Identification of structural modules within the transcription factor IRF4 controlling T helper cell subtype diversification

我们多年的前期工作表明，转录因子IRF4在不同亚型的辅助性T细胞的分化过程中起着至关重要的作用。在分工中，它们的生理功能在于调节生物体内不同的免疫途径，例如防御细胞外细菌(Th17)、细胞内病毒(Th1)或蠕虫(Th2、Th9)。另一方面，这些亚型的失衡会导致多发性硬化症、哮喘或结肠炎等疾病。与IRF4不同的是，对于每个子类型(如GATA3、STAT6、PU.1或RORgT)，还有其他特定和必要的TF。这种转铁蛋白的缺失导致了对特定T细胞亚型介导的疾病的选择性抵抗。已知IRF4与这些其他TF在物理上相互作用。因此，我们假设IRF4的结构和功能模块存在，每个模块只与其他TF中的一个结合，从而具体贡献其单独的功能。这样的模块应该可以被IRF4突变体识别，在这些突变体中，只有部分功能选择性地丢失。在第一个资助期，我们产生了40个IRF4突变体并对其进行了部分鉴定。我们已经成功地描述了两个具有部分功能的突变。前者表现为IRF4过度活跃，主要表现为Th17功能增强，后者表现为Th9升高，Th17完全抑制。在本项目中，我们首先想要进一步描述这40个突变体的特征。此外，我们想通过以下方式更深入地分析上述两个突变体：a)产生具有这些突变的小鼠；b)分析这些小鼠中的免疫细胞表型；c)分析这些小鼠中上述疾病的病程；d)分析那些与各自的突变体发生物理作用的其他TF，因此是可共沉淀的；e)澄清由突变引起的IRF4结构的各自变化；f)利用这些信息来鉴定小分子抑制物，这些信息会引起带有未突变的IRF4的T细胞中类似的功能丧失，因此可能与各自的模块结合。在未来，这些抑制剂应该被评估为治疗上述疾病的药物。

As our many years of preliminary work show, the transcription factor (TF) IRF4 is crucially involved in the differentiation of different subtypes of T helper cells. In a division of labour, their physiological function lies in the regulation of different immune pathways in the organism, for example in the defense against extracellular bacteria (Th17), intracellular viruses (Th1) or worms (Th2, Th9). On the other hand, an imbalance in these subtypes leads to diseases such as multiple sclerosis, asthma or colitis. In contrast to IRF4, there are other TFs that are specific and essential for each of the subtypes (such as GATA3, STAT6, PU.1 or RORgT). Loss of such a TF leads to selective resistance to the disease mediated by the particular T cell subtype. IRF4 is known to physically interact with these other TFs. We therefore hypothesize that structural and functional modules of IRF4 exist which each bind to only one of the other TFs and thus specifically contribute to its individual function. Such modules should be identifiable by IRF4 mutants in which only partial functions are selectively lost. In the first funding period, we generated and partially characterized 40 mutants of IRF4. We have succeeded in describing two mutations with partial functions. In the first, there is hyperactivity of IRF4, mainly increasing Th17 function, while in the other, Th9 is increased and Th17 is completely suppressed. In the present project we would first like to further characterize the 40 mutants. Furthermore, we want to analyze the mentioned two mutants in much greater depth by a) generating mice with these mutations, b) analyzing the immune cell phenotypes in these mice, c) analyzing the course of the mentioned diseases in these mice d) the analysis of those other TFs that physically interact with the respective mutants and are therefore co-precipitable, e) the clarification of the respective change in IRF4 structure by the mutation and f) use of this information for the identification of small molecule inhibitors, which provoke a similar loss of function in T cells with unmutated IRF4 and therefore presumably bind to the respective module. In the future, such inhibitors should be evaluated as therapeutic agents for the diseases mentioned.