DF/HCC Program Affiliation

Cancer Cell Biology
Cancer Immunology

Research Abstract

Field of Study: Signal transduction, Transcription and gene regulation, Ca signalling, Chromatin and other DNA:protein interactions, Growth factors/ hormones, Intracellular signalling and cell:cell interactions
Approach: Biochemistry, Molecular biology, Cell biology

Signalling to transcription: The NFAT transcription factor controls cell lineage specification in many different cell types. In immune cells, it controls both lymphocyte activation and tolerance. The first step of NFAT activation involves calcium entry through “CRAC” channels in the plasma membrane. The increase in intracellular calcium causes activation of the phosphatase calcineurin, which dephosphorylates NFAT and causes it to translocate from the cytoplasm to the nucleus. Using a genome-wide RNAi screen -based approaches to identify regulators of the Ca/ calcineurin/ NFAT pathway, we have identified CRAC channel components as well as a triad of kinases that deactivate NFAT (CK1, GSK3 and DYRK). We have shown that a mutation in a component of the CRAC channel pathway, Orai1, is responsible for the genetic defect in two severe combined immunodeficiency patients whose T lymphocytes lack the ability to open “CRAC” Ca channels in response to depletion of intracellular Ca stores. (i) We are further investigating the pathway of calcium influx, particularly the mechanism by which depletion of calcium stores is coupled to CRAC channel opening. (ii) We are investigating the biochemical and structural aspects of calcineurin-NFAT interaction and NFAT activation as a result of dephosphorylation.

The immune system as a model for understanding cell differentiation: Chronic stimulation with antigen and cytokines results in the differentiation of uncommitted precursor T cells into “effector” T cells with specific functions in the immune response. As expected, different classes of effector T cells are distinguished by specific patterns of gene expression, and deviation from balanced representation of the different effector classes is associated with diseases such as autoimmunity, asthma and allergy. (i) We are defining the regulatory regions and nuclear processes that control chromatin accessibility of selected lineage-specific genes, leading to their activation or silencing in characteristic T cell subsets. (ii) T cells lacking either Dicer or the siRNA exonuclease mExo are skewed in the balance of T cell differentiation and lineage-specific gene expression, and we are also asking how microRNAs affect this balance.

Mechanisms of peripheral immune tolerance: (i) We have shown that the transcriptional profile of activated T cells is driven by cooperative complexes of NFAT with its transcriptional partners Fos and Jun, while NFAT in the absence of Fos and Jun drives a transcriptional programme that is associated with anergy (a cell-intrinsic unresponsive state). Anergic T cells express characteristic patterns of negative regulators, ranging from tyrosine phosphatases and inhibitory cell surface receptors to transcriptional repressors and proteins involved in ubiquitin-mediated degradation. We are investigating the negative feedback mechanisms that operate in anergic T cells and defining the proteins and processes involved. (ii) Autoimmune responses are further suppressed by a special class of regulatory T cells which express high levels of the transcription factor FOXP3. We have shown that T regulatory function depends on a complex of NFAT with FOXP3 on DNA. We are investigating the transcriptional programmes and differentiation pathways involved in development and function of regulatory T cells.

References:

Gwack Y, Sharma S, Nardone J, Tanasa B, Iuga A, Srikanth S, Okamura H, Bolton D, Feske S, Hogan PG, Rao A. 2006. A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT. Nature 441, 646-650.

Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel S-H, Tanasa B, Hogan PG, Lewis RS, Daly M, Rao A. 2006. A mutation in Orai1 causes immune deficiency by abrogating store-operated Ca2+ entry and CRAC channel

Publications

• Djuretic IM, Levanon D, Negreanu V, Groner Y, Rao A, Ansel KM.Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells.Nat Immunol 2007 Feb;8(2):145-53.
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• Wu Y, Borde M, Heissmeyer V, Feuerer M, Lapan AD, Stroud JC, Bates DL, Guo L, Han A, Ziegler SF, Mathis D, Benoist C, Chen L, Rao A.FOXP3 Controls Regulatory T Cell Function through Cooperation with NFAT.Cell 2006 Jul 28;126(2):375-87.
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• Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, Tanasa B, Hogan PG, Lewis RS, Daly M, Rao A.A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.Nature 2006 May 11;441(7090):179-85.
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• Gwack Y, Sharma S, Nardone J, Tanasa B, Iuga A, Srikanth S, Okamura H, Bolton D, Feske S, Hogan PG, Rao A.A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT.Nature 2006 Jun 1;441(7093):646-50.
  16511445
• Muljo SA, Ansel KM, Kanellopoulou C, Livingston DM, Rao A, Rajewsky K.Aberrant T cell differentiation in the absence of Dicer.J Exp Med 2005 Jul 18;202(2):261-9.
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• Macián F, García-Cózar F, Im SH, Horton HF, Byrne MC, Rao A.Transcriptional mechanisms underlying lymphocyte tolerance.Cell 2002 Jun 14;109(6):719-31.
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• Feske S, Giltnane J, Dolmetsch R, Staudt LM, Rao A.Gene regulation mediated by calcium signals in T lymphocytes.Nat Immunol 2001 Apr;2(4):316-24.
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• Okamura H, Aramburu J, García-Rodríguez C, Viola JP, Raghavan A, Tahiliani M, Zhang X, Qin J, Hogan PG, Rao A.Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity.Mol Cell 20
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• Agarwal S, Avni O, Rao A.Cell-type-restricted binding of the transcription factor NFAT to a distal IL-4 enhancer in vivo.Immunity 2000 Jun;12(6):643-52.
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• Aramburu J, Yaffe MB, López-Rodríguez C, Cantley LC, Hogan PG, Rao A.Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A.Science 1999 Sep 24;285(5436):2129-33.
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• Lopez-Rodríguez C, Aramburu J, Rakeman AS, Rao A.NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun.Proc Natl Acad Sci U S A 1999 Jun 22;96(13):7214-9.
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• Agarwal S, Rao A.Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation.Immunity 1998 Dec;9(6):765-75.
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• Aramburu J, Garcia-Cózar F, Raghavan A, Okamura H, Rao A, Hogan PG.Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT.Mol Cell 1998 Apr;1(5):627-37.
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