，提示Foxp3通过劫持由Treg细胞活化或分化产生的DNA结合蛋白与染色质结合，该过程可通过Foxp3直接与DNA结合得以稳定，从而根据免疫微环境动态调控Treg细胞功能。实验设计概述：我们开发了一种比较蛋白质组学方法，借助过氧化物酶介导的邻近连接技术呈现核蛋白的空间信息（PSI），以此揭示由调节性T细胞谱系特异性因子Foxp3所结合的顺式调控元件的蛋白组分。将该方法与染色质免疫共沉淀测序（ChIP sequencing）相结合，我们完成了高效免疫沉淀与ChIP测序实验。