title: Global DNA hypomethylation prevents consolidation of differentiation programs and allows reversion to the embryonic stem cell state.
abstract: DNA methylation patterns change dynamically during mammalian development and lineage specification, yet scarce information is available about how DNA methylation affects gene expression profiles upon differentiation. Here we determine genome-wide transcription profiles during undirected differentiation of severely hypomethylated (Dnmt1⁻/⁻) embryonic stem cells (ESCs) as well as ESCs completely devoid of DNA methylation (Dnmt1⁻/⁻;Dnmt3a⁻/⁻;Dnmt3b⁻/⁻ or TKO) and assay their potential to transit in and out of the ESC state. We find that the expression of only few genes mainly associated with germ line function and the X chromosome is affected in undifferentiated TKO ESCs. Upon initial differentiation as embryoid bodies (EBs) wild type, Dnmt1⁻/⁻ and TKO cells downregulate pluripotency associated genes and upregulate lineage specific genes, but their transcription profiles progressively diverge upon prolonged EB culture. While Oct4 protein levels are completely and homogeneously suppressed, transcription of Oct4 and Nanog is not completely silenced even at late stages in both Dnmt1⁻/⁻ and TKO EBs. Despite late wild type and Dnmt1⁻/⁻ EBs showing a much higher degree of concordant expression, after EB dissociation and replating under pluripotency promoting conditions both Dnmt1⁻/⁻ and TKO cells, but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation seems not to be critical for initial activation of differentiation programs, it is crucial for permanent restriction of developmental fate during differentiation.
authors.affiliation: Department of Biology II, Ludwig Maximilians University Munich, Planegg-Martinsried, Germany.
authors.forename: Christine S
authors.initials: CS
authors.lastname: Schmidt
authors.forename: Sebastian
authors.initials: S
authors.lastname: Bultmann
authors.forename: Daniela
authors.initials: D
authors.lastname: Meilinger
authors.forename: Benedikt
authors.initials: B
authors.lastname: Zacher
authors.forename: Achim
authors.initials: A
authors.lastname: Tresch
authors.forename: Kerstin C
authors.initials: KC
authors.lastname: Maier
authors.forename: Christian
authors.initials: C
authors.lastname: Peter
authors.forename: Dietmar E
authors.initials: DE
authors.lastname: Martin
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.forename: Fabio
authors.initials: F
authors.lastname: Spada
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000172:deficiency / Q000235:genetics; D019175:DNA Methylation; D058732:Embryoid Bodies / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology; D044127:Epigenesis, Genetic; D055786:Gene Knockout Techniques; D016678:Genome; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D053605:Leukemia Inhibitory Factor / Q000502:physiology; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D015398:Signal Transduction; D059467:Transcriptome
chemical_list: D018398:Homeodomain Proteins; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619381:Dnmt1 protein, mouse