Role of ROR??t in the transcriptional regulatory network underlying Th17 lineage

描述（由申请人提供）：该申请涉及广泛的挑战领域（08）：基因组学，以及特定的挑战主题08- dk -107：核受体介导的功能性转录单位组装。核受体ROR(t)在免疫系统的发育和功能中起着关键作用。最近，我们已经证明了在被称为Th17细胞的炎症细胞因子产生CD4 t辅助细胞谱系的分化中需要ROR(t)。这些产生IL-17和il -22的细胞对于清除病原体非常重要，特别是在粘膜表面，并且是自身免疫性疾病诱导组织炎症的主要因素。ROR(t)的表达不仅是必需的，还足以诱导Th17表型细胞的发育，这表明这种核受体是一个关键的谱系定义转录因子。基因消融ROR(t)或使用高通量筛选中鉴定的小分子抑制其转录活性可消除小鼠模型中的自身免疫性疾病，这表明更好地了解ROR(t)功能将导致人类炎症性疾病治疗的新方法。虽然它在Th17细胞谱系规范中起着核心作用，但ROR(t)与其他几个转录因子（包括STAT3、IRF-4、Ahr、Runx1/CBF¿和ROR）协同工作，这些转录因子也被证明对Th17细胞的分化和功能至关重要或有贡献。在这个应用中，我们建议结合因子依赖转录组分析（RNA-Seq）和全基因组核因子占用和染色质修饰分析（ChIP-Seq）来阐明控制Th17细胞分化和功能的转录调控网络。我们已经产生或获得了每个相关转录因子基因突变的小鼠，从这些小鼠的T细胞中进行转录组和占用分析将有助于深入了解转录因子与ROR3t活性的合作相互作用和相互依赖性。此外，我们的目标是表征Th17谱系细胞中含有ROR3t的转录复合物。为此，我们将采用生化策略，串联亲和纯化和质谱分析，使用标记ROR(t或Runx1转导的原代小鼠Th17细胞。这种方法也将用于鉴定可能与这种转录调控复合物相关的非编码rna。ncrna的分析将与转录组研究和组蛋白修饰检查（例如H3 K4- K36结构域）获得的信息相结合，以确定最有可能在Th17分化程序中发挥重要作用的ncrna。总之，这些实验将扩大我们对ror3t介导的基因调控的分子机制的认识，并将发现与Th17细胞介导的发病机制相关的炎症性疾病的潜在治疗靶点，包括炎症性肠病、类风湿性关节炎、多发性硬化症、糖尿病、牛皮癣和哮喘。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (08): Genomics, and specific Challenge Topic 08-DK-107: Nuclear Receptor Mediated Assembly of Functional Transcriptional Units. The nuclear receptor ROR(t plays a critical role in the development and function of the immune system. Recently, we have demonstrated a requirement for ROR(t in the differentiation of a lineage of inflammatory cytokine-producing CD4 T helper cells termed Th17 cells. These IL-17- and IL-22-producing cells are important for the clearance of pathogens, particularly at mucosal surfaces, and are major contributors to the induction of tissue inflammation in autoimmune disease. In addition to being necessary, ROR(t expression is also sufficient to induce the development of cells with a Th17 phenotype, highlighting this nuclear receptor as a critical lineage-defining transcription factor. Genetic ablation of ROR(t or inhibition of its transcriptional activity using small molecules identified in a high throughput screen abrogated autoimmune disease in mouse models, suggesting that a better understanding of ROR(t function will result in novel approaches towards therapy in human inflammatory diseases. Although it has a central role in Th17 cell lineage specification, ROR(t collaborates with several other transcription factors that have also been shown to be essential for, or to contribute to, differentiation and function of Th17 cells; these include STAT3, IRF-4, Ahr, Runx1/CBF¿, and ROR¿. In this application we propose to combine factor-dependent transcriptome analysis (RNA-Seq) with genome-wide analyses of nuclear factor occupancy and chromatin modifications (ChIP-Seq) to elucidate the transcriptional regulatory network governing Th17 cell differentiation and function. We have generated or obtained mice with mutations in each of the relevant transcription factor genes, and transcriptome and occupancy analysis in T cells from these mice will provide insight into cooperative interactions and interdependency in the activity of the transcription factors with ROR3t. Further, we aim to characterize ROR3t- containing transcriptional complexes in Th17 lineage cells. To this end, we will employ a biochemical strategy, Tandem Affinity Purification followed by mass spectrometric analysis, using primary mouse Th17 cells transduced with tagged ROR(t or Runx1. This approach will also be used to identify non-coding RNAs that may be associated with such transcriptional regulatory complexes. Analysis of the ncRNAs will be coupled to information attained from the transcriptome study and from examination of histone modifications (e.g. H3 K4- K36 domains) to identify those ncRNAs most likely to have important roles in the Th17 differentiation program. Taken together, these experiments will expand our knowledge of the molecular mechanisms governing ROR3t-mediated gene regulation, and will uncover potential therapeutic targets in inflammatory diseases linked to Th17 cell-mediated pathogenesis, including inflammatory bowel diseases, rheumatoid arthritis, multiple sclerosis, diabetes, psoriasis, and asthma. PUBLIC HEALTH RELEVANCE: Recently, we have demonstrated a requirement for the transcription factor ROR(t in the differentiation of a lineage of inflammatory immune cells termed CD4 T helper-17 cells. These cells are important for the clearance of certain pathogens during host defense reactions, and are major contributors to the induction of tissue inflammation in autoimmune disease; Th17 cells are thought to be particularly involved in inflammatory bowel disease, arthritis, and multiple sclerosis. The proposed research proposes to uncover the molecular mechanisms of ROR(t activity, as well as uncover potential therapeutic targets in inflammatory diseases.