Regulated Accessibility of a cytokine gene locus.

Summary

Principal Investigator: Anjana Rao
Abstract: DESCRIPTION (provided by applicant): This application extends work performed during the previous project period, in which we investigated the mechanism of chromatin-based regulation of cytokine genes in the mouse Th2 locus, which encodes the Il4, Il13, Rad50 and Il5 genes. We showed that Th1 and Th2 differentiation were accompanied by long-range chromatin changes in activated as well as silenced cytokine genes, as judged by changes in DNase I hypersensitive (DH) sites, DNA methylation and histone modification;explored the transcriptional and chromatin-based programs underlying CD8 T cell differentiation;and defined the functions of multiple regulatory regions in the Il4, Ifng and Il10 loci. Related studies were performed in many other labs, resulting in our current detailed and comprehensive understanding of the transcriptional and chromatin-level changes that form the molecular basis for the various directions of CD4 and CD8 T cell differentiation. During this project period, we will broaden the scope of our studies beyond an exclusive analysis of the Th2 cytokine locus. We recently discovered that the TET proteins TET1, TET2 and TET3 constitute a new family of 2-oxoglutarate (2OG)- and Fe(II)-dependent oxygenases, that convert 5- methylcytosine (5mC) to 5-hydroxymethylcytosine (hmC) in DNA. Moreover, we demonstrated that hmC is a physiological constituent of mammalian DNA, and that hmC levels and TET mRNA levels are both regulated in T cells and several other cell types. These are exciting findings because hydroxylation of 5mC alters DNA methylation status in a hitherto unprecedented way, and because DNA methylation is relevant to several fields including mammalian development, cancer, aging, cell lineage specification, genome defense and stem cell function. In this proposal, we focus on the question of whether TET proteins and hmC have a role in cell differentiation and cell lineage specification, using T cells as a tractable model system for studying cell differentiation in culture as well as in vivo. Our overall goal is to define the biological roles of Tet-family proteins in T cells and potentially other cells of the immune system, taking advantage of our long familiarity with gene regulation in naive, activated and differentiating T cells. In Aim 1, we will analyze hmC levels and Tet occupancy at relevant genes in naive, activated and differentiating murine CD4 and CD8 T cells. In Aim 2, we will use mice with conditional deletions of Tet1, Tet2 and Tet3 genes to examine the importance of Tet proteins for T cell function. These proposed studies should advance our understanding of the biological functions of hmC and Tet proteins, and provide a new perspective into how DNA methylation status might regulate gene expression. PUBLIC HEALTH RELEVANCE: No more than 2-3 sentences, describe the relevance of this research to public health. In this section be succinct and use plain language that can be understood by a general, lay audience. In addition to the four major bases in the DNA alphabet - A, C, G and T - there is also a very minor base known as 5-methylcytosine (5mC) that has a disproportionately crucial role. This base is produced from the major base cytosine (C) by attaching a methyl group to its "5" position. Interference with cytosine methylation can lead to a number of developmental abnormalities and genetic diseases. We have recently identified a new class of proteins known as TET proteins that convert 5-methylcytosine to a variant known as 5-hydroxymethylcytosine (hmC). TET proteins have been linked to cancer, and the modified cytosine base (hmC) that they produce is found at high levels in mouse embryonic stem cells. In this proposal we plan to investigate the role of TET proteins in T cells, a major class of cells in the immune system that protect the organism from attack by bacteria, viruses and other pathogens including cancer cells.
Funding Period: 1999-07-01 - 2014-11-30
more information: NIH RePORT

Top Publications

  1. ncbi Foxp1 is an essential transcriptional regulator of B cell development
    Hui Hu
    Department of Pathology, Harvard Medical School, CBR Institute for Biomedical Research, Boston, Massachusetts 02115, USA
    Nat Immunol 7:819-26. 2006
  2. pmc RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation
    Elisa Närvä
    Turku Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, Finland
    Stem Cells 30:452-60. 2012
  3. pmc Interleukin-4 production by follicular helper T cells requires the conserved Il4 enhancer hypersensitivity site V
    Pandurangan Vijayanand
    Pulmonary and Critical Care Division, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
    Immunity 36:175-87. 2012
  4. pmc Nontargeted nucleotide analysis based on benzoylhistamine labeling-MALDI-TOF/TOF-MS: discovery of putative 6-oxo-thymine in DNA
    Poguang Wang
    Pharmaceutical Science and Barnett Institute, Northeastern University, Boston, Massachusetts 02115, United States
    Anal Chem 84:3811-9. 2012
  5. pmc PGC7, H3K9me2 and Tet3: regulators of DNA methylation in zygotes
    Jinsuk Kang
    La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA
    Cell Res 23:6-9. 2013
  6. pmc Selective inhibition of CD4+ T-cell cytokine production and autoimmunity by BET protein and c-Myc inhibitors
    Hozefa S Bandukwala
    La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA
    Proc Natl Acad Sci U S A 109:14532-7. 2012
  7. pmc D-2-hydroxyglutarate produced by mutant IDH1 perturbs collagen maturation and basement membrane function
    Masato Sasaki
    The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada
    Genes Dev 26:2038-49. 2012
  8. pmc The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine
    Yun Huang
    La Jolla Institute for Allergy and Immunology, La Jolla, California, USA
    Nat Protoc 7:1897-908. 2012
  9. pmc The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    William A Pastor
    La Jolla Institute for Allergy and Immunology, La Jolla, California, USA
    Nat Protoc 7:1909-17. 2012
  10. pmc High-resolution nucleosome mapping of targeted regions using BAC-based enrichment
    Erbay Yigit
    Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
    Nucleic Acids Res 41:e87. 2013

Detail Information

Publications36

  1. ncbi Foxp1 is an essential transcriptional regulator of B cell development
    Hui Hu
    Department of Pathology, Harvard Medical School, CBR Institute for Biomedical Research, Boston, Massachusetts 02115, USA
    Nat Immunol 7:819-26. 2006
    ..Our results identify Foxp1 as an essential participant in the transcriptional regulatory network of B lymphopoiesis...
  2. pmc RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation
    Elisa Närvä
    Turku Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, Finland
    Stem Cells 30:452-60. 2012
    ..Thus, we hypothesize that L1TD1 is part of the L1TD1-RHA-LIN28 complex that could influence levels of OCT4. Our results strongly suggest that L1TD1 has an important role in the regulation of stemness...
  3. pmc Interleukin-4 production by follicular helper T cells requires the conserved Il4 enhancer hypersensitivity site V
    Pandurangan Vijayanand
    Pulmonary and Critical Care Division, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
    Immunity 36:175-87. 2012
    ..Thus, Tfh and Th2 cells utilize distinct but overlapping molecular mechanisms to regulate Il4, a finding with important implications for understanding the molecular basis of allergic diseases...
  4. pmc Nontargeted nucleotide analysis based on benzoylhistamine labeling-MALDI-TOF/TOF-MS: discovery of putative 6-oxo-thymine in DNA
    Poguang Wang
    Pharmaceutical Science and Barnett Institute, Northeastern University, Boston, Massachusetts 02115, United States
    Anal Chem 84:3811-9. 2012
    ..The method provides deoxynucleotide-specific detection, accurate measurement of molecular ions, high sensitivity, semiquantitation, and, to the extent studied to date, normalization of response within a factor of <3...
  5. pmc PGC7, H3K9me2 and Tet3: regulators of DNA methylation in zygotes
    Jinsuk Kang
    La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA
    Cell Res 23:6-9. 2013
    ....
  6. pmc Selective inhibition of CD4+ T-cell cytokine production and autoimmunity by BET protein and c-Myc inhibitors
    Hozefa S Bandukwala
    La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA
    Proc Natl Acad Sci U S A 109:14532-7. 2012
    ..Our study demonstrates that inhibiting the functions of BET-family proteins during early T-cell differentiation causes long-lasting suppression of the proinflammatory functions of Th1 cells...
  7. pmc D-2-hydroxyglutarate produced by mutant IDH1 perturbs collagen maturation and basement membrane function
    Masato Sasaki
    The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada
    Genes Dev 26:2038-49. 2012
    ..Our study presents strong in vivo evidence that the D2HG produced by the mutant Idh1 enzyme is responsible for the above effects...
  8. pmc The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine
    Yun Huang
    La Jolla Institute for Allergy and Immunology, La Jolla, California, USA
    Nat Protoc 7:1897-908. 2012
    ..Moreover, it does not enrich for CA and CT repeats, as noted for 5hmC DNA IP using antibodies to 5hmC. The anti-CMS protocol takes 3 d to complete...
  9. pmc The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    William A Pastor
    La Jolla Institute for Allergy and Immunology, La Jolla, California, USA
    Nat Protoc 7:1909-17. 2012
    ..GLIB is capable of quantitatively tagging and precipitating fragments containing a single 5hmC molecule. Sample preparation and GLIB can be conducted in 2-3 d...
  10. pmc High-resolution nucleosome mapping of targeted regions using BAC-based enrichment
    Erbay Yigit
    Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
    Nucleic Acids Res 41:e87. 2013
    ..This method is widely applicable to mapping many features of chromatin and discerning its landscape in large genomes at unprecedented resolution...
  11. pmc DNA methylation and methylcytosine oxidation in cell fate decisions
    Kian Peng Koh
    KU Leuven Department of Development and Regeneration and Stem Cell Institute Leuven, 3000 Leuven, Belgium
    Curr Opin Cell Biol 25:152-61. 2013
    ....
  12. pmc TETonic shift: biological roles of TET proteins in DNA demethylation and transcription
    William A Pastor
    Sanford Consortium for Regenerative Medicine and La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, California 92037, USA
    Nat Rev Mol Cell Biol 14:341-56. 2013
    ....
  13. pmc Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells
    Kathryn Blaschke
    Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Obstetrics and Gynecology and Center for Reproductive Sciences, University of California San Francisco, San Francisco, California 94143, USA
    Nature 500:222-6. 2013
    ..Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells...
  14. pmc Global epigenomic reconfiguration during mammalian brain development
    Ryan Lister
    Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
    Science 341:1237905. 2013
    ....
  15. pmc Intronic non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees
    Pablo Cingolani
    Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI 48201, USA
    BMC Genomics 14:666. 2013
    ..By doing this, we have performed the first whole genome analyses of DNA modifications at non-CG sites in honey bees and correlated the effects of these DNA modifications on gene expression and alternative mRNA splicing...
  16. pmc Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice
    Myunggon Ko
    Division of Signaling and Gene Expression, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA
    Proc Natl Acad Sci U S A 108:14566-71. 2011
    ..Our data indicate that Tet2 has a critical role in regulating the expansion and function of HSCs, presumably by controlling 5hmC levels at genes important for the self-renewal, proliferation, and differentiation of HSCs...
  17. pmc Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A
    Anna M Jankowska
    Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA
    Blood 118:3932-41. 2011
    ..Various mutant genotype combinations were observed, indicating molecular heterogeneity in CMML. Our results suggest that molecular defects affecting distinct pathways can lead to similar clinical phenotypes...
  18. ncbi Chromosome transfer activates and delineates a locus control region for perforin
    Matthew E Pipkin
    Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
    Immunity 26:29-41. 2007
    ..Thus, LCR function is central for regulating the developmental and activation-specific PRF1 promoter activity characteristic of NK cells and CTL...
  19. ncbi Delivering the kiss of death: progress on understanding how perforin works
    Matthew E Pipkin
    CBR Institute for Biomedical Research, Harvard Medical School, Boston MA 02115, USA
    Curr Opin Immunol 19:301-8. 2007
    ..Recently, an alternative model has been proposed that involves active target cell collaboration with perforin to deliver granzymes and direct the target cell to an apoptotic, rather than necrotic, death...
  20. pmc Mouse Eri1 interacts with the ribosome and catalyzes 5.8S rRNA processing
    K Mark Ansel
    Immune Disease Institute, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA
    Nat Struct Mol Biol 15:523-30. 2008
    ..8S rRNA 3' end processing. Taken together, our findings indicate that Eri1 catalyzes the final trimming step in 5.8S rRNA processing, functionally and spatially connecting this regulator of RNAi with the basal translation machinery...
  21. pmc Regulation of CD45 alternative splicing by heterogeneous ribonucleoprotein, hnRNPLL
    Shalini Oberdoerffer
    Department of Pathology, Immune Disease Institute, Harvard Medical School, Boston, MA 02115, USA
    Science 321:686-91. 2008
    ..Induction of hnRNPLL during hematopoietic cell activation and differentiation may allow cells to rapidly shift their transcriptomes to favor proliferation and inhibit cell death...
  22. pmc MicroRNA-221-222 regulate the cell cycle in mast cells
    Ramon J Mayoral
    Institute for Research in Biomedicine, Bellinzona, Switzerland
    J Immunol 182:433-45. 2009
    ..Our study provides further insights on miR-221-222 transcriptional regulation as well as evidences that miR-221-222 regulate cell cycle checkpoints in mast cells in response to acute activation stimuli...
  23. pmc Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs
    Fernando Cruz-Guilloty
    Harvard Medical School and the Immune Disease Institute, Boston, MA 02115, USA
    J Exp Med 206:51-9. 2009
    ..Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs...
  24. pmc Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1
    Mamta Tahiliani
    Department of Pathology, Harvard Medical School and Immune Disease Institute, 200 Longwood Avenue, Boston, MA 02115, USA
    Science 324:930-5. 2009
    ..hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference-mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC...
  25. pmc Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids
    Lakshminarayan M Iyer
    National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA
    Cell Cycle 8:1698-710. 2009
    ....
  26. pmc Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells
    Matthew E Pipkin
    Harvard Medical School, Boston, MA 02115, USA Immune Disease Institute, Boston, MA 02115, USA
    Immunity 32:79-90. 2010
    ..Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development...
  27. pmc The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing
    Yun Huang
    Department of Pathology, Harvard Medical School and Immune Disease Institute, Boston, Massachusetts, United States of America
    PLoS ONE 5:e8888. 2010
    ..Since 5-hmC reacts with bisulfite to yield cytosine 5-methylenesulfonate (CMS), we asked how DNA containing 5-hmC behaves in bisulfite sequencing...
  28. ncbi Regulation of gene expression in peripheral T cells by Runx transcription factors
    Ivana M Djuretic
    Department of Pathology, Harvard Medical School and Immune Disease Institute and Program in Cellular and Molecular Medicine, Children s Hospital Boston, Boston, Massachusetts, USA
    Adv Immunol 104:1-23. 2009
    ..Here, we review gene regulation by Runx proteins in T lymphocytes, with a focus on their recently emerging roles in the development and function of peripheral CD4+ and CD8+ T lineages...
  29. ncbi The transcriptional control of the perforin locus
    Matthew E Pipkin
    Department of Signaling and Gene Expression, The La Jolla Institute of Allergy and Immunology, La Jolla, CA 92037, USA
    Immunol Rev 235:55-72. 2010
    ..Our review emphasizes how studies of PRF1/Prf1 gene transcription can illuminate not only the mechanisms of cytotoxic lymphocyte differentiation but also some basic principles of transcriptional regulation...
  30. pmc Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2
    Myunggon Ko
    Department of Pathology, Harvard Medical School, Immune Disease Institute and Program in Cellular and Molecular Medicine, Children s Hospital Boston, Boston, Massachusetts 02115, USA
    Nature 468:839-43. 2010
    ..Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs...
  31. pmc Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells
    Kian Peng Koh
    Immune Disease Institute and Program in Cellular and Molecular Medicine, Children s Hospital Boston, Boston, MA 02115, USA
    Cell Stem Cell 8:200-13. 2011
    ..Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs...
  32. pmc Structure of a domain-swapped FOXP3 dimer on DNA and its function in regulatory T cells
    Hozefa S Bandukwala
    Immune Disease Institute and Program in Cellular and Molecular Medicine, Children s Hospital, Boston, MA 02115, USA
    Immunity 34:479-91. 2011
    ..We conclude that FOXP3-mediated suppressor function requires dimerization through the forkhead domain and that mutations in the dimer interface can lead to the systemic autoimmunity observed in IPEX patients...
  33. pmc Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells
    William A Pastor
    Harvard Medical School, Immune Disease Institute and Program in Cellular and Molecular Medicine, Children s Hospital Boston, Boston, Massachusetts 02115, USA
    Nature 473:394-7. 2011
    ..Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the 'poised' chromatin signature found at developmentally-regulated genes in ES cells...
  34. pmc Distinct roles of the methylcytosine oxidases Tet1 and Tet2 in mouse embryonic stem cells
    Yun Huang
    Divisions of Signalling and Gene Expression and Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037
    Proc Natl Acad Sci U S A 111:1361-6. 2014
    ..Together, the data point to a complex interplay between Tet1 and Tet2 in mESC, and to distinct roles for these two proteins in regulating promoter, exon, and polyadenylation site usage in cells. ..