Roles of nucleoporins in RORgammat-dependent immune functions

 描述（由申请方提供）：配体依赖性转录因子RORγt调节淋巴细胞的发育和功能，包括Th 17细胞和3型先天淋巴细胞（ILC 3），其在粘膜屏障保护和介导多种自身免疫性炎性疾病中发挥核心作用。通过小分子抑制剂靶向RORγt是一种已在动物模型中成功验证的策略，并正在开发用于人类自身免疫性疾病的治疗应用，包括炎症性肠病、银屑病、类风湿性关节炎和潜在的多发性硬化症。尽管许多目标 虽然已经鉴定了参与Th 17细胞分化的RORγt和其他关键转录因子的作用，但尚未研究RORγt在其他细胞类型（例如胸腺细胞、ILC 3和淋巴组织诱导细胞）的基因调控程序中的作用，也未研究在免疫系统外表达的密切相关的RORγ同种型的作用。更好地了解RORγt如何执行细胞类型特异性功能可能有助于开发更好的策略，以选择性地抑制其在所需细胞和组织中的活性。我们已经使用遗传学和蛋白质组学筛选来鉴定在Th 17细胞中有助于RORγ t依赖性靶基因调控的分子，这些分子通过与RORγt复合物的结合而间接或直接地起作用。在这两个屏幕中确定的分子包括核孔蛋白，最近被牵连作为具有核质以及核膜中的作用。我们建议研究Nup 98和Nup 153的作用，这两种蛋白质与RORγt相互作用并有助于大量RORγt靶基因的表达。在初步研究中，我们已经确定了RORγt中消除与Nup 153相互作用的突变。具有这些突变之一的小鼠具有受损的Th 17细胞诱导，但正常的T细胞发育。我们将用Th 17细胞扩展我们的研究，并且还研究RORγ t-核孔蛋白相互作用在TCR-γδ-17细胞和ILC 3的分化和功能中以及在淋巴组织诱导细胞和胸腺细胞的发育中的作用。对于第一个目标，我们将描述RORγt与Nup 153和Nup 98相互作用的生化基础，并确定这种相互作用如何影响RORγt转录复合物的形成以及极化Th 17细胞中每个因子的DNA占有率。我们还将研究配体结合对RORγt与其配偶体结合的影响，利用小分子拮抗剂和我们的发现，即Nup 98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，我们将向小鼠生殖系中引入相关突变，并检查对体外和体内产生的Th 17细胞以及其他RORγ t依赖性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RORγt或其伴侣蛋白中具有各种点突变的小鼠，以确定在Th 17介导的自身免疫性疾病或ILC 3依赖性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RORγt细胞类型选择性靶向筛选。 功能协调发展的

 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.