DAXX safeguards pericentromeric heterochromatin formation in embryonic stem cells

AbstractDNA methylation is essential for heterochromatin formation and repression of DNA repeat transcription, both of which are essential for genome integrity. Loss of DNA methylation is associated with disease, including cancer, but is also required for development. Alternative pathways to maintain heterochromatin are thus needed to limit DNA damage accumulation. Here, we find that DAXX, an H3.3 chaperone, protects pericentromeric heterochromatin and is essential for embryonic stem cells (ESCs) maintenance in the ground-state of pluripotency. Upon DNA demethylation-mediated damage, DAXX relocalizes to pericentromeric regions, and recruits PML and SETDB1, thereby promoting heterochromatin formation. In the absence of DAXX, the 3D-architecture and physical properties of pericentric heterochromatin are disrupted, resulting in derepression of major satellite DNA. Using epigenome editing tools, we demonstrate that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX and H3.3 unite DNA damage response and heterochromatin maintenance in ESCs.

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RNA interference in mammalian DNA methylation1This review is part of Special Issue entitled Asilomar Chromatin and has undergone the Journal’s usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o11-050 ◽

2012 ◽

Vol 90 (1) ◽

pp. 70-77 ◽

Cited By ~ 1

Author(s):

Jeffrey R. Mann ◽

Deidre M. Mattiske

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Dna Methyltransferase ◽

Germ Line ◽

Peer Review Process ◽

Embryonic Stem ◽

Cell Types ◽

Heterochromatin Formation ◽

Nonspecific Effects

RNAi and Dicer-dependent siRNAs are required for constitutive heterochromatin formation in fission yeast and for establishing DNA methylation at repetitive elements in plants. In the mammalian male germ line, DICER1-independent piRNAs are required for the full establishment of DNA methylation of dispersed repetitive transposable elements. However, in other mammalian cell types, no clear picture has yet emerged of the role of RNAi in establishing heterochromatin and DNA methylation. In mouse embryonic stem cells, which remain viable on loss of DICER1 and ablation of RNAi, while no firm evidence has been obtained for defective heterochromatin formation, there are indications of defective DNA methylation. The latter has been attributed to an indirect effect of reduced DNA methyltransferase (DNMT) activity due to a loss of miRNA-mediated gene regulation. However, it is unclear whether the reductions in DNMT activity were sufficient to affect DNA methylation. We consider it equally likely that the defects in DNA methylation that can be observed in DICER1-deficient embryonic stem cells are the result of nonspecific effects related to RNAi loss aside from reduced DNMT activity.

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SETDB1 prevents TET2-dependent activation of IAP retroelements in naïve embryonic stem cells

10.1101/193854 ◽

2017 ◽

Author(s):

Özgen Deniz ◽

Lorenzo de la Rica ◽

Kevin C. L. Cheng ◽

Dominik Spensberger ◽

Miguel R. Branco

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Dna Demethylation ◽

Endogenous Retroviruses ◽

Preimplantation Embryos ◽

Dependent Manner ◽

Epigenetic Mechanisms

BackgroundEndogenous retroviruses (ERVs), which are responsible for 10% of spontaneous mouse mutations, are kept under control via several epigenetic mechanisms. The H3K9 histone methyltransferase SETDB1 is essential for ERV repression in embryonic stem cells (ESCs), with DNA methylation also playing an important role. It has been suggested that SETDB1 protects ERVs from TET- dependent DNA demethylation, but the relevance of this mechanism for ERV expression remains unclear. Moreover, previous studies have been performed in primed ESCs, which are not epigenetically or transcriptionally representative of preimplantation embryos.ResultsWe used naïve ESCs to investigate the role of SETDB1 in ERV regulation and, in particular, its relationship with TET-mediated DNA demethylation. Naïve ESCs show an increased dependency on SETDB1 for ERV silencing when compared to primed ESCs, including at the highly mutagenic intracisternal A particles (IAPs). We found that, in the absence of SETDB1, TET2 activates IAP elements in a catalytic-dependent manner. Surprisingly, however, TET2 does not drive changes in DNA methylation levels at IAPs, suggesting that it regulates these transposons indirectly. Instead, SETDB1 depletion leads to a TET2-dependent loss of H4R3me2s, which is indispensable for IAP silencing during epigenetic reprogramming.ConclusionsOur results demonstrate a novel and unexpected role for SETDB1 in protecting IAPs from TET2-dependent histone arginine demethylation.

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The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage

Nucleic Acids Research ◽

10.1093/nar/27.16.3276 ◽

1999 ◽

Vol 27 (16) ◽

pp. 3276-3282 ◽

Cited By ~ 65

Author(s):

P. P. H. Van Sloun ◽

J. G. Jansen ◽

G. Weeda ◽

L. H. F. Mullenders ◽

A. A. van Zeeland ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Embryonic Stem ◽

Genotoxic Effects ◽

Nucleotide Excision

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Replication Stress and Telomere Dysfunction Are Present in Cultured Human Embryonic Stem Cells

Cytogenetic and Genome Research ◽

10.1159/000441245 ◽

2015 ◽

Vol 146 (4) ◽

pp. 251-260 ◽

Cited By ~ 3

Author(s):

Christine Janson ◽

Kristine Nyhan ◽

John P. Murnane

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Chromosome Instability ◽

Embryonic Stem ◽

Replication Stress ◽

Activin A ◽

Fragile Sites ◽

Telomere Dysfunction

Replication stress causes DNA damage at fragile sites in the genome. DNA damage at telomeres can initiate breakage-fusion-bridge cycles and chromosome instability, which can result in replicative senescence or tumor formation. Little is known about the extent of replication stress or telomere dysfunction in human embryonic stem cells (hESCs). hESCs are grown in culture with the expectation of being used therapeutically in humans, making it important to minimize the levels of replication stress and telomere dysfunction. Here, the hESC line UCSF4 was cultured in a defined medium with growth factor Activin A, exogenous nucleosides, or DNA polymerase inhibitor aphidicolin. We used quantitative fluorescence in situ hybridization to analyze individual telomeres for dysfunction and observed that it can be increased by aphidicolin or Activin A. In contrast, adding exogenous nucleosides relieved dysfunction, suggesting that telomere dysfunction results from replication stress. Whether these findings can be applied to other hESC lines remains to be determined. However, because the loss of telomeres can lead to chromosome instability and cancer, we conclude that hESCs grown in culture for future therapeutic purposes should be routinely checked for replication stress and telomere dysfunction.

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DNA methylation on N6-adenine in mammalian embryonic stem cells

Nature ◽

10.1038/nature17640 ◽

2016 ◽

Vol 532 (7599) ◽

pp. 329-333 ◽

Cited By ~ 291

Author(s):

Tao P. Wu ◽

Tao Wang ◽

Matthew G. Seetin ◽

Yongquan Lai ◽

Shijia Zhu ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem

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Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination

Stem Cell Reports ◽

10.1016/j.stemcr.2017.10.002 ◽

2017 ◽

Vol 9 (5) ◽

pp. 1660-1674 ◽

Cited By ~ 12

Author(s):

Kalpana Mujoo ◽

Raj K. Pandita ◽

Anjana Tiwari ◽

Vijay Charaka ◽

Sharmistha Chakraborty ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Homologous Recombination ◽

Dna Damage Repair ◽

Embryonic Stem ◽

Damage Repair ◽

Induced Pluripotent

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Corrigendum: p53 is essential for DNA methylation homeostasis in naïve embryonic stem cells, and its loss promotes clonal heterogeneity

Genes & Development ◽

10.1101/gad.319863.118 ◽

2018 ◽

Vol 32 (19-20) ◽

pp. 1358-1358

Author(s):

Ayala Tovy ◽

Adam Spiro ◽

Ryan McCarthy ◽

Zohar Shipony ◽

Yael Aylon ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Clonal Heterogeneity

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Effects of a hypomagnetic field on DNA methylation during the differentiation of embryonic stem cells

Scientific Reports ◽

10.1038/s41598-018-37372-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 6

Author(s):

Soonbong Baek ◽

Hwan Choi ◽

Hanseul Park ◽

Byunguk Cho ◽

Siyoung Kim ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Hypomagnetic Field

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Cops5 safeguards genomic stability of embryonic stem cells through regulating cellular metabolism and DNA repair

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915079117 ◽

2020 ◽

Vol 117 (5) ◽

pp. 2519-2525 ◽

Cited By ~ 1

Author(s):

Peng Li ◽

Lulu Gao ◽

Tongxi Cui ◽

Weiyu Zhang ◽

Zixin Zhao ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Genomic Stability ◽

Cellular Metabolism ◽

M Cell ◽

Pluripotency Marker ◽

Damage Repair ◽

Pluripotency Maintenance

The highly conserved COP9 signalosome (CSN), composed of 8 subunits (Cops1 to Cops8), has been implicated in pluripotency maintenance of human embryonic stem cells (ESCs). Yet, the mechanism for the CSN to regulate pluripotency remains elusive. We previously showed that Cops2, independent of the CSN, is essential for the pluripotency maintenance of mouse ESCs. In this study, we set out to investigate how Cops5 and Cops8 regulate ESC differentiation and tried to establish Cops5 and Cops8 knockout (KO) ESC lines by CRISPR/Cas9. To our surprise, no Cops5 KO ESC clones were identified out of 127 clones, while three Cops8 KO ESC lines were established out of 70 clones. We then constructed an inducible Cops5 KO ESC line. Cops5 KO leads to decreased expression of the pluripotency marker Nanog, proliferation defect, G2/M cell-cycle arrest, and apoptosis of ESCs. Further analysis revealed dual roles of Cops5 in maintaining genomic stability of ESCs. On one hand, Cops5 suppresses the autophagic degradation of Mtch2 to direct cellular metabolism toward glycolysis and minimize reactive oxygen species (ROS) production, thereby reducing endogenous DNA damage. On the other hand, Cops5 is required for high DNA damage repair (DDR) activities in ESCs. Without Cops5, elevated ROS and reduced DDR activities lead to DNA damage accumulation in ESCs. Subsequently, p53 is activated to trigger G2/M arrest and apoptosis. Altogether, our studies reveal an essential role of Cops5 in maintaining genome integrity and self-renewal of ESCs by regulating cellular metabolism and DDR pathways.

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