Genomes and Genes
Chemical modulators of Tet-family proteins
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
- Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acidsLakshminarayan 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....
- STIM1 gates the store-operated calcium channel ORAI1 in vitroYubin 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....
- The behaviour of 5-hydroxymethylcytosine in bisulfite sequencingYun 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...
- Molecular basis of calcium signaling in lymphocytes: STIM and ORAIPatrick 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...
- Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2Myunggon 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...
- Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cellsKian 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...
- Structure of a domain-swapped FOXP3 dimer on DNA and its function in regulatory T cellsHozefa 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...
- Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cellsWilliam 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...
- Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in miceMyunggon 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...