Molecular Mechanisms of Major histocompatibility complex (MHC) gene regulation

主要组织相容性复合体 (MHC) 基因调控的分子机制

• 批准号: RGPIN-2016-05455
• 负责人: Steimle, Viktor
• 金额: $ 2.26万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=709690
• 关键词: Molecular; Mechanisms; Major; histocompatibility; complex

My research program is focused on the multiple regulatory mechanisms that control major histocompatibility complex (MHC; HLA in human) class I (MHC-I) and class II (MHC-II) gene and protein expression. MHC molecules are antigen-presenting molecules displaying peptide antigens to T cells, and are thus of central importance for the adaptive immune response. MHC-dependent immune responses are intricately regulated. The MHC-II transactivator CIITA, a member of the NLR (nucleotide binding and leucine rich repeats) family of proteins, is the master regulator of MHC-II gene expression. We have found recently that protein turnover of CIITA is directly linked to its capacity to activate transcription. The first ten amino acids of CIITA isoform III act as a portable degron and transactivation sequence. The N-terminal end of CIITA isoform III is responsible for increased interaction with components of the transcription machinery. These experiments reveal a novel function of free N-terminal ends of proteins in degradation-dependent transcriptional activation. We have recently shown that NLRC5 is important for MHC-I expression. Intriguingly, NLRC5 is also an NLR protein, a family of proteins that are predominantly involved in innate immune responses. Thus far, CIITA and NLRC5 are the only NLR proteins that are transcriptional regulators. Using domain-swap experiments, we have shown that the NLRC5 N-terminal domain (NTD) behaves as a bona fide transcriptional activation domain, although its mechanism of transactivation is unknown. In collaboration with the group of Dr. T. Kufer, we propose to use our domain-swap constructs to analyze chromatin modifications at endogenous MHC-I and MHC-II promoters via ChIP (chromatin IP) experiments. We will use the NLRC5 NTD as a substrate to identify binding partners by affinity purification and mass spectrometry. Recently we have found that there is a crosstalk between IFN- induced HLA-II activation and estrogen receptor (ER) signaling. We propose to analyze the molecular mechanisms of this crosstalk. Bio-informatics analysis of ChIp-seq data revealed in vivo binding of ER at several distal sites around the CIITA gene, which in part overlap with distal regulatory elements of IFN- induced CIITA expression. At a number of sites, several potentially relevant transcription factors, such as Er, FoxA, Stat1, IRF1 and BRG1 bind in close proximity. We will carry out a ChIp-seq and RNA-seq analysis for ER, FoxA1 and IRF1 in breast cancer cells treated simultaneously with IFN- and E2. Functional tests of potential regulators will be carried out by shRNA-mediated knockdowns. Potential regulatory sites will be analyzed by somatic knockouts of potential endogenous distal regulatory sites. Taken together these experiments will inform us on the interactions of important signaling pathways in general and on the regulation of the CIITA gene in particular.

我的研究项目主要集中在控制主要组织相容性复合体（MHC;人类HLA）I类（MHC-I）和II类（MHC-II）基因和蛋白质表达的多种调控机制。MHC分子是向T细胞展示肽抗原的抗原呈递分子，因此对于适应性免疫应答至关重要。MHC依赖性免疫应答受到复杂的调节。MHC-II反式激活因子CIITA是NLR（核苷酸结合和富含亮氨酸重复序列）蛋白家族的成员，是MHC-II基因表达的主要调节因子。我们最近发现CIITA的蛋白质周转与其激活转录的能力直接相关。CIITA同种型III的前10个氨基酸充当便携式降解决定子和反式激活序列。CIITA同种型III的N-末端负责增加与转录机制组分的相互作用。这些实验揭示了蛋白质的游离N-末端在降解依赖的转录激活中的新功能。 我们最近发现NLRC 5对MHC-I表达很重要。有趣的是，NLRC 5也是一种NLR蛋白，一种主要参与先天免疫反应的蛋白质家族。到目前为止，CIITA和NLRC 5是唯一的NLR蛋白是转录调节因子。使用结构域交换实验，我们已经表明，NLRC 5 N-末端结构域（NTD）的行为作为一个真正的转录激活域，虽然它的反式激活的机制是未知的。与T博士的团队合作。Kufer，我们建议使用我们的结构域交换结构，通过ChIP（染色质IP）实验分析内源性MHC-I和MHC-II启动子的染色质修饰。我们将使用NLRC 5 NTD作为底物，通过亲和纯化和质谱法鉴定结合伴侣。 最近，我们发现IFN-诱导的HLA-II激活和雌激素受体（ER）信号传导之间存在串扰。我们建议分析这种串扰的分子机制。ChIp-seq数据的生物信息学分析揭示了ER在CIITA基因周围的几个远端位点的体内结合，其与IFN诱导的CIITA表达的远端调控元件部分重叠。在许多位点，几个潜在相关的转录因子，如Er，FoxA，Stat 1，IRF 1和BRG 1紧密结合。我们将对同时用IFN-γ和E2处理的乳腺癌细胞中的ER、FoxA 1和IRF 1进行ChIp-seq和RNA-seq分析。潜在调节剂的功能测试将通过shRNA介导的敲除进行。将通过潜在内源性远端调控位点的体细胞敲除来分析潜在调控位点。总之，这些实验将告知我们的相互作用的重要信号转导途径，特别是对CIITA基因的调控。