Differential maintenance and de novo methylating activity by three DNA methyltransferases in aging and immortalized fibroblasts

Genomic methylation, which influences many cellular processes such as gene expression and chromatin organization, generally declines with cellular senescence although some genes undergo paradoxical hypermethylation during cellular aging and immortalization. To explore potential mechanisms for this process, we analyzed the methylating activity of three DNA methyltransferases (Dnmts) in aging and immortalized WI‐38 fibroblasts. Overall maintenance methylating activity by the Dnmts greatly decreased during cellular senescence. In immortalized WI‐38 cells, maintenance methylating activity was similar to that of normal young cells. Combined de novo methylation activity of the Dnmts initially decreased but later increased as WI‐38 cells aged and was strikingly elevated in immortalized cells. To further elucidate the mechanisms for changes in DNA methylation in aging and immortalized cells, the individual Dnmts were separated and individually assessed for maintenance and de novo methylating activity. We resolved three Dnmt fractions, one of which was the major maintenance methyltransferase, Dnmt1, which declined steadily in activity with cellular senescence and immortalization. However, a more basic Dnmt, which has significant de novo methylating activity, increased markedly in activity in aging and immortalized cells. We have identified this methyltransferase as Dnmt3b which has an important role in neoplastic transformation but its role in cellular senescence and immortalization has not previously been reported. An acidic Dnmt we isolated also had increased de novo methylating activity in senescent and immortalized WI‐38 cells. These studies indicate that reduced genome‐wide methylation in aging cells may be attributed to attenuated Dnmt1 activity but that regional or gene‐localized hypermethylation in aging and immortalized cells may be linked to increased de novo methylation by Dnmts other than the maintenance methyltransferase. J. Cell. Biochem. 84: 324–334, 2002. © 2001 Wiley‐Liss, Inc.

[1]  James R. Smith,et al.  DNA Methyltransferase Inhibition in Normal Human Fibroblasts Induces a p21-dependent Cell Cycle Withdrawal* , 2001, The Journal of Biological Chemistry.

[2]  T. Bestor,et al.  Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA. , 2001, Nucleic acids research.

[3]  K. Davies,et al.  Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I— effects of proliferative senescence , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[4]  Ulf Hellman,et al.  Purification, characterization and amino‐acid sequence analysis of a thermostable, low molecular mass endo‐β‐1,4‐glucanase from blue mussel, Mytilus edulis , 2000 .

[5]  A. Bird,et al.  Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Macleod,et al.  Characterization of the Human DNA Methyltransferase Splice Variant Dnmt1b* , 2000, The Journal of Biological Chemistry.

[7]  R. Craver,et al.  Separation of hemoglobin variants with similar charge by capillary isoelectric focusing: Value of isoelectric point for identification of common and uncommon hemoglobin variants , 2000, Electrophoresis.

[8]  K. Kasai,et al.  Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection , 2000, Electrophoresis.

[9]  T. Bestor,et al.  Cytosine methylation and human cancer. , 2000, Current opinion in oncology.

[10]  C. Wijmenga,et al.  The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[11]  N. Tommerup,et al.  Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene , 1999, Nature.

[12]  D. Haber,et al.  DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development , 1999, Cell.

[13]  X. Chen,et al.  Two major forms of DNA (cytosine-5) methyltransferase in human somatic tissues. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  E. Li,et al.  Cloning, expression and chromosome locations of the human DNMT3 gene family. , 1999, Gene.

[15]  M. Szyf,et al.  Downregulation of DNA (cytosine-5-)methyltransferase is a late event in NGF-induced PC12 cell differentiation. , 1999, Brain research. Molecular brain research.

[16]  Peter A. Jones,et al.  Cancer-epigenetics comes of age , 1999, Nature Genetics.

[17]  K. Robertson,et al.  The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. , 1999, Nucleic acids research.

[18]  R. Jaenisch,et al.  A Short DNA Methyltransferase Isoform Restores Methylation In Vivo * , 1998, The Journal of Biological Chemistry.

[19]  S. Baylin,et al.  Aging and DNA methylation in colorectal mucosa and cancer. , 1998, Cancer research.

[20]  T. Zglinicki.  Telomeres: Influencing the Rate of Aging , 1998 .

[21]  T. Zglinicki,et al.  Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts. , 1998, The journals of gerontology. Series A, Biological sciences and medical sciences.

[22]  L. Poirier,et al.  Elevated expression and altered pattern of activity of DNA methyltransferase in liver tumors of rats fed methyl-deficient diets. , 1998, Carcinogenesis.

[23]  M. Szyf,et al.  Multiple Isoforms of DNA Methyltransferase Are Encoded by the Vertebrate Cytosine DNA Methyltransferase Gene* , 1998, The Journal of Biological Chemistry.

[24]  E. Li,et al.  Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases , 1998, Nature Genetics.

[25]  C. Hutchison,et al.  Analysis in Escherichia coli of the effects of in vivo CpG methylation catalyzed by the cloned murine maintenance methyltransferase. , 1998, Biochemical and biophysical research communications.

[26]  J. Herman,et al.  Alterations in DNA methylation: a fundamental aspect of neoplasia. , 1998, Advances in cancer research.

[27]  T. Bestor,et al.  DNA (cytosine-5)-methyltransferases in mouse cells and tissues. Studies with a mechanism-based probe. , 1997, Journal of molecular biology.

[28]  C. Hutchison,et al.  Control of methylation spreading in synthetic DNA sequences by the murine DNA methyltransferase. , 1997, Journal of molecular biology.

[29]  Stefan U Kass,et al.  DNA methylation directs a time-dependent repression of transcription initiation , 1997, Current Biology.

[30]  R. Jaenisch,et al.  De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. , 1996, Development.

[31]  A. Riggs,et al.  Dynamic methylation adjustment and counting as part of imprinting mechanisms. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[32]  S. Baylin,et al.  Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[33]  R. Beart,et al.  Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. , 1995, Cancer research.

[34]  C Roskelley,et al.  A biomarker that identifies senescent human cells in culture and in aging skin in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[35]  C. Hutchison,et al.  Mammalian DNA (Cytosine-5-)-methyltransferase Expressed in Escherichia coli, Purified and Characterized (*) , 1995, The Journal of Biological Chemistry.

[36]  B. Ames,et al.  Oxidative DNA damage and senescence of human diploid fibroblast cells. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Szyf,et al.  Regulation of the DNA Methyltransferase by the Ras-AP-1 Signaling Pathway (*) , 1995, The Journal of Biological Chemistry.

[38]  S. Baylin,et al.  Stabilization of DNA methyltransferase levels and CpG island hypermethylation precede SV40-induced immortalization of human fibroblasts. , 1994, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[39]  S. Baylin,et al.  Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon , 1994, Nature Genetics.

[40]  T. Tollefsbol,et al.  Mechanisms for methylation-mediated gene silencing and aging. , 1993, Medical hypotheses.

[41]  K. Chen,et al.  A specific CCAAT-binding protein, CBP/tk, may be involved in the regulation of thymidine kinase gene expression in human IMR-90 diploid fibroblasts during senescence. , 1993, The Journal of biological chemistry.

[42]  Cooney Ca,et al.  Are somatic cells inherently deficient in methylation metabolism? A proposed mechanism for DNA methylation loss, senescence and aging. , 1993 .

[43]  A. Razin,et al.  Gene methylation patterns and expression. , 1993, EXS.

[44]  T. Bestor,et al.  Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. , 1992, The EMBO journal.

[45]  Rudolf Jaenisch,et al.  Targeted mutation of the DNA methyltransferase gene results in embryonic lethality , 1992, Cell.

[46]  P. Hornsby,et al.  Demethylation of satellite I DNA during senescence of bovine adrenocortical cells in culture. , 1992, Mutation research.

[47]  S. Baylin,et al.  High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[48]  R. Adams.  DNA methylation. The effect of minor bases on DNA-protein interactions. , 1990, The Biochemical journal.

[49]  B. Li,et al.  Attenuated induction of heat shock gene expression in aging diploid fibroblasts. , 1989, The Journal of biological chemistry.

[50]  R. Singhal,et al.  DNA methylation in aging of mice , 1987, Mechanisms of Ageing and Development.

[51]  G. Margison,et al.  The in vitro lifespan of MRC-5 cells is shortened by 5-azacytidine-induced demethylation. , 1987, Experimental cell research.

[52]  P. Jones,et al.  DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture. , 1986, The Journal of biological chemistry.

[53]  V. L. Wilson,et al.  DNA methylation decreases in aging but not in immortal cells. , 1983, Science.

[54]  R. Burdon,et al.  Mouse DNA methylase: methylation of native DNA. , 1979, Biochimica et biophysica acta.

[55]  R. Schimke,et al.  Protein degradation in human fibroblasts (WI-38). Effects of aging, viral transformation, and amino acid analogs. , 1976, The Journal of biological chemistry.

[56]  E. Scarano,et al.  DNA Methylation , 1973, Nature.

[57]  R. Schimke,et al.  Catalase turnover in human diploid cell cultures. , 1972, Experimental cell research.

[58]  L. Hayflick.  THE LIMITED IN VITRO LIFETIME OF HUMAN DIPLOID CELL STRAINS. , 1965, Experimental cell research.