Roles of nucleoporins in RORgammat-dependent immune functions

核孔蛋白在 RORγ 依赖性免疫功能中的作用

• 批准号: 9174895
• 负责人: Dan Littman
• 金额: $ 42.38万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-15 至 2020-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9174895
• 关键词: ATAC-seq; Affect; Amino Acids; Anabolism; Animal Model; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Bacteria; Biochemical; CRISPR/Cas technology; Cell Differentiation process; Cell Line; Cells; Cholesterol; Chromatin; Citrobacter rodentium; Colitis; Complex; Crohn' s disease; DNA; DNA Binding; Development; Disease; EP300 gene; Enzymes; Experimental Autoimmune Encephalomyelitis; Funding; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Germ Lines; Helper-Inducer T-Lymphocyte; Human; IRF4 gene; Immune response; Immune system; Impairment; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Insecta; Interleukin-17; Intestines; Lamina Propria; Leukocytes; Ligand Binding; Ligands; Lymphocyte Function; Lymphoid; Lymphoid Cell; Lymphoid Tissue; Mediating; Mesentery; Metabolism; Modeling; Molecular; Multiple Sclerosis; Mus; Mutation; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nuclear Receptors; Nucleoplasm; Organogenesis; Output; Pathway interactions; Peripheral; Point Mutation; Pore Proteins; Process; Property; Protein Isoforms; Proteins; Proteomics; Psoriasis; RNA Polymerase II; Recruitment Activity; Regulator Genes; Rheumatoid Arthritis; Role; T cell differentiation; T-Cell Development; T-Lymphocyte; Therapeutic; Thymocyte Development; Tissues; Toxic effect; Transcriptional Regulation; Transgenic Mice; Treatment Efficacy; cell type; cholesterol biosynthesis; cytokine; design; gain of function; immune function; in vivo; inhibitor/antagonist; interleukin-22; loss of function; lymph nodes; mutant; orphan nuclear receptor ROR-gamma; programs; public health relevance; response; small molecule; small molecule inhibitor; thymocyte; transcription factor; transcriptome sequencing

 DESCRIPTION (provided by applicant): The ligand-dependent transcription factor RORγt regulates the development and functions of lymphocytes, including Th17 cells and type 3 innate lymphoid cells (ILC3) that have central roles in mucosal barrier protection and in mediating multiple autoimmune inflammatory diseases. Targeting of RORγt by small molecule inhibitors is a strategy that has been successfully validated in animal models and is being developed for therapeutic application in human autoimmune diseases, including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and, potentially, multiple sclerosis. Although many of the targets of RORγt and other key transcription factors involved in Th17 cell differentiation have been identified, the roles of RORγt in the gene regulatory programs of other cell types, e.g. thymocytes, ILC3, and lymphoid tissue inducer cells, have not been studied, nor have the roles of the closely-related RORγ isoform that is expressed outside of the immune system. A better understanding of how RORγt executes cell type-specific functions may facilitate development of better strategies to selectively inhibit its activities in the desired cells and tissues. We have ued genetic and proteomics screens to identify molecules that contribute to RORγt-dependent target gene regulation in Th17 cells, either indirectly or directly by associating with RORγt complexes. Molecules identified in both screens include nuclear pore proteins that have been recently implicated as having roles in the nucleoplasm as well as the nuclear envelope. We propose to study the roles of Nup98 and Nup153, proteins that interact with RORγt and contribute to the expression of a large number of RORγt target genes. In preliminary studies, we have identified mutations in RORγt that abrogate interaction with Nup153. Mice with one of these mutations have impaired Th17 cell induction but normal T cell development. We will extend our studies with Th17 cells, and also study the role of the RORγt-nucleoporin interactions in differentiation and function of TCR-γδ-17 cells and ILC3 and in the development of lymphoid tissue inducer cells and thymocytes. For the first aim, we will characterize the biochemical basis of the interaction of RORγt with Nup153 and Nup98 and will determine how this interaction influences formation of RORγt transcriptional complexes and the DNA occupancy of each factor in polarized Th17 cells. We will also examine the effect of ligand binding on association of RORγt with its partners, taking advantage of small molecule antagonists and our finding that Nup98 regulates enzymes involved in cholesterol biosynthesis, which may thus influence endogenous ligand availability. In Aim 2, we will introduce relevant mutations into the mouse germ line and examine the effect on chromatin accessibility and gene expression programs in Th17 cells generated in vitro and in vivo and in other RORγt-dependent cells. In Aim 3, we will use mice with the various point mutations in RORγt or its partner proteins to determine the effects in models of Th17-mediated autoimmune disease or ILC3-dependent barrier protection. Together, these studies will help in the design of better screens for cell type-selective targeting of RORγt functions.

 描述(申请人提供)：配体依赖的转录因子RoRγt调节淋巴细胞的发育和功能，包括Th17细胞和3型固有淋巴样细胞(ILC3)，它们在粘膜屏障保护和介导多种自身免疫性炎症性疾病中发挥核心作用。通过小分子抑制剂靶向rorγt是一种已经在动物模型中成功验证的策略，并正在开发用于治疗人类自身免疫性疾病，包括炎症性肠病、牛皮癣、类风湿性关节炎，以及潜在的多发性硬化症。尽管许多目标 RORγt和其他参与Th1 7细胞分化的关键转录因子的关系已经确定，但还没有研究RORγt在其他类型细胞如胸腺细胞、ILC3和淋巴组织诱导细胞等基因调控程序中的作用，也没有研究在免疫系统外表达的密切相关的RORγ亚型的作用。更好地了解RoRγt如何执行细胞类型特定的功能可能有助于开发更好的策略来选择性地抑制其在所需细胞和组织中的活动。我们已经使用遗传和蛋白质组学筛选来识别在Th17细胞中间接或直接通过与RoRγt复合体相关联而对RoRγt依赖的靶基因调控做出贡献的分子。在这两种筛选中发现的分子包括核孔蛋白，这些蛋白最近被认为与核质和核膜有关。我们建议研究与RoRγt相互作用的Nup98和Nup153蛋白的作用，它们有助于大量RoRγt靶基因的表达。在初步研究中，我们已经确定了RoRγt中的突变，这些突变取消了与Nup153的相互作用。带有这些突变之一的小鼠会损害Th17细胞的诱导，但T细胞发育正常。我们将扩展我们对Th17细胞的研究，并研究RoR-γt-核孔蛋白相互作用在Tcr-γδ-17细胞和ILC3分化和功能以及在淋巴组织诱导细胞和胸腺细胞发育中的作用。对于第一个目标，我们将表征RoRγt与Nup153和Nup98相互作用的生化基础，并将确定这种相互作用如何影响RoRγt转录复合体的形成以及每个因子在极化的Th17细胞中的占据。我们还将利用小分子拮抗剂的优势，研究配体结合对RoRγt与其伙伴结合的影响，并发现Nup98调节参与胆固醇生物合成的酶，从而可能影响内源性配体的可用性。在目标2中，我们将相关突变引入小鼠生殖系，并检测其对体外和体内产生的Th17细胞以及其他依赖RoRγt的细胞中染色质可及性和基因表达程序的影响。在目标3中，我们将使用具有不同点突变的rorγt或其伙伴蛋白的小鼠来确定其在Th17介导的自身免疫性疾病或ILC3依赖的屏障保护模型中的作用。总之，这些研究将有助于设计更好的针对RORγt的细胞类型选择性靶向的筛选 功能。