Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine

Genome-wide erasure of DNA cytosine-5 methylation has been reported to occur along the paternal pronucleus in fertilized oocytes in an apparently replication-independent manner, but the mechanism of this reprogramming process has remained enigmatic. Recently, considerable amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5-methylcytosine (5mC) by TET proteins, have been detected in certain mammalian tissues. 5hmC has been proposed as a potential intermediate in active DNA demethylation. Here, we show that in advanced pronuclear-stage zygotes the paternal pronucleus contains substantial amounts of 5hmC but lacks 5mC. The converse is true for the maternal pronucleus, which retains 5mC but shows little or no 5hmC signal. Importantly, 5hmC persists into mitotic one-cell, two-cell, and later cleavage-stage embryos, suggesting that 5mC oxidation is not followed immediately by genome-wide removal of 5hmC through excision repair pathways or other mechanisms. This conclusion is supported by bisulfite sequencing data, which shows only limited conversion of modified cytosines to cytosines at several gene loci. It is likely that 5mC oxidation is carried out by the Tet3 oxidase. Tet3, but not Tet1 or Tet2, was expressed at high levels in oocytes and zygotes, with rapidly declining levels at the two-cell stage. Our results show that 5mC oxidation is part of the early life cycle of mammals.

[1]  H. Shibuya,et al.  DNA demethylation for hormone-induced transcriptional derepression , 2011, Nature.

[2]  Peter A. Jones,et al.  DNA methylation and cellular reprogramming. , 2010, Trends in cell biology.

[3]  Yi Zhang,et al.  Active DNA demethylation: many roads lead to Rome , 2010, Nature Reviews Molecular Cell Biology.

[4]  Yi Zhang,et al.  Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification , 2010, Nature.

[5]  A. Orth,et al.  Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen , 2010, Proceedings of the National Academy of Sciences.

[6]  Guoping Fan,et al.  DNA methylation in cell differentiation and reprogramming: an emerging systematic view. , 2010, Regenerative medicine.

[7]  M. Surani,et al.  Genome-Wide Reprogramming in the Mouse Germ Line Entails the Base Excision Repair Pathway , 2010, Science.

[8]  H. Schöler,et al.  Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes , 2010, The EMBO journal.

[9]  Swati Kadam,et al.  Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine , 2010, Nucleic acids research.

[10]  M. Pellegrini,et al.  Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency , 2010, Nature.

[11]  Helen M. Blau,et al.  Reprogramming towards pluripotency requires AID-dependent DNA demethylation , 2010, Nature.

[12]  P. Geyer,et al.  Faculty Opinions recommendation of A role for the elongator complex in zygotic paternal genome demethylation. , 2010 .

[13]  David R. Liu,et al.  The Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing , 2010, PloS one.

[14]  David R. Liu,et al.  Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1 , 2009, Science.

[15]  N. Heintz,et al.  The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain , 2009, Science.

[16]  Israel Steinfeld,et al.  Developmental programming of CpG island methylation profiles in the human genome , 2009, Nature Structural &Molecular Biology.

[17]  C. Sapienza,et al.  Conserved DNA methylation in Gadd45a-/- mice , 2009, Epigenetics.

[18]  U. Hoffmann‐Rohrer,et al.  TAF12 recruits Gadd45a and the nucleotide excision repair complex to the promoter of rRNA genes leading to active DNA demethylation. , 2009, Molecular cell.

[19]  S. Henikoff,et al.  DNA demethylation by DNA repair. , 2009, Trends in genetics : TIG.

[20]  B. Cairns,et al.  DNA Demethylation in Zebrafish Involves the Coupling of a Deaminase, a Glycosylase, and Gadd45 , 2008, Cell.

[21]  G. Pfeifer,et al.  Haploid male germ cell‐ and oocyte‐specific Mbd3l1 and Mbd3l2 genes are dispensable for early development, fertility, and zygotic DNA demethylation in the mouse , 2008, Developmental dynamics : an official publication of the American Association of Anatomists.

[22]  T. Bestor,et al.  The Colorful History of Active DNA Demethylation , 2008, Cell.

[23]  A. Bird,et al.  DNA methylation landscapes: provocative insights from epigenomics , 2008, Nature Reviews Genetics.

[24]  Albert Jeltsch,et al.  Cyclical DNA methylation of a transcriptionally active promoter , 2008, Nature.

[25]  G. Pfeifer,et al.  GADD45A Does Not Promote DNA Demethylation , 2008, PLoS genetics.

[26]  W. Reik.  Stability and flexibility of epigenetic gene regulation in mammalian development , 2007, Nature.

[27]  M. Surani,et al.  Genetic and Epigenetic Regulators of Pluripotency , 2007, Cell.

[28]  Peter A. Jones,et al.  The Epigenomics of Cancer , 2007, Cell.

[29]  Christof Niehrs,et al.  Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation , 2007, Nature.

[30]  L. Sowers,et al.  Endogenous cytosine damage products alter the site selectivity of human DNA maintenance methyltransferase DNMT1. , 2007, Cancer research.

[31]  Jian-Kang Zhu,et al.  Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Rudolf Jaenisch,et al.  Nuclear reprogramming and pluripotency , 2006, Nature.

[33]  Jon Penterman,et al.  DEMETER DNA Glycosylase Establishes MEDEA Polycomb Gene Self-Imprinting by Allele-Specific Demethylation , 2006, Cell.

[34]  U. Bunz.  How Are Alkynes Scrambled? , 2005, Science.

[35]  W. Reik,et al.  Active demethylation of the paternal genome in the mouse zygote , 2000, Current Biology.

[36]  T. Haaf,et al.  Spatial Separation of Parental Genomes in Preimplantation Mouse Embryos , 2000, The Journal of cell biology.

[37]  J. Walter,et al.  Embryogenesis: Demethylation of the zygotic paternal genome , 2000, Nature.

[38]  M Plachot,et al.  Chromosome methylation patterns during mammalian preimplantation development. , 1998, Genes & development.

[39]  G. Teebor,et al.  5-Hydroxymethylcytosine DNA glycosylase activity in mammalian tissue. , 1988, Biochemical and biophysical research communications.

[40]  R Holliday,et al.  DNA modification mechanisms and gene activity during development , 1975, Science.

[41]  Satoshi Tanaka,et al.  PGC7/Stella protects against DNA demethylation in early embryogenesis , 2007, Nature Cell Biology.

[42]  E. Li,et al.  Establishment and maintenance of DNA methylation patterns in mammals. , 2006, Current topics in microbiology and immunology.

[43]  A. Bird,et al.  Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). , 2004, Nucleic acids research.

[44]  Wendy Dean,et al.  Dynamic reprogramming of DNA methylation in the early mouse embryo. , 2002, Developmental biology.

[45]  M. A. Goldman,et al.  Mammalian X chromosome inactivation. , 1992, Molecular genetic medicine.

[46]  Arthur D. Riggs,et al.  X inactivation, differentiation, and DNA methylation. , 1975, Cytogenetics and cell genetics.