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: 9010056
• 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/9010056
• 关键词: Adipocytes; Adipose tissue; Animals; Attenuated; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Binding; Biochemical; Bone Growth; Bone Marrow; Boxing; CD8B1 gene; 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; Histocompatibility Testing; 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; Protein Isoforms; Proteins; Proteomics; Psoriasis; RNA Helicase; Recombinant Proteins; Regulation; Resistance; Rheumatoid Arthritis; Role; Site; Staging; 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; improved; in vitro Assay; in vivo; insight; interest; microbiota; mutant; orphan nuclear receptor ROR-gamma; polarized cell; programs; targeted treatment; thymocyte; transcription factor; transcriptomics

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.

项目概要/摘要 核受体 RORγt 指导 T 细胞的分化，在多种自身免疫性疾病中发挥关键作用 和炎症性疾病。因此，正在开发 RORγt 的药理学靶向来治疗疾病 例如牛皮癣、类风湿性关节炎和炎症性肠病，这些疾病被认为是由 大部分由 Th17 细胞和产生 IL-17 细胞因子的相关细胞组成。然而，RORγt 还有多个其他 在多种细胞类型中发挥作用，包括介导胸腺细胞的存活和调节胸腺细胞的发育 淋巴组织诱导（LTi）细胞，引导二级和三级淋巴组织的形成，以及 保护上皮屏障的 3 型先天淋巴细胞 (ILC3)。此外，RORγ，一种密切相关的亚型 由同一基因编码，在多种组织中具有代谢功能，包括肝脏和脂肪细胞。如何 RORγ 和 RORγt 在不同的细胞类型中发挥作用，以指导不同的转录和功能程序。 明白了。 RORγt 和多种其他转录因子调控的转录网络 已在体外用细胞因子组合极化的 Th17 细胞中进行了研究，但 RORγ/γt 在 Th17 中如何发挥作用 细胞和其他细胞类型在体内的作用尚不清楚。我们使用蛋白质组学方法来表征 控制不同细胞类型中 RORγ/γt 功能的大分子复合物，并鉴定出 DDX5（一种 DEAD- box RNA 解旋酶，与 Th17 细胞中的 RORγt 相关。在 T 细胞中缺乏 DDX5 的小鼠中， 多个 RORγt 靶基因减弱，动物对 Th17 细胞介导的炎症具有​​抵抗力 疾病。我们还发现了与 DDX5 和 RORγt 相关的长非编码 (lnc) RNA Rmrp，两者均位于 Th17 细胞以及所有三种成分均在体外合成时。 Rmrp 突变导致软骨毛 发育不全（CHH），一种隐性人类遗传病，其特征是骨骼生长异常、免疫 肠道内的缺乏和神经元发育不良。表达野生型，但不表达 CHH 突变体 Rmrp 以 DDX5 依赖性方式促进 Th17 细胞分化，表明这是一种罕见的 lncRNA 反式中的函数。我们的结果表明，Rmrp 与 DDX5 的结合促进了 DDX5 与 RORγt 和 RORγt 靶基因的共激活。我们在具体目标 1 中建议确定基因靶标，其 表达依赖于 DDX5、Rmrp 和 RORγt，使用全基因组转录组学、染色质 使用体外极化或体内产生的野生 Th17 细胞进行可及性和染色质占据研究 型和突变型动物。在具体目标 2 中，我们将使用生化方法确定这些 分子之间相互作用，以及这种相互作用是否仅限于 Th17 细胞或存在于其他细胞中 RORγ/γt 依赖性细胞。在具体目标 3 中，我们将描述 Th17 细胞中相互作用的影响—— 介导的炎症、ILC3 依赖性屏障保护以及胸腺细胞和淋巴细胞的发育 组织。我们的结果将有助于更好地理解 RORγt 的功能，这可能有助于设计 针对自身免疫性疾病和 CHH 患者免疫缺陷的更具体的治疗方法。