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

New York University School Of Medicine, New York NY

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Abstract

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[unreadable], and ROR[unreadable]. 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.