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

项目总结/摘要 核受体RORγt指导在多种自身免疫性疾病中起关键作用的T细胞分化 和炎症性疾病。因此，正在开发RORγt的药物靶向治疗疾病 如银屑病、类风湿性关节炎和炎症性肠病，这些疾病被认为是由 大部分是Th 17细胞和产生IL-17细胞因子的相关细胞。然而，RORγt具有多个其他 在不同的细胞类型中发挥作用，包括介导胸腺细胞的存活和调节 淋巴组织诱导（LTi）细胞，其引导二级和三级淋巴组织的形成，以及 保护上皮屏障的3型先天淋巴样细胞（ILC 3）。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，指导不同的转录和功能程序， 明白由RORγt和多种其他转录因子调控的转录网络具有 在体外用细胞因子组合极化的Th 17细胞中进行了研究，但RORγ/γt如何在Th 17细胞中起作用， 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 在不同细胞类型中控制RORγ/γt功能的大分子复合物，并鉴定了DDX 5，一种死亡的 box RNA解旋酶，与Th 17细胞中的RORγt相关。在T细胞中缺乏DDX 5的小鼠中， 多个RORγt靶基因被减弱，动物对Th 17细胞介导的炎症反应具有抗性， 疾病我们还发现了与DDX 5和RORγt相关的长非编码（lnc）RNA Rmrp，两者均在 Th 17细胞和当所有三种组分在体外合成时。Rmrp突变导致软骨-毛发 发育不全（CHH）是一种隐性遗传性疾病，其特征是骨生长异常，免疫 缺乏症和肠神经元发育不良。野生型而非CHH突变体Rmrp的表达 以DDX 5依赖的方式促进Th 17细胞分化，表明这是一种罕见的lncRNA， 我们的研究结果表明，Rmrp与DDX 5的结合促进了DDX 5与 RORγt和RORγt靶基因的共激活。我们在具体目标1中提出， 表达依赖于DDX 5、Rmrp和RORγt，使用全基因组转录组学、染色质 用体外极化或体内产生的来自野生型的Th 17细胞进行的可接近性和染色质占有研究 类型和突变动物。在具体目标2中，我们将使用生物化学方法来确定，这些方法如何 分子之间相互作用，以及这种相互作用是否限于Th 17细胞或存在于其他细胞中， RORγ/γ t依赖性细胞。在具体目标3中，我们将描述Th 17细胞中相互作用的影响， 介导的炎症，ILC 3依赖性屏障保护，以及胸腺细胞和淋巴细胞的发育 组织中我们的研究结果将有助于更好地理解RORγt的功能，这可能有助于设计 用于自身免疫性疾病和CHH患者的免疫缺陷的更特异性的治疗剂。

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.