Regulatory mechanisms governing Th17 cell effector identity and plasticity

项目总结 辅助性T细胞(Th)是一种表达CD4的T淋巴细胞，可分化为几个亚类以支持不同的 免疫反应。在Th亚群分化的多样性选项中，产生IL-17A的炎性细胞 Th17细胞因其相对较高的固有可塑性而脱颖而出。事实上，这个子集 正常发挥粘膜免疫功能的细菌和真菌很容易采用其他T细胞的特征 环境条件更改时的辅助对象子集。虽然此功能在以下情况下是有利的 清除感染，调节失调的Th17细胞功能与许多自身免疫有关 疾病，包括炎症性肠病、多发性硬化症和类风湿性关节炎。此外， 在炎症性疾病的背景下表现出的Th17细胞可塑性往往具有Th1样的特征，例如 IFNG或T-bet的表达，也与病理增加相关。在我们目前资助的研究中， 我们发现JunB-T-bet轴是调控Th17细胞谱系稳定性的调控网络的中心节点 与可塑性的对比。而影响Th17细胞识别和转录程序的几个调节剂 灵活性已被确定，但对三维基因组结构的调节器知之甚少 这在细胞类型和状态之间是不同的，是基因调控的关键决定因素。因此，我们假设 家族特异性因子对染色质确认的调节是控制CD4T细胞效应器的关键 转换。在这里，我们建议综合评价染色质构型的动态变化 以及体内控制Th17细胞效应器转换的顺式基因组元件。为此，在目标1中，我们将 应用遗传命运图谱小鼠模型、全球染色质构象分析和表观基因组图谱 在Th17细胞可塑性小鼠模型中描述染色质环路动力学的工具 实验性自身免疫性脑脊髓炎(EAE)。我们将确定循环对Th17细胞的贡献 通过评估染色质组织蛋白和谱系调节的作用实现塑料转化 转录因子。在目标2中，我们计划确定调控Th17细胞可塑性的顺式调控网络。 为此，我们将应用对EAE中Th细胞的scATACseq剖析来识别与可塑性相关的顺式元件。我们 将使用新颖的体内高通量报告分析和 CRISPR/Cas9表观基因组筛选。这将确定新的监管网络、监管机构和基因靶点 对Th17细胞效应器的可塑性至关重要。综上所述，拟议的工作将填补知识的一个重要空白 关于控制Th17细胞稳定性和可塑性的分子机制。

PROJECT SUMMARY T helper (Th) cells are CD4-expressing T lymphocytes that diversify into several subclasses to support distinct immune responses. Among the diversity of Th subset differentiation options, IL-17A-producing inflammatory Th17 cells stand out as unique by virtue of their relatively high level of inherent plasticity. Indeed, this subset that normally functions in mucosal immunity against bacteria and fungi can easily adopt features of other T helper subsets when environmental conditions change. While this feature can be advantageous during the clearance of an infection, dysregulated Th17 cell function has been implicated in numerous autoimmune conditions, including Inflammatory Bowel Disease, Multiple Sclerosis, and Rheumatoid Arthritis. Moreover, Th17 cell plasticity exhibited in the context of inflammatory disease tends to take on Th1-like traits, such as expression of IFNg or T-bet, that are also associated with increased pathology. In our current funded studies, we identified a JunB-T-bet axis as a central node in the regulatory network governing Th17 cell lineage stability versus plasticity. While several regulators influencing the transcriptional program of Th17 cell identity and flexibility have been identified, less is known about the regulators of the three-dimensional genome architecture that differs between cell types and states and is a critical determinant of gene regulation. Thus, we hypothesize that regulation of chromatin confirmation by lineage-specific factors is key to the control of CD4 T cell effector conversions. Here, we propose to comprehensively evaluate the dynamic changes in chromatin configuration and the cis genomic elements that govern Th17 cell effector conversions in vivo. To this end, in Aim 1, we will apply genetic fate-mapping mouse models, global chromatin conformation assays, and epigenomic profiling tools to characterize the chromatin looping dynamics during Th17 cell plasticity in a mouse model of experimental autoimmune encephalomyelitis (EAE). We will determine the contribution of looping to Th17 cell plastic conversion by assessing the role of both chromatin organizing proteins and lineage-regulating transcription factors. In Aim 2, we plan to determine the cis regulatory network that governs Th17 cell plasticity. For this, we will apply scATACseq profiling of Th cells in EAE to identify plasticity-associated cis elements. We will validate candidates for activity and function using novel in vivo high throughput reporter assays and CRISPR/Cas9 epigenomic screens. This will identify new regulatory networks, regulators, and gene targets critical to Th17 cell effector plasticity. Taken together, the proposed work will fill an important gap in knowledge concerning the molecular mechanism governing stability versus plasticity of Th17 cells.