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

描述（由申请人提供）：本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：核受体介导的功能转录单位组装。核受体ROR（t）在免疫系统的发育和功能中起关键作用。最近，我们已经证明了ROR β t在称为Th 17细胞的炎性产生烟碱的CD 4辅助性T细胞谱系的分化中的需要。这些产生IL-17和IL-22的细胞对于病原体的清除是重要的，特别是在粘膜表面，并且是诱导自身免疫性疾病中的组织炎症的主要贡献者。除了必需之外，ROR β 1表达还足以诱导具有Th 17表型的细胞的发育，突出了这种核受体作为关键的谱系限定转录因子。在小鼠模型中，使用高通量筛选中鉴定的小分子进行ROR β的遗传消除或其转录活性的抑制消除了自身免疫性疾病，这表明更好地理解ROR β功能将导致治疗人类炎性疾病的新方法。虽然ROR β在Th 17细胞谱系特化中具有核心作用，但它与其他几种转录因子协作，这些转录因子也被证明是Th 17细胞分化和功能所必需的或有助于Th 17细胞的分化和功能;这些转录因子包括STAT 3、IRF-4、Ahr、Runx 1/CBF β和ROR β。在本申请中，我们提出将联合收割机因子依赖性转录组分析（RNA-Seq）与核因子占用和染色质修饰的全基因组分析（ChIP-Seq）相结合，以阐明控制Th 17细胞分化和功能的转录调控网络。我们已经产生或获得了在每个相关转录因子基因中具有突变的小鼠，并且来自这些小鼠的T细胞中的转录组和占据分析将提供对转录因子与ROR 3 t的活性中的协作相互作用和相互依赖性的洞察。此外，我们的目标是表征在Th 17谱系细胞中含有ROR 3 t的转录复合物。为此，我们将采用生物化学策略，串联亲和纯化，然后进行质谱分析，使用用标记的ROR（t或Runx 1）转导的原代小鼠Th 17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的分析将与从转录组研究和组蛋白修饰（例如H3 K4- K36结构域）检查获得的信息相结合，以鉴定那些最可能在Th 17分化程序中具有重要作用的ncRNA。总之，这些实验将扩大我们对ROR 3 t介导的基因调控的分子机制的认识，并将揭示与Th 17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，银屑病和哮喘。 公共卫生相关性：最近，我们已经证明了转录因子ROR（t）在称为CD 4 T辅助细胞-17细胞的炎性免疫细胞谱系的分化中的需要。这些细胞对于宿主防御反应期间清除某些病原体非常重要，并且是诱导自身免疫性疾病中组织炎症的主要贡献者; Th 17细胞被认为特别参与炎症性肠病，关节炎和多发性硬化症。这项研究旨在揭示ROR活性的分子机制，以及发现炎症性疾病的潜在治疗靶点。

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.