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

正如我们多年的前期工作所示，转录因子（TF）IRF4在不同亚型T辅助细胞的分化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中不同的免疫途径，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，这些亚型的不平衡导致疾病，如多发性硬化症，哮喘或结肠炎。与IRF4相反，还有其他TF对每个亚型（如GATA 3，STAT 6，PU. 1或RORgT）具有特异性和必需性。这种TF的缺失导致对由特定T细胞亚型介导的疾病的选择性抗性。已知IRF4与这些其他TF发生物理相互作用。因此，我们假设存在IRF4的结构和功能模块，每个模块仅与其他TF中的一个结合，从而特异性地促进其个体功能。这样的模块应该是可识别的IRF4突变体，其中只有部分功能被选择性丢失。在第一个资助期内，我们产生并部分表征了40个IRF4突变体。我们已经成功地描述了两个突变的部分功能。在第一种情况下，存在IRF 4的过度活跃，主要增加Th17功能，而在另一种情况下，Th9增加而Th17完全被抑制。在本项目中，我们首先要进一步表征40个突变体。此外，我们希望通过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）分析这些小鼠中所述疾病的过程，d）分析与相应突变体物理相互作用并因此可共沉淀的那些其它TF，e）通过突变阐明IRF4结构的相应变化，和f）使用该信息鉴定小分子抑制剂，其在具有未突变的IRF4的T细胞中引起类似的功能丧失，因此推测与相应模块结合。在未来，这种抑制剂应被评估为所述疾病的治疗剂。

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.