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

项目摘要/摘要 核受体RoRγt指导在多发性自身免疫中起关键作用的T细胞的分化 和炎症性疾病。因此，rorγt的药理靶向正在被开发用于治疗疾病。 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 很大一部分是由产生IL-17细胞因子的Th17细胞和相关细胞产生的。然而，RoRγ测试具有多个其他 在不同类型的细胞中的功能，包括调节胸腺细胞的生存和调节 淋巴组织诱导物(LTI)细胞，引导第二级和第三级淋巴组织的形成，以及 3型先天淋巴样细胞(ILC3)，保护上皮屏障。此外，RoRγ，一种密切相关的亚型 由同一基因编码，在包括肝脏和脂肪细胞在内的多个组织中具有代谢功能。多么 RoRγ和RoRγt在不同细胞类型中的功能不同，以指导不同的转录和功能程序 明白了。受rorγt和其他多种转录因子调控的转录网络 已在体外用多种细胞因子组合极化的Th17细胞中进行了研究，但在Th17中rorγ/γt是如何发挥作用的 体内的细胞和其他细胞类型尚不清楚。我们已经使用蛋白质组学方法来表征 调控RoRγ/γt在不同细胞类型中功能的大分子复合体，并鉴定了一种死亡的 盒核糖核酸解旋酶，与Th17细胞中的RoRγt相关。在T细胞缺乏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，这两个基因都在 Th17细胞，以及这三种成分在体外合成的时间。Rmrp基因突变导致软骨-头发 发育不全(CHH)，一种隐性人类遗传病，特征是骨生长异常，免疫 肠道缺乏和神经元发育不良。野生型而非CHH突变体Rmrp的表达 以DDX5依赖的方式促进Th17细胞分化，表明这是一种罕见的 在事务中的功能。我们的结果表明，Rmrp与DDX5的结合促进了DDX5与 RoRγt和共激活RoRγt靶基因。我们建议在特定目标1中识别其基因靶点 表达依赖于ddx5，rmrp和rorγt，使用全基因组转录，染色质 体外极化或体内产生的野生Th17细胞的可及性和染色质占有率研究 类型和突变的动物。在具体目标2中，我们将使用生化方法来确定这些 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依赖细胞。在特定的目标3中，我们将表征Th17细胞中相互作用的影响- 介导的炎症、ILC3依赖的屏障保护以及胸腺细胞和淋巴样细胞的发育 纸巾。我们的结果将对RoRγt的功能有更好的理解，这可能有助于设计 针对自身免疫性疾病和慢性肝炎患者免疫缺陷的更具体的治疗方法。

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.