scholarly journals DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation

2002 ◽  
Vol 22 (21) ◽  
pp. 7572-7580 ◽  
Author(s):  
Matthew C. Lorincz ◽  
Dirk Schübeler ◽  
Shauna R. Hutchinson ◽  
David R. Dickerson ◽  
Mark Groudine
Keyword(s):  
Dna Methylation ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
De Novo ◽  
Embryonic Stem ◽  
Dna Methyltransferases ◽  
The Stability ◽  
Murine Erythroleukemia ◽  
De Novo Methylation

ABSTRACT DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.

scholarly journals Establishment and Maintenance of Genomic Methylation Patterns in Mouse Embryonic Stem Cells by Dnmt3a and Dnmt3b

2003 ◽  
Vol 23 (16) ◽  
pp. 5594-5605 ◽  
Author(s):  
Taiping Chen ◽  
Yoshihide Ueda ◽  
Jonathan E. Dodge ◽  
Zhenjuan Wang ◽  
En Li
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Single Copy ◽  
Dna Methyltransferases ◽  
Methylation Patterns ◽  
Genomic Methylation ◽  
De Novo Methylation

ABSTRACT We have previously shown that the DNA methyltransferases Dnmt3a and Dnmt3b carry out de novo methylation of the mouse genome during early postimplantation development and of maternally imprinted genes in the oocyte. In the present study, we demonstrate that Dnmt3a and Dnmt3b are also essential for the stable inheritance, or “maintenance,” of DNA methylation patterns. Inactivation of both Dnmt3a and Dnmt3b in embryonic stem (ES) cells results in progressive loss of methylation in various repeats and single-copy genes. Interestingly, introduction of the Dnmt3a, Dnmt3a2, and Dnmt3b1 isoforms back into highly demethylated mutant ES cells restores genomic methylation patterns; these isoforms appear to have both common and distinct DNA targets, but they all fail to restore the maternal methylation imprints. In contrast, overexpression of Dnmt1 and Dnmt3b3 failed to restore DNA methylation patterns due to their inability to catalyze de novo methylation in vivo. We also show that hypermethylation of genomic DNA by Dnmt3a and Dnmt3b is necessary for ES cells to form teratomas in nude mice. These results indicate that genomic methylation patterns are determined partly through differential expression of different Dnmt3a and Dnmt3b isoforms.

scholarly journals Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory

2021 ◽  
Vol 118 (38) ◽  
pp. e2109475118
Author(s):  
Masaki Kinoshita ◽  
Meng Amy Li ◽  
Michael Barber ◽  
William Mansfield ◽  
Sabine Dietmann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Fate ◽  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dna Methyltransferases ◽  
Bhlh Transcription Factor ◽  
Mammalian Development

Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed toward production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2. Ascl2 encodes a bHLH transcription factor expressed in the placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2. However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.

scholarly journals Dnmt1 has de novo activity targeted to transposable elements

2021 ◽  
Author(s):  
Chuck Haggerty ◽  
Helene Kretzmer ◽  
Christina Riemenschneider ◽  
Abhishek Sampath Kumar ◽  
Alexandra L. Mattei ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Critical Role ◽  
Embryonic Stem ◽  
Genetically Engineered ◽  
Genomic Recruitment ◽  
Methylation Activity ◽  
De Novo Methylation

AbstractDNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

scholarly journals Analysis of genomic DNA methylation and gene transcription modulation induced by DNMT3A deficiency in HEK293 cells

2020 ◽  
Author(s):  
Mengxiao Zhang ◽  
Jiaxian Wang ◽  
Qiuxiang Tian ◽  
Lei Feng ◽  
Hui Yang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Growth ◽  
Transcriptional Repression ◽  
Catalytic Domain ◽  
De Novo ◽  
Epigenetic Modification ◽  
Cell Metabolism ◽  
Dna Methyltransferases ◽  
Hek293 Cells ◽  
Signal Pathways

AbstractDNA methylation is an important epigenetic modification associated with transcriptional repression, and plays key roles in normal cell growth as well as oncogenesis. Among the three main DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), DNMT3A mediates de novo DNA methylation with partial functional redundancy with DNMT3B. However, the general effects of DNMT3A and its downstream gene regulation profile are yet to be unveiled. In the present study, we used CRISPR/Cas9 technology to successfully create DNMT3A deficient human embryonic kidney cell line HEK293, with frameshift mutations in its catalytic domain. Our results showed that the cell growth slowed down in DNMT3A knockout cells. UPLC-MS analysis of DNMT3A deficient cells showed that the genome-level DNA methylation was reduced by 21.5% and led to an impairment of cell proliferation as well as a blockage of MAPK and PI3K-Akt pathways. Whole genome RNA-seq revealed that DNMT3A knockout up-regulated expression of genes and pathways related to cell metabolism but down-regulated those involved in ribosome function, which explained the inhibition of cell growth and related signal pathways. Further, bisulfite DNA treatment showed that DNMT3A ablation reduced the methylation level of DNMT3B gene as well, indicating the higher DNMT3B activity and thereby explaining those down-regulated profiles of genes.

QSER1 protects DNA methylation valleys from de novo methylation

Science ◽  
2021 ◽  
Vol 372 (6538) ◽  
pp. eabd0875 ◽  
Author(s):  
Gary Dixon ◽  
Heng Pan ◽  
Dapeng Yang ◽  
Bess P. Rosen ◽  
Therande Jashari ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Embryonic Stem ◽  
Central Question ◽  
Mammalian Development ◽  
Uncharacterized Gene ◽  
Pathological Conditions ◽  
Genome Wide ◽  
A Genome ◽  
De Novo Methylation

DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.

scholarly journals 135DNA METHYLATION PROFILES IN THE PREIMPLANTATION PORCINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 190
Author(s):  
S. Yeo ◽  
Y.-K. Kang ◽  
D.-B Koo ◽  
J.-S Han ◽  
W.-K Chang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Paternal Genome ◽  
Active Demethylation ◽  
Morula Stage ◽  
De Novo Methylation

DNA methylation at CpG dinucleotides is an important epigenetic regulation process, which is associated with gene expression without any change in DNA sequence. During early development of the mouse embryo, dynamic changes in DNA methylation of the genome occur. After fertilization, active demethylation occurs on the paternal genome followed by passive demethylation until morula stage and then de novo methylation at the blastocyst stage. This study was designed to investigate changes in DNA methylation of in vivo- and in vitro-fertilized (IVF) porcine embryos. DNA methylation states were observed in preimplantation porcine embryos by using an immunofluorescence method after staining with an antibody against 5-methylcytosine. In contrast to the data from mouse embryos, active demethylation of the genome from the paternal pronucleus was not observed in the porcine embryos. Also, no passive demethylation was detected in in vivo- and IVF-derived embryos until the morula stage. Moreover, differential de novo methylation was not shown on the genome of the inner cell mass. Whole genomes of inner cell mass and trophectoderm cells were fully methylated. Our results demonstrate that DNA methylation of porcine embryos is different from that of mouse embryos during preimplantation development, suggesting that the machinery to regulate DNA methylation may be species-specific in mammals.

scholarly journals BAP1 activity regulates PcG occupancy and global chromatin condensation counteracting diffuse PCGF3/5-dependent H2AK119ub1 deposition

2020 ◽  
Author(s):  
Eric Conway ◽  
Federico Rossi ◽  
Simone Tamburri ◽  
Eleonora Ponzo ◽  
Karin Johanna Ferrari ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Chromatin Condensation ◽  
Embryonic Stem ◽  
Apparent Contradiction ◽  
Major Function ◽  
Genome Wide ◽  
Cancer Types ◽  
Precise Relationship ◽  
The Stability

AbstractBAP1 is recurrently mutated or deleted in a large number of diverse cancer types, including mesothelioma, uveal melanoma and hepatocellular cholangiocarcinoma. BAP1 is the catalytic subunit of the Polycomb Repressive De-Ubiquitination complex (PR-DUB) which removes PRC1 mediated H2AK119ub1. We and others have shown that H2AK119ub1 is essential for maintaining transcriptional repression and contributes to PRC2 chromatin recruitment. However, the precise relationship between BAP1 and PRC1 remains mechanistically elusive. Using embryonic stem cells, we show that a major function of BAP1 is to restrict H2AK119ub1 deposition to target sites. This increases the stability of PcG complexes with their targets and prevents diffuse accumulation of H2AK119ub1 and H3K27me3 modifications. Loss of BAP1 results in a broad increase in H2AK119ub1 levels that are primarily dependent on PCGF3/5-PRC1 complexes with a mechanism that is reminiscent of X-chromosome inactivation. Increased genome-wide H2AK119ub1 levels titrates away PRC2 from its targets and stimulates diffuse H3K27me3 accumulation across the genome. This decreases the activity of PcG repressive machineries at physiological targets and induces a general compaction of the entire chromatin. Our findings provide evidences for a unifying model that resolves the apparent contradiction between BAP1 catalytic activity and its role in vivo, uncovering molecular vulnerabilities that could be useful for BAP1-related pathologies.

scholarly journals Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

2000 ◽  
Vol 20 (20) ◽  
pp. 7419-7426 ◽  
Author(s):  
Sara R. Cherry ◽  
D. Biniszkiewicz ◽  
L. van Parijs ◽  
D. Baltimore ◽  
R. Jaenisch
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Transcriptional Repression ◽  
Fluorescent Protein ◽  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Stem

ABSTRACT Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

scholarly journals Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yanchun Pan ◽  
Takuji Daito ◽  
Yo Sasaki ◽  
Yong Hee Chung ◽  
Xiaoyun Xing ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cortical Neurons ◽  
Transcriptional Repression ◽  
Cytosine Methylation ◽  
Critical Role ◽  
Dna Methyltransferases ◽  
Mrna Levels ◽  
Mutant Huntingtin ◽  
Striatal Neurons

Abstract Although epigenetic abnormalities have been described in Huntington’s disease (HD), the causal epigenetic mechanisms driving neurodegeneration in HD cortex and striatum remain undefined. Using an epigenetic pathway-targeted drug screen, we report that inhibitors of DNA methyltransferases (DNMTs), decitabine and FdCyd, block mutant huntingtin (Htt)-induced toxicity in primary cortical and striatal neurons. In addition, knockdown of DNMT3A or DNMT1 protected neurons against mutant Htt-induced toxicity, together demonstrating a requirement for DNMTs in mutant Htt-triggered neuronal death and suggesting a neurodegenerative mechanism based on DNA methylation-mediated transcriptional repression. Inhibition of DNMTs in HD model primary cortical or striatal neurons restored the expression of several key genes, including Bdnf, an important neurotrophic factor implicated in HD. Accordingly, the Bdnf promoter exhibited aberrant cytosine methylation in mutant Htt-expressing cortical neurons. In vivo, pharmacological inhibition of DNMTs in HD mouse brains restored the mRNA levels of key striatal genes known to be downregulated in HD. Thus, disturbances in DNA methylation play a critical role in mutant Htt-induced neuronal dysfunction and death, raising the possibility that epigenetic strategies targeting abnormal DNA methylation may have therapeutic utility in HD.

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