Differentiation of Embryonic Stem Cells and Oxidative DNA Damage / DNA Repair Systems

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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DAXX safeguards pericentromeric heterochromatin formation in embryonic stem cells

10.1101/2021.04.28.441827 ◽

2021 ◽

Author(s):

Antoine Canat ◽

Adeline Veillet ◽

Robert Illingworth ◽

Emmanuelle Fabre ◽

Pierre Therizols

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Pericentric Heterochromatin ◽

Dna Demethylation ◽

Pericentromeric Heterochromatin ◽

Major Satellite ◽

Heterochromatin Formation

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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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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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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Systems Biology Approach Identifies the Kinase Csnk1a1 as a Regulator of the DNA Damage Response in Embryonic Stem Cells

Science Signaling ◽

10.1126/scisignal.2003208 ◽

2013 ◽

Vol 6 (259) ◽

pp. ra5-ra5 ◽

Cited By ~ 20

Author(s):

J. Carreras Puigvert ◽

L. von Stechow ◽

R. Siddappa ◽

A. Pines ◽

M. Bahjat ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Systems Biology ◽

Embryonic Stem Cells ◽

Dna Damage Response ◽

Embryonic Stem ◽

Damage Response

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Distinct Regulatory Mechanisms and Functions for p53-Activated and p53-Repressed DNA Damage Response Genes in Embryonic Stem Cells

Molecular Cell ◽

10.1016/j.molcel.2012.01.020 ◽

2012 ◽

Vol 46 (1) ◽

pp. 30-42 ◽

Cited By ~ 164

Author(s):

Mangmang Li ◽

Yunlong He ◽

Wendy Dubois ◽

Xiaolin Wu ◽

Jianxin Shi ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Dna Damage Response ◽

Embryonic Stem ◽

Regulatory Mechanisms ◽

Damage Response

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Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

The Journal of Cell Biology ◽

10.1083/jcb.201408106 ◽

2015 ◽

Vol 208 (5) ◽

pp. 513-520 ◽

Cited By ~ 29

Author(s):

Jianhua Xiong ◽

Dilyana Todorova ◽

Ning-Yuan Su ◽

Jinchul Kim ◽

Pei-Jen Lee ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Dna Damage Response ◽

Embryonic Stem ◽

Ataxia Telangiectasia Mutated ◽

Mouse Escs ◽

Chromatin Remodeling Complex ◽

Damage Response ◽

Underlying Mechanisms

Mouse embryonic stem cells (ESCs) are genetically more stable than somatic cells, thereby preventing the passage of genomic abnormalities to their derivatives including germ cells. The underlying mechanisms, however, remain largely unclear. In this paper, we show that the stemness factor Sall4 is required for activating the critical Ataxia Telangiectasia Mutated (ATM)–dependent cellular responses to DNA double-stranded breaks (DSBs) in mouse ESCs and confer their resistance to DSB-induced cytotoxicity. Sall4 is rapidly mobilized to the sites of DSBs after DNA damage. Furthermore, Sall4 interacts with Rad50 and stabilizes the Mre11–Rad50–Nbs1 complex for the efficient recruitment and activation of ATM. Sall4 also interacts with Baf60a, a member of the SWI/SNF (switch/sucrose nonfermentable) ATP-dependent chromatin-remodeling complex, which is responsible for recruiting Sall4 to the site of DNA DSB damage. Our findings provide novel mechanisms to coordinate stemness of ESCs with DNA damage response, ensuring genomic stability during the expansion of ESCs.

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