ATR expands embryonic stem cell fate potential in response to replication stress (original) (raw)
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STEM CELLS, 2012
Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise to all the somatic cells of the developing fetus. Any defects that occur in their genome or epigenome would have devastating consequences. Genetic and epigenetic change in human ESCs appear to be an inevitable consequence of long-term culture, driven by selection of variant cells that have a higher propensity for self-renewal rather than either differentiation or death. Mechanisms underlying the potentially separate events of mutation and subsequent selection of variants are poorly understood. Here, we show that human ESCs and their malignant counterpart, embryonal carcinoma (EC) cells, both fail to activate critical S-phase checkpoints when exposed to DNA replication inhibitors and commit to apoptosis instead. Human ESCs and EC cells also fail to form replication protein A, cH2AX, or RAD51 foci or load topoisomerase (DNA) II binding protein 1 onto chromatin in response to replication inhibitors. Furthermore, direct measurements of single-stranded DNA (ssDNA) show that these cells fail to generate the ssDNA regions in response to replication stress that are necessary for the activation of checkpoints and the initiation of homologous recombination repair to protect replication fork integrity and restart DNA replication. Taken together, our data suggest that pluripotent cells control genome integrity by the elimination of damaged cells through apoptosis rather than DNA repair, and therefore, mutations or epigenetic modifications resulting in an imbalance in cell death control could lead to genetic instability.
Nature communications, 2016
Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propos...
Activation of DNA damage response signaling in mouse embryonic stem cells
Cell Cycle, 2008
Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G 1 /S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated ATM kinase and histone H2AX (γH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of γH2AX staining. Exposure of mESC to γ-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional γH2AX foci, which disappeared thereafter. To decrease the background of γH2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G 2 /M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for γH2AX. Irradiation induced the formation of γH2AX foci both in G 2-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G 1 /S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for ATM kinase, was phosphorylated in response to γ-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating ATM and inducing γH2AX foci formation, which are necessary for the activation of DNA damage response.
DNA Damage-Sensing Kinases Mediate the Mouse 2-Cell Embryo's Response to Genotoxic Stress
Biology of Reproduction, 2011
A critical function of cells is the maintenance of their genomic integrity. A family of phosphoinositide-3-kinase-related protein kinases, which includes ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) kinases, play key roles in sensing DNA damage. ATM and ATR were demonstrated in the cleavage stages of mouse embryo development. Genotoxic stress was imposed by exposure to ultraviolet (UV) radiation (causes DNA strand breaks) or cisplatin (causes strand crosslinks). UV irradiation or cisplatin treatment of 2-cell embryos in the G 2 phase of the cell cycle caused DNA damage as defined by increased phosphorylation of the H2A histone family, member X (H2AFX; previously H2AX) variant. UV irradiation caused a stable G 2 -M arrest, and cisplatin treatment allowed progression through mitosis followed by activation of a G 1 -S checkpoint. Both checkpoints were transformation-related protein 53independent. Caffeine (inhibits both ATM and ATR), but not KU55933 (ATM-selective inhibitor), reversed the G 2 -M block induced by UV, inferring a primary role for ATR in sensing this form of DNA damage. Caffeine and KU55933 were equally effective in reversing the cisplatin-induced G 1 -S block, implicating ATM as the primary sensing enzyme. Breaching of either checkpoint by treatment with caffeine or KU55933 allowed embryos to progress through several further cell cycles, yet none developed to blastocysts. The results show, to our knowledge for the first time, that the G 2 -M and G 1 -S cell-cycle checkpoints in the early embryo are differentially regulated by ATM and ATR in response to genotoxic stress and that they act as an initial point for containment of genomic damage. Under conditions of extensive or persistent DNA damage, the demise of the embryo is the ultimate method of protecting genomic integrity. apoptosis, ataxia telangiectasia and Rad3 related, ataxia telangiectasia mutated, cell cycle, checkpoint, DNA damage, early development, embryo, H2AFX, transformation-related protein 53, tumor suppressor gene
Development (Cambridge, England), 2015
A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twof...
ATRX Dysfunction Induces Replication Defects in Primary Mouse Cells
PLoS ONE, 2014
The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells.
The progressive accumulation of DNA damage is thought to be one of the driving forces that initiates ageing. However, the nature of the damage that arises endogenously is still ill-defined. A known source of endogenous damage is replicative stress (RS), which is intrinsically associated to DNA replication and prevented mainly by the ATR kinase. Here, we have developed a murine model of the human Seckel Syndrome characterized by a severe deficiency in ATR. Seckel mice suffer high levels of RS during embryogenesis when proliferation is widespread, but which decrease to marginal levels in postnatal life. In spite of this decrease, adult Seckel mice present accelerated ageing, which is further aggravated in the absence of p53 due to a further increase of RS. Together, these results support the concept that endogenous RS, particularly in utero, contributes to the onset of ageing in postnatal life and this is counterbalanced by the RS-limiting role of the checkpoint proteins ATR and p53. The accumulation of DNA damage can have important consequences that limit the lifespan of mammalian organisms such as ageing or cancer. On one hand, one of the current theories of ageing is based on the accumulation of DNA damage1. Accordingly, signals of an activated DDR have been shown to increase on aged tissues and stem cells (SC)2 , 3, and a number of murine models with impaired DNA repair show features of premature ageing4. On the other hand, damaged DNA is the source of the mutations that drive malignant transformation. Therefore, it is not surprising that organisms have evolved complex signalling pathways to protect their DNA. In particular, the so-called DNA damage response (DDR) starts with the Correspondence should be addressed to O.F. (ofernandez@cnio.es) Phone: +34-91-7328000, Ext: 3480.
ATR: a master conductor of cellular responses to DNA replication stress
Trends in Biochemical Sciences, 2011
The integrity of the genome is constantly challenged by intrinsic and extrinsic genotoxic stresses that damage DNA. The cellular responses to DNA damage are orchestrated by DNA damage signaling pathways, also known as DNA damage checkpoints. These signaling pathways play crucial roles in detecting DNA damage, regulating DNA repair, and coordinating DNA repair with other cellular processes. In vertebrates, the ATM-and Rad3-related (ATR) kinase plays a key role in the response to a broad spectrum of DNA damage and DNA replication stress. In this article, we will discuss the recent findings on how ATR is activated by DNA damage, and how it protects the genome against interference with DNA replication.
Stalled replication forks within heterochromatin require ATRX for protection
Cell Death and Disease, 2016
Expansive growth of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered neocortex, while also placing a heavy burden on proteins that regulate chromatin packaging and genome integrity. This problem is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. ATRX gene mutations cause a severe intellectual disability disorder (α-thalassemia mental retardation X-linked (ATRX) syndrome; OMIM no. 301040), characterized by microcephaly, urogenital abnormalities and α-thalassemia. Although the ATRX protein is required for the maintenance of repetitive DNA within heterochromatin, how this translates to disease pathogenesis remain poorly understood and was a focus of this study. We demonstrate that Atrx FoxG1Cre forebrain-specific conditional knockout mice display poly(ADP-ribose) polymerase-1 (Parp-1) hyperactivation during neurogenesis and generate fewer late-born Cux1-and Brn2positive neurons that accounts for the reduced cortical size. Moreover, DNA damage, induced Parp-1 and Atm activation is elevated in progenitor cells and contributes to their increased level of cell death. ATRX-null HeLa cells are similarly sensitive to hydroxyurea-induced replication stress, accumulate DNA damage and proliferate poorly. Impaired BRCA1-RAD51 colocalization and PARP-1 hyperactivation indicated that stalled replication forks are not efficiently protected. DNA fiber assays confirmed that MRE11 degradation of stalled replication forks was rampant in the absence of ATRX or DAXX. Indeed, fork degradation in ATRX-null cells could be attenuated by treatment with the MRE11 inhibitor mirin, or exacerbated by inhibiting PARP-1 activity. Taken together, these results suggest that ATRX is required to limit replication stress during cellular proliferation, whereas upregulation of PARP-1 activity functions as a compensatory mechanism to protect stalled forks, limiting genomic damage, and facilitating late-born neuron production.