Chemical modulators of Tet-family proteins

Summary

Principal Investigator: Anjana Rao
Abstract: DESCRIPTION (provided by applicant): The methylation status of DNA influences many biological processes during mammalian development, including retrotransposon silencing, X-inactivation and the asymmetric expression of parentally imprinted genes. In mammalian cells, DNA methylation occurs almost exclusively as symmetrical methylation of cytosine in the context of the dinucleotide CpG. 5-methylcytosine (5mC) is found at high levels at repetitive elements, telomeres and the inactive X-chromosome in females, and its presence correlates with diminished gene expression. In cancer, DNA methylation has been linked to aberrant silencing of tumor suppressor genes;in the central nervous system (CNS), it has been implicated, among other things, in learning, memory and synaptic plasticity. We have recently discovered that the TET proteins TET1, TET2 and TET3 constitute a new family of 2-oxoglutarate (2OG)- and Fe(II)-dependent dioxygenases that catalyse hydroxylation of 5mC to 5-hydroxymethylcytosine (hmC) in DNA. hmC levels and TET expression/ activity appear to be tightly regulated. (i) hmC is present in genomic DNA of undifferentiated ES cells but not differentiated cell types, and hmC levels diminish upon ES cell differentiation. (ii) TET1 is an MLL partner in acute myeloid and lymphoid leukemia (AML, ALL), and TET2 is implicated in myelodysplastic syndromes and AML. (iii) In the CNS, hmC is present at high levels in cerebellar Purkinje neurons;moreover, TET1 and TET2 mRNA are expressed in the brain, with TET1 mRNA being particularly high in Purkinje cells and hippocampal pyramidal cells. Together these data suggest potential functions of TET proteins and hmC in (a) pluripotency and stem cell function;(b) oncogenic transformation, especially of haematopoietic cells;and (c) motor control, learning and memory. Here we propose to perform two separate high-throughput screens to identify small-molecule activators and inhibitors of TET-family proteins. The first is a completely in vitro screen in which we will use recombinant catalytic domains of TET1, TET2 or TET3 expressed in insect cells to convert 5mC to hmC in methylated double-stranded DNA oligonucleotides. The appearance of hmC will be monitored by using an antibody to hmC. The second is a cell-based screen which relies on the fact that overexpression of TET1, TET2 or TET3 catalytic domains in HEK293 cells yields cellular phenotypes that are readily assayed by high-throughput imaging: loss of 5mC staining, acquisition of hmC staining, and increased nuclear size. Together these studies should identify small molecules that modulate the functions of this new and interesting class of enzymes, and may be used as reagents to investigate their biological functions in cells and in organisms. PUBLIC HEALTH RELEVANCE: In addition to the four major bases in the DNA alphabet - A, C, G and T - there is also a minor fifth base, 5-methylcytosine (5mC) that has a disproportionately important role. Abnormal production, distribution and recognition of 5mC have been linked to developmental abnormalities and genetic diseases, including Rett syndrome, an autism spectrum disorder. We recently identified a new family of proteins, the TET proteins that convert 5mC to a sixth base, 5-hydroxymethylcytosine (hmC). TET proteins have been linked to cancer, and hmC is found at high levels in embryonic stem cells and certain classes of neuronal cells. In this proposal we will devise methods to find chemical activators and inhibitors of TET proteins. These molecules will be useful for further investigations into the biological functions of TET proteins, and may eventually be used in the clinic to treat Rett syndrome and other diseases of DNA methylation.
Funding Period: ----------------2009 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. 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
  2. pmc STIM1 gates the store-operated calcium channel ORAI1 in vitro
    Yubin Zhou
    Immune Disease Institute and Program in Cellular and Molecular Medicine, Children s Hospital, Boston, Massachusetts, USA
    Nat Struct Mol Biol 17:112-6. 2010
  3. 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
  4. pmc Molecular basis of calcium signaling in lymphocytes: STIM and ORAI
    Patrick G Hogan
    Department of Pathology, Harvard Medical School, Immune Disease Institute, Children s Hospital Boston, Massachusetts 02115, USA
    Annu Rev Immunol 28:491-533. 2010
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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

Scientific Experts

  • Anjana Rao
  • Yun Huang
  • Myunggon Ko
  • Mamta Tahiliani
  • Hozefa S Bandukwala
  • Kian Peng Koh
  • William A Pastor
  • Patrick G Hogan
  • George Q Daley
  • Edward D Lamperti
  • Utz J Pape
  • X Shirley Liu
  • Suneet Agarwal
  • Jungeun An
  • Yubin Zhou
  • L Aravind
  • Lakshminarayan M Iyer
  • Sridhar Rao
  • Yevgeny Brudno
  • Gustavo Mostoslavsky
  • Joseph R Ecker
  • James C Stroud
  • Scott J Rodig
  • Akiko Yabuuchi
  • Bianca Barboza
  • Lin Chen
  • Klaus Rajewsky
  • Kerrianne Cunniff
  • Riitta Lahesmaa
  • Stuart H Orkin
  • Elizabeth C Thompson
  • Sahasransu Mahapatra
  • Yongheng Chen
  • Christophe Benoist
  • Erin M McLoughlin
  • Hope R Henderson
  • Yongqing Wu
  • Cesar A Sommer
  • Ryan Hastie
  • Julie Nardone
  • Ryan Lister
  • Patrice M Milos
  • Angeliki Tsangaratou
  • Sergei B Koralov
  • Srimoyee Ghosh
  • Diane Mathis
  • Philipp Kapranov
  • Markus Feuerer
  • Asta Laiho
  • Rebecca Ganetzky
  • Anna M Jankowska
  • Masatsugu Oh-hora
  • Danya Machnes
  • David R Liu
  • Jochen Zimmer
  • Paul Meraner
  • Antonio Stura
  • Jaroslaw P Maciejewski
  • Yinghua Shen
  • Richard S Lewis
  • Hyoung T Kwon

Detail Information

Publications9

  1. 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
    ....
  2. pmc STIM1 gates the store-operated calcium channel ORAI1 in vitro
    Yubin Zhou
    Immune Disease Institute and Program in Cellular and Molecular Medicine, Children s Hospital, Boston, Massachusetts, USA
    Nat Struct Mol Biol 17:112-6. 2010
    ....
  3. 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...
  4. pmc Molecular basis of calcium signaling in lymphocytes: STIM and ORAI
    Patrick G Hogan
    Department of Pathology, Harvard Medical School, Immune Disease Institute, Children s Hospital Boston, Massachusetts 02115, USA
    Annu Rev Immunol 28:491-533. 2010
    ..In this review, we discuss selected aspects of Ca(2+) signaling in cells of the immune system, focusing on the roles of STIM and ORAI proteins in store-operated Ca(2+) entry...
  5. 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...
  6. 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...
  7. 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...
  8. 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...
  9. 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...