Determination of chromatin protein dynamics in CD8 T cells

CD8 T细胞中染色质蛋白动力学的测定 CD8 T细胞对于抗病毒和抗肿瘤免疫至关重要。这些细胞如何对刺激做出反应， 为了治疗目的而操纵它们的效应器功能已经在深入研究中。是 广泛接受T细胞抗原受体（TCR）和辅助受体信号传导驱动几乎所有的关键过程 通过基因表达的转录和表观遗传调节，CD8 T细胞分化和功能。因素 调节TCR信号转导或靶基因表达可能在决定CD8 T细胞分化和免疫功能。尽管直接介导TCR和辅助受体的蛋白质 信号转导已经被很好地定义，调节靶基因表达的核蛋白仍然是 进行全面调查。需要无偏见的方法来全面揭示核蛋白 调节TCR和共受体依赖性基因表达，可能导致鉴定新的 控制CD8 T细胞分化和功能的因子决定抗病毒和抗肿瘤免疫应答。 最近，CRISPR筛选提供了前所未有的效率，可以系统地描述基因表达的调控因子。 CD8 T细胞分化和功能。然而，这种方法并没有揭示出 候选因子和信号通路，并且还受到可用实验读数的严重限制。 我们推断，确定CD8 T细胞中活性基因启动子和增强子的动态蛋白， 在TCR和共受体刺激后，这将是一种解决方案，因为蛋白质在这些细胞中被募集或耗尽， 在这个过程中的区域将揭示新的关键监管机构。为了确定动态染色质蛋白， 感兴趣的基因组区域，我们最近开发了一种比较蛋白质组学方法， 在另一项研究中，在初步实验中，我们将这种方法应用于小鼠原发性 CD8 T细胞，并分析了由组蛋白H3赖氨酸标记的活性增强子和启动子处的动态蛋白 27乙酰化（H3K27ac），因为这种信号传导似乎作用于这些遗传修饰。 元件，以控制CD8 T细胞中的重要功能基因（如细胞因子和Myc）， 组蛋白乙酰化途径。该实验揭示了已知的TCR信号转导子和许多新的TCR信号转导子。 代表性过高或过低的因素。然后，我们进行了体内合并CRISPR筛选， 敲除这些动态蛋白，发现了几种肿瘤浸润的正性和负性调节因子， CD8 T细胞。在这些结果的基础上，我们建议进一步确定在CD8染色质蛋白动力学 T细胞，并确定其在调节诱导型基因表达和CD8 T细胞抗肿瘤活性中的作用。 总之，我们将采用我们开发的一种新的蛋白质组学方法来确定信号依赖的染色质 蛋白质动力学，以无偏地鉴定CD8 T细胞功能的新型调节剂。这些因素可以作为新的 目的是改善基于CD8 T细胞的免疫疗法。

Determination of chromatin protein dynamics in CD8 T cells CD8 T cells are crucial for antiviral and antitumor immunity. How these cells respond to stimulations and how to manipulate their effector function for therapeutic purposes have been under intensively investigations. It is widely accepted that T cell antigen receptor (TCR) and co-receptor signaling drives almost all the key processes of CD8 T cell differentiation and function via transcriptional and epigenetic regulation of gene expression. Factors modulating TCR signal transduction or target gene expression presumably play important roles in dictating CD8 T cell differentiation and immunological functions. Although proteins directly mediating TCR and co-receptor signal transduction have been well defined, the nuclear proteins modulating target gene expression remain to be fully investigated. Unbiased approaches are required to comprehensively reveal the nuclear proteins regulating TCR and co-receptor dependent gene expression, potentially leading to the identification of novel factors governing CD8 T cell differentiation and function that determine antiviral and antitumor immune response. Recently CRISPR screening offers an unprecedented efficiency to systematically delineate the regulators of CD8 T cell differentiation and function. However, this method does not uncover the mechanistic linkage between candidate factors and signaling pathways and is also severely constrained by available experimental readouts. We reasoned that defining the dynamic proteins of the active gene promoters and enhancers in CD8 T cells upon TCR and co-receptor stimulation would be a solution, because proteins recruited to or depleted at these regions during this process would reveal novel key regulators. To determine the dynamic chromatin proteins at genomic regions of interest, we recently developed a comparative proteomics method using antibody-guided proximity biotinylation in a separate study. In preliminary experiments, we applied this method to mouse primary CD8 T cells and profiled the dynamic proteins at active enhancers and promoters marked by histone H3 lysine 27 acetylation (H3K27ac) upon TCR and co-stimulation, because this signaling appears to act on these genetic elements to control important functional genes in CD8 T cells (such as cytokines and Myc) by coordinating with the histone acetylation pathway. This experiment revealed known TCR signal transducers and a number of novel factors overrepresented or underrepresented. We then performed an in vivo pooled CRISPR screening by knocking out these dynamic proteins and discovered several positive and negative regulators of tumor infiltrating CD8 T cells. On the basis of these results, we propose to further define the chromatin protein dynamics in CD8 T cells and determine their roles in modulating inducible gene expression and CD8 T cell antitumor activity. Overall, we will employ a new proteomics method, developed by us, to determine signal dependent chromatin protein dynamics to unbiasedly identify novel regulators of CD8 T cell function. These factors may serve as new targets to improve CD8 T cell based immunotherapies.