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      Hematopoietic Stem and Progenitor Cells Acquire Distinct DNA-Hypermethylation During in vitro Culture

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          Abstract

          Hematopoietic stem and progenitor cells (HPCs) can be maintained in vitro, but the vast majority of their progeny loses stemness during culture. In this study, we compared DNA-methylation (DNAm) profiles of freshly isolated and culture-expanded HPCs. Culture conditions of CD34+ cells - either with or without mesenchymal stromal cells (MSCs) - had relatively little impact on DNAm, although proliferation is greatly increased by stromal support. However, all cultured HPCs - even those which remained CD34+ - acquired significant DNA-hypermethylation. DNA-hypermethylation occurred particularly in up-stream promoter regions, shore-regions of CpG islands, binding sites for PU.1, HOXA5 and RUNX1, and it was reflected in differential gene expression and variant transcripts of DNMT3A. Low concentrations of DNAm inhibitors slightly increased the frequency of colony-forming unit initiating cells. Our results demonstrate that HPCs acquire DNA-hypermethylation at specific sites in the genome which is relevant for the rapid loss of stemness during in vitro manipulation.

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          Most cited references 49

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          Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.

          DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. 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.
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            DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

            The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease.
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              Genome-wide methylation analysis of human colon cancer reveals similar hypo- and hypermethylation at conserved tissue-specific CpG island shores

              Alterations in DNA methylation (DNAm) in cancer have been known for 25 years, including hypomethylation of oncogenes and hypermethylation of tumor suppressor genes1. However, most studies of cancer methylation have assumed that functionally important DNAm will occur in promoters, and that most DNAm changes in cancer occur in CpG islands2,3. Here we show that most methylation alterations in colon cancer occur not in promoters, and also not in CpG islands but in sequences up to 2 kb distant which we term “CpG island shores.” CpG island shore methylation was strongly related to gene expression, and it was highly conserved in mouse, discriminating tissue types regardless of species of origin. There was a surprising overlap (45-65%) of the location of colon cancer-related methylation changes with those that distinguished normal tissues, with hypermethylation enriched closer to the associated CpG islands, and hypomethylation enriched further from the associated CpG island and resembling non-colon normal tissues. Thus, methylation changes in cancer are at sites that vary normally in tissue differentiation, and they are consistent with the epigenetic progenitor model of cancer4, that epigenetic alterations affecting tissue-specific differentiation are the predominant mechanism by which epigenetic changes cause cancer.
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                Author and article information

                Affiliations
                [1]Helmholtz-Institute for Biomedical Engineering, RWTH University Medical School, Aachen, Germany
                [2]Institute for Biomedical Technology – Cell Biology, RWTH University Medical School, Aachen, Germany
                [3]Department of Obstetrics and Gynecology, RWTH University Medical School, Aachen, Germany
                [4]Interdisciplinary Centre for Clinical Research (IZKF), RWTH University Medical School, Aachen, Germany
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                28 November 2013
                2013
                : 3
                24284763
                3842544
                srep03372
                10.1038/srep03372
                Copyright © 2013, Macmillan Publishers Limited. All rights reserved

                This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/

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