Roles of DDX5 and its associated long non-coding RNAs in RORgt-mediated host immune system functions

DDX5及其相关长非编码RNA在RORgt介导的宿主免疫系统功能中的作用

• 批准号: 9179587
• 负责人: Dan Littman
• 金额: $ 53.63万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9179587
• 关键词: Adipocytes; Adipose tissue; Animals; Attenuated; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Binding; Biochemical; Bone Growth; Bone Marrow; CD8-Positive T-Lymphocytes; Cartilage; Cartilage Diseases; Cell Differentiation process; Cell Lineage; Cells; Cellular Immunity; Chimera organism; Chromatin; Circadian Rhythms; Citrobacter rodentium; Colitis; Complex; Crohn' s disease; Defect; Dependence; Dependency; Development; Disadvantaged; Disease; Dysplasia; Epithelial; Evaluation; Experimental Autoimmune Encephalomyelitis; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genome; Hair; Helper-Inducer T-Lymphocyte; Hematopoietic; Hepatocyte; Hereditary Disease; Human; Human Genetics; Immune; Immune system; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Interleukin-17; Intestines; Knock-in; Liver; Lymphoid Cell; Lymphoid Tissue; Macromolecular Complexes; Mediating; Metabolic; Modeling; Multiple Sclerosis; Mus; Mutant Strains Mice; Mutation; Neurons; Nuclear; Nuclear Receptors; Nucleotides; Pathogenesis; Pathway interactions; Patients; Pharmacology; Protein Isoforms; Proteins; Proteomics; Psoriasis; RNA Helicase; Recombinant Proteins; Regulation; Resistance; Rheumatoid Arthritis; Role; Site; T cell differentiation; T-Lymphocyte; Therapeutic; Thymocyte Development; Tissues; Transcription Coactivator; Transgenic Organisms; Untranslated RNA; base; cell type; conditional mutant; cytokine; design; energy balance; genome-wide; immune system function; improved; in vitro Assay; in vivo; insight; interest; microbiota; mutant; orphan nuclear receptor ROR-gamma; polarized cell; programs; targeted treatment; thymocyte; transcription factor; transcriptomics; γδ T cells

PROJECT SUMMARY/ABSTRACT The nuclear receptor RORγt directs the differentiation of T cells that have critical roles in multiple autoimmune and inflammatory diseases. Pharmacologic targeting of RORγt is hence being developed to treat diseases such as psoriasis, rheumatoid arthritis, and inflammatory bowel disease that are thought to be mediated in large part by Th17 cells and related cells that produce IL-17 cytokines. However, RORγt has multiple other functions in diverse cell types, including mediating survival of thymocytes and regulating the development of lymphoid tissue inducer (LTi) cells, that guide the formation of secondary and tertiary lymphoid tissues, and of type 3 innate lymphoid cells (ILC3) that protect epithelial barriers. In addition, RORγ, a closely related isoform encoded by the same gene, has metabolic functions in multiple tissues, including liver and adipocytes. How RORγ and RORγt function in different cell types to direct distinct transcriptional and functional programs is not understood. The transcriptional networks regulated by RORγt and multiple other transcription factors have been studied in Th17 cells polarized in vitro with combinations of cytokines, but how RORγ/γt functions in Th17 cells and other cell types in vivo is not yet known. We have used a proteomics approach to characterize macromolecular complexes that govern RORγ/γt functions in different cell types and identified DDX5, a DEAD- box RNA helicase, as associated with RORγt in Th17 cells. In mice deficient for DDX5 in T cells, expression of multiple RORγt target genes is attenuated and the animals are resistant to Th17 cell-mediated inflammatory disease. We also found the long noncoding (lnc) RNA Rmrp associated with DDX5 and with RORγt, both in Th17 cells and when all three components were synthesized in vitro. Mutations in Rmrp result in cartilage-hair hypoplasia (CHH), a recessive human genetic disease characterized by abnormal bone growth, immune deficiency and neuronal dysplasia in the intestine. Expression of wild-type, but not a CHH mutant, Rmrp promoted Th17 cell differentiation in a DDX5-dependent manner, indicating that this is a rare lncRNA that functions in trans. Our results suggest that Rmrp binding to DDX5 promotes the interaction of DDX5 with RORγt and coactivation of RORγt target genes. We propose in Specific Aim 1 to identify gene targets whose expression is dependent on DDX5, Rmrp, and RORγt, using genome-wide transcriptomics, chromatin accessibility, and chromatin occupancy studies with in vitro polarized or in vivo generated Th17 cells from wild type and mutant animals. In Specific Aim 2, we will determine, using biochemical approaches, how these molecules interact with each other and whether this interaction is restricted to Th17 cells or is present in other RORγ/γt-dependent cells. In Specific Aim 3, we will characterize the influence of the interactions in Th17 cell- mediated inflammation, ILC3-dependent barrier protection, and development of thymocytes and lymphoid tissues. Our results will provide a better understanding of the functions of RORγt, which may facilitate design of more specific therapeutics for autoimmune disease and for immune deficiency in CHH patients.

项目摘要/摘要 核接收器RORγT指导在多个自身免疫中具有关键作用的T细胞的分化 和炎症性疾病。因此，RORγT的药理靶向是为了治疗疾病 例如牛皮癣，类风湿关节炎和炎症性肠病，被认为是在 由Th17细胞和产生IL-17细胞因子的相关细胞的很大一部分。但是，RORγT有多个 潜水细胞类型的功能，包括介导胸腺细胞存活和调节 淋巴组织诱导剂（LTI）细胞，指导次级和三级淋巴组织的形成以及 3型先天淋巴样细胞（ILC3），可保护上皮屏障。另外，RORγ，密切相关的同工型 由同一基因编码，在包括肝脏和脂肪细胞在内的多个时机中具有代谢功能。如何 RORγ和RORγT在不同的细胞类型中的功能指导不同的转录和功能程序不是 理解。由RORγT和其他多个转录因子调节的转录网络具有 在Th17细胞中研究了细胞因子组合的体外极化，但RORγ/γT在TH17中的功能如何 细胞和其他体内细胞类型尚不清楚。我们已经使用蛋白质组学方法来表征 负责RORγ/γT在不同细胞类型中起作用并确定DDX5的大分子复合物 盒子RNA解旋酶，与Th17细胞中的RORγT相关。在T细胞中针对DDX5的小鼠中，表达 多种RORγT靶基因被减弱，动物对Th17细胞介导的炎症具有​​抗性 疾病。我们还发现了与DDX5和RORγT相关的长未编码（LNC）RNA RMRP Th17细胞以及所有三个成分在体外合成时。 RMRP的突变导致软骨头发 低流质（CHH），一种隐性人类遗传疾病，其特征是异常骨骼生长，免疫 肠道缺乏和神经元发育不良。野生型的表达，但不表达CHH突变体RMRP 以DDX5依赖性的方式促进了Th17细胞分化，表明这是一个罕见的lncRNA trans的功能。我们的结果表明，RMRP与DDX5结合促进了DDX5与 RORγT和RORγT靶基因的共激活。我们建议在特定目标1中识别其基因靶标的 表达依赖于DDX5，RMRP和RORγT，使用全基因组转录组学，染色质 可访问性，以及体外极化或体内的染色质占用研究，从野生产生了Th17细胞 类型和突变动物。在特定目标2中，我们将使用生化方法确定这些方法 分子相互相互作用，这种相互作用是否仅限于Th17细胞还是存在于其他相互作用 RORγ/γT依赖性细胞。在特定目标3中，我们将表征Th17细胞中相互作用的影响 介导的注射，ILC3依赖性屏障保护以及胸腺细胞和淋巴样的发展 组织。我们的结果将更好地了解RORγT的功能，这可能有助于设计 CHH患者的自身免疫性疾病和免疫缺乏症的更具体疗法。