CIITA and MHC II Transcription

CIITA 和 MHC II 转录

• 批准号: 6683552
• 负责人: Boris Matija PETERLIN
• 金额: $ 15.15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6683552
• 关键词: MHC class II antigen; acetylation; antigen presentation; antigen presenting cell; biological signal transduction; gene expression; gene targeting; genetic manipulation; genetic promoter element; genetic transcription; genetically modified animals; immunity; interferon gamma; laboratory mouse; major histocompatibility complex; phosphorylation; posttranslational modifications; protein localization; protein sequence; protein structure function; transcription factor; ubiquitin

DESCRIPTION (provided by applicant): Transcription of major histocompatibility complex class II (MHCII) genes and thus antigen processing and presentation (APP) to CD4+ T helper cells and their education of MHCII-positive B cells for immunoglobulin class switching and hypermutation is carefully regulated in development and in tissues. Four separate transcription factors are dedicated solely to the transcription of structural MHCII genes, DMA/DMB and the invariant chain (Ii). Of these, the class II transactivator, CIITA plays a key role in turning these genes on and off following the administration of IFN-gamma. Recently, we discovered that in addition to IFN-gamma, antigen presenting cells (APC) require another signal that phosphorylates CIITA. Phosphorylation of CIITA leads to its oligomerization, accumulation and increased activity on MHCII promoters. CIITA is also not a DNA-binding protein but is somehow attracted to the MHCII enhanceosome, which assembles on conserved upstream sequences in these promoters. This grant will investigate the precise signals that are required for the full activity of CIITA, what posttranslational modifications occur and how they create super-CIITA complexes that trigger the full APP. First, nucleocytoplasmic shuttling of CIITA will be investigated in great detail. Precise NES and NLS will be mapped and confirmed functionally. Next, phosphorylation, acetylation and ubiquitylation of CIITA will be investigated in great detail. These sites will be mutated appropriately to resemble constitutively phosphorylated and acetylated forms of CIITA. Additionally, they will be changed so that they cannot be modified posttranslationally. Ubiquitin will be fused directly to CIITA. Forced oligomers of CIITA will also be made. All these modified CIITA proteins will be examined for binding the MHCII enhanceosome in vitro and for function in vivo. Moreover, kinases, HATs, HDACs, ubiquitin ligases that participate in these processes will be revealed. Finally, a super-CIITA protein will be created that does not require this second stimulus for full activity. This mutant CIITA protein/s will then program any cell for APP. It will be introduced into cells and into the murine germline, in place of the endogenous CIITA gene (knock-in) and with P1 or P4 promoters of CIITA for expression in hematopoetic and nonhematopoetic cells, respectively, for induction of full APP following the administration of IFN-gamma. Collectively, these studies will reveal the normal physiology of CIITA, why it oligomerizes and shuttles, and create new tools to manipulate the immune response in health and disease.

描述（由申请人提供）：主要组织相容性复合物 II 类 (MHCII) 基因的转录以及抗原加工和呈递 (APP) 到 CD4+ T 辅助细胞，以及它们对 MHCII 阳性 B 细胞进行免疫球蛋白类别转换和超突变的教育受到仔细调节在发育和组织中。四个独立的转录因子专门用于结构 MHCII 基因、DMA/DMB 和不变链 (Ii) 的转录。其中，II 类反式激活因子 CIITA 在施用 IFN-γ 后打开和关闭这些基因方面发挥着关键作用。最近，我们发现除了 IFN-γ 之外，抗原呈递细胞 (APC) 还需要另一种磷酸化 CIITA 的信号。 CIITA 的磷酸化导致其寡聚化、积累并增加对 MHCII 启动子的活性。 CIITA 也不是 DNA 结合蛋白，但以某种方式被 MHCII 增强体吸引，该增强体在这些启动子中的保守上游序列上组装。这笔资助将研究 CIITA 完整活动所需的精确信号、发生哪些翻译后修饰以及它们如何创建触发完整 APP 的超级 CIITA 复合物。首先，将详细研究 CIITA 的核质穿梭。精确的 NES 和 NLS 将在功能上进行映射和确认。接下来，将详细研究 CIITA 的磷酸化、乙酰化和泛素化。这些位点将被适当突变以类似于 CIITA 的组成型磷酸化和乙酰化形式。此外，它们将被更改，以便它们不能在翻译后被修改。泛素将直接与 CIITA 融合。 CIITA的强制低聚物也将被制造。将检查所有这些修饰的 CIITA 蛋白在体外与 MHCII 增强体的结合以及在体内的功能。此外，参与这些过程的激酶、HAT、HDAC、泛素连接酶也将被揭示。最后，将产生一种超级 CIITA 蛋白质，其不需要第二次刺激即可发挥全部活性。然后，这种突变的 CIITA 蛋白将为任何细胞编程 APP。它将被引入细胞和小鼠种系中，代替内源 CIITA 基因（敲入），并与 CIITA 的 P1 或 P4 启动子一起分别在造血和非造血细胞中表达，以便在给药后诱导完整的 APP IFN-γ。总的来说，这些研究将揭示 CIITA 的正常生理学、为什么它寡聚和穿梭，并创造新的工具来操纵健康和疾病中的免疫反应。