Molecular Mechanisms of Major histocompatibility complex (MHC) gene regulation

我的研究方向是控制主要组织相容性复合体（MHC；人HLA） I类（MHC-I）和II类（MHC-II）基因和蛋白表达的多种调控机制。MHC分子是抗原呈递分子，向T细胞展示肽抗原，因此对适应性免疫反应至关重要。依赖mhc的免疫反应受到复杂的调节。MHC-II反激活子CIITA是NLR（核苷酸结合和富亮氨酸重复序列）蛋白家族的一员，是MHC-II基因表达的主要调控因子。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异构体III的前10个氨基酸是一个可移植的降解和转激活序列。CIITA亚型III的n端负责增加与转录机制成分的相互作用。这些实验揭示了蛋白质的游离n端在降解依赖性转录激活中的新功能。***我们最近发现NLRC5对MHC-I的表达很重要。有趣的是，NLRC5也是NLR蛋白，这是一个主要参与先天免疫反应的蛋白家族。到目前为止，CIITA和NLRC5是仅有的NLR蛋白是转录调节因子。通过结构域交换实验，我们已经证明NLRC5 n端结构域（NTD）作为一个真正的转录激活结构域，尽管其转激活机制尚不清楚。与T. Kufer博士合作，我们建议通过ChIP（染色质IP）实验，使用我们的结构域交换结构来分析内源性MHC-I和MHC-II启动子的染色质修饰。我们将使用NLRC5 NTD作为底物，通过亲和纯化和质谱鉴定结合伙伴。***最近我们发现IFN诱导的HLA-II激活与雌激素受体（ER）信号传导之间存在串扰。我们建议对这种串扰的分子机制进行分析。ChIp-seq数据的生物信息学分析显示，在CIITA基因周围的几个远端位点存在ER结合，这些位点与IFN诱导CIITA表达的远端调控元件部分重叠。在许多位点上，一些潜在的相关转录因子，如Er、FoxA、Stat1、IRF1和BRG1紧密结合。我们将对IFN-和E2同时处理的乳腺癌细胞中的ER、FoxA1和IRF1进行ChIp-seq和RNA-seq分析。潜在调节因子的功能测试将通过shrna介导的敲低进行。潜在的调控位点将通过体细胞敲除潜在的内源性远端调控位点来分析。综上所述，这些实验将告诉我们重要信号通路的相互作用，特别是CIITA基因的调控。*****

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.*****