DNMT1 regulates the timing of DNA methylation by DNMT3 in an enzymatic activity-dependent manner in mouse embryonic stem cells

DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.

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MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells

Nature Structural & Molecular Biology ◽

10.1038/nsmb.1391 ◽

2008 ◽

Vol 15 (3) ◽

pp. 259-267 ◽

Cited By ~ 334

Author(s):

Lasse Sinkkonen ◽

Tabea Hugenschmidt ◽

Philipp Berninger ◽

Dimos Gaidatzis ◽

Fabio Mohn ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

De Novo ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Transcriptional Repressors

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PRC2 Is Required to Maintain Expression of the Maternal Gtl2-Rian-Mirg Locus by Preventing De Novo DNA Methylation in Mouse Embryonic Stem Cells

Cell Reports ◽

10.1016/j.celrep.2015.07.053 ◽

2015 ◽

Vol 12 (9) ◽

pp. 1456-1470 ◽

Cited By ~ 40

Author(s):

Partha Pratim Das ◽

David A. Hendrix ◽

Effie Apostolou ◽

Alice H. Buchner ◽

Matthew C. Canver ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

De Novo ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells

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Lipid rafts integrity is essential for the prolactin-induced mitogenesis in mouse embryonic stem cells

The International Journal of Developmental Biology ◽

10.1387/ijdb.210194dm ◽

2021 ◽

Author(s):

Sophia Karouzaki ◽

Charoula Peta ◽

Emmanouella Tsirimonaki ◽

George Leondaritis ◽

Kostas Vougas ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Lipid Rafts ◽

Membrane Lipids ◽

Close Association ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Mass Spectrometry Analysis ◽

Signalling Pathways ◽

Dependent Manner

Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constrains and regulation applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using twodimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergentfree preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs are in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.

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MOV10 facilitates messenger RNA decay in an N6-methyladenosine (m6A) dependent manner to maintain the mouse embryonic stem cells state

10.1101/2021.08.11.456030 ◽

2021 ◽

Author(s):

Majid Mehravar ◽

Yogesh Kumar ◽

Moshe Olshansky ◽

Dhiru Bansal ◽

Craig Dent ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Biology ◽

Messenger Rna ◽

Mrna Decay ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Beta Catenin ◽

Dependent Manner ◽

Leukaemia Virus

N6-methyladenosine (m6A) is the most predominant internal mRNA modification in eukaryotes, recognised by its reader proteins (so-called m6A-readers) for regulating subsequent mRNA fates, such as splicing, export, localisation, decay, stability, and translation to control several biological processes. Although a few m6A-readers have been identified, yet the list is incomplete. Here, we identify a new m6A-reader protein, Moloney leukaemia virus 10 homologue (MOV10), in mouse embryonic stem cells (mESCs). MOV10 recognises m6A-containing mRNAs with a conserved GGm6ACU motif. Mechanistic studies uncover that MOV10 facilitates mRNA decay of its bound m6A- containing mRNAs in an m6A-dependent manner within the cytoplasmic processing bodies (P-bodies). Furthermore, MOV10 decays the Gsk-3beta mRNA through m6A that stabilises the BETA-CATENIN expression of a WNT/BETA-CATENIN signalling pathway to regulate downstream NANOG expression for maintaining the mESC state. Thus, our findings reveal how a newly identified m6A-reader, MOV10 mediates mRNA decay via m6A that impact embryonic stem cell biology.

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278 EFFECT OF XENOESTROGENS ON THE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab278 ◽

2009 ◽

Vol 21 (1) ◽

pp. 236

Author(s):

E.-M. Jeung ◽

K.-C. Choi ◽

E.-B. Jeung

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Embryoid Bodies ◽

Time Dependent ◽

Dependent Manner ◽

Cardiomyocyte Differentiation ◽

Differentiation Markers ◽

Oct4 Expression

Endocrine disruptors (ED) may have adverse impacts on reproductive and immune systems in human and wild animals. It has been shown that octyl-phenol (OP) and nonyl-phenol (NP) have estrogenicity in estrogen-responding cells or tissues. In this study, we further investigated the effect(s) of OP and NP on the expression of undifferentiation and differentiation markers in mouse embryonic stem cells (ESC), which function as an important factor in the differentiation of ESC into cardiomyocytes. Mouse ESC were cultured in hanging drops to form embryoid bodies (EB). The medium was replaced with phenol red-free DMEM/F-12 supplemented with 5% charcoal-dextran-stripped FBS. The ESC were treated with OP, NP (1Ã-10-6 and 1Ã-10-7 M) or 17β-estradiol (E2; 1Ã-10-8 and 1Ã-10-9 M) in a time-dependent manner (1, 2 and 3 days), and EB were treated with identical concentrations for 4 and 8 days, respectively. High increasing doses of OP and NP were employed in this study because a binding affinity of ED to estrogen receptors (ER) is about 1000 less than that of E2. We determined the mRNA expression of undifferentiation markers (Oct4, Sox2 and Zfp206) and cardiomyocyte differentiation markers (cardiac alpha-MHC, beta-MHC and myosin light chain isoform-2V) using real-time PCR. In ESC, undifferentiation markers were identified. It is of interest that treatment with OP, NP or E2 induced a significant increase (1.4 5.5-fold) in Oct4 expression at the transcription levels according to a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2 and Zfp206 genes in ESC, suggesting that OP and NP may play a role as an Oct4 enhancer in ESC. In addition, both undifferentiation and cardiomyocyte differentiation markers were identified in EB. Treatment with OP and NP induced a significant increase in the expression of Oct4, Sox2 and Zfp206 genes at the transcription levels in a dose-dependent manner for 4 days, whereas Oct4 expression was only induced at these doses for 8 days. In contrast, cardiomyocyte differentiation markers were reduced by these ED in EB. Taken together, these results suggest that OP and NP play a role as a positive regulator in the undifferentiation process of ESC and EB, and maintenance and differentiation of mouse ESC.

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Targeting of De Novo DNA Methylation Throughout the Oct-4 Gene Regulatory Region in Differentiating Embryonic Stem Cells

PLoS ONE ◽

10.1371/journal.pone.0009937 ◽

2010 ◽

Vol 5 (4) ◽

pp. e9937 ◽

Cited By ~ 51

Author(s):

Rodoniki Athanasiadou ◽

Dina de Sousa ◽

Kevin Myant ◽

Cara Merusi ◽

Irina Stancheva ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

De Novo ◽

Regulatory Region ◽

Embryonic Stem ◽

Gene Regulatory

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DNA methylation in mouse embryonic stem cells and development

Cell and Tissue Research ◽

10.1007/s00441-007-0537-9 ◽

2007 ◽

Vol 331 (1) ◽

pp. 31-55 ◽

Cited By ~ 27

Author(s):

Tom Latham ◽

Nick Gilbert ◽

Bernard Ramsahoye

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells

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ZD7288, a blocker of the HCN channel family, increases doubling time of mouse embryonic stem cells and modulates differentiation outcomes in a context-dependent manner

SpringerPlus ◽

10.1186/s40064-016-1678-7 ◽

2016 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Anna Omelyanenko ◽

Petra Sekyrova ◽

Michael Andäng

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Doubling Time ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Dependent Manner ◽

Hcn Channel ◽

Context Dependent

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Identification of Novel miRNAs in Mouse Embryonic Stem Cells, Embryonic Fibroblasts, and Reprogrammed Pluripotent Cells

10.1101/817395 ◽

2019 ◽

Author(s):

Botao Zhao ◽

Chunsun Fan

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

De Novo ◽

Embryonic Stem ◽

Induced Pluripotent Stem Cell ◽

Mouse Embryonic Stem Cells ◽

Pluripotent Cells ◽

Embryonic Fibroblasts ◽

Species Specific

AbstractMicroRNAs (miRNAs) are a class of non-coding small RNAs that function in almost every known cellular activity. MiRNAs play an important role in gene regulation that controls embryonic stem cell (ESC) pluripotency and differentiation, as well as induced pluripotent stem cell (iPSC) reprogramming. In this study, we identified nine novel miRNAs by mining the deep sequencing dataset from mouse embryonic stem cells, mouse embryonic fibroblasts (MEF) and three kinds of reprogrammed pluripotent cells. Most of them are non-conserved but species-specific and cell-specific miRNAs. Eight miRNAs are derived from gene introns, including a “mirtron” miRNA, miR-novel-41. We also showed that miR-novel-27 is a mouse-specific miRNA and the 5′ arm of its precursor hairpin, embedding the mature miR-novel-27, uniquely exists in mouse species but not in any other Placentalia animals. Notably, the 5′ arm of the pre-miR-novel-27 hairpin shows nearly perfect palindrome to the 3′ arm suggesting that it was generated by inverted duplication of the 3′ arm. By this mechanism, the pre-miR-novel-27 hairpin was de novo gained in the mouse genome. This is a new type of de novo miRNA emergence mechanism in animals, which we called “inverted local half hairpin duplication” here. In addition, very limited nucleotide mutants accumulated on the newly emerged 5′ arm since its birth suggesting an especially young evolutionary history of the miR-novel-27 gene.

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