Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts
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Curated by eLife
Evaluation Summary:
This interesting genetics study shows that two well known tumor suppressor genes, ATR and Chk1, have a new function in sensing oxidative stress agents. The study is good quality and the results generally support the rather novel conclusions. It should be of interest in the fields of cancer genetics and cell biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Progenitors of the thoracic tracheal system of adult Drosophila (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ataxia telangiectasia mutated and rad3-related kinase (ATR)-dependent phosphorylation of checkpoint kinase 1 (Chk1) that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018; Kizhedathu et al., 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an H 2 O 2 generating dual oxidase. ROS quenching by overexpression of superoxide dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of H 2 O 2 in minutes. The findings presented reveal that H 2 O 2 activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.
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Evaluation Summary:
This interesting genetics study shows that two well known tumor suppressor genes, ATR and Chk1, have a new function in sensing oxidative stress agents. The study is good quality and the results generally support the rather novel conclusions. It should be of interest in the fields of cancer genetics and cell biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
In prior work, the authors identified a requirement for an ATR-dependent activation of Chk1 for the maintenance of G2 arrest in larval tracheoblasts. Absent the activity of ATR or pChk1, larval tracheoblasts re-enter the cell cycle early. Here the authors were attempting to build on their prior work by determining the mechanism by which ATR is regulated in larval tracheoblasts. In large measure, the authors are successful in this endeavor, finding that ROS generated by Duox are required to activate ATR. However, what regulates the timing of Duox expression and the precise molecular mechanism by which ROS activates ATR remains unresolved. The closely related ATM kinase has previously been shown to be directly regulated by ROS, through the formation of disulfide bridges. The authors' modeling suggest a …
Reviewer #1 (Public Review):
In prior work, the authors identified a requirement for an ATR-dependent activation of Chk1 for the maintenance of G2 arrest in larval tracheoblasts. Absent the activity of ATR or pChk1, larval tracheoblasts re-enter the cell cycle early. Here the authors were attempting to build on their prior work by determining the mechanism by which ATR is regulated in larval tracheoblasts. In large measure, the authors are successful in this endeavor, finding that ROS generated by Duox are required to activate ATR. However, what regulates the timing of Duox expression and the precise molecular mechanism by which ROS activates ATR remains unresolved. The closely related ATM kinase has previously been shown to be directly regulated by ROS, through the formation of disulfide bridges. The authors' modeling suggest a different mechanism may be at play.
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Reviewer #2 (Public Review):
This paper follows up on a previous eLife paper from the lab in which they showed that multiple Wnts control ATR expression to mediate cell cycle arrest of the trachea stem cells that will repopulate the tracheal system during pupal development. Here they show that activity, but not expression, of ATR is regulated by phosphorylation in response to high reactive oxygens species (ROS) generated by the enzyme Duox, an H2O2 generating-Dual Oxidase. Duox is required for high ROS levels in the trachea cells and suppression of high ROS levels by overexpression of super oxide dismutase causes precocious proliferation. Duox regulation of ATR and cell cycle arrest is independent of DNA-damage or activation of ATRIP, TOPBP1 or Claspin. Exogenous H2O2 can block excessive proliferation and restore ATR phosphorylation …
Reviewer #2 (Public Review):
This paper follows up on a previous eLife paper from the lab in which they showed that multiple Wnts control ATR expression to mediate cell cycle arrest of the trachea stem cells that will repopulate the tracheal system during pupal development. Here they show that activity, but not expression, of ATR is regulated by phosphorylation in response to high reactive oxygens species (ROS) generated by the enzyme Duox, an H2O2 generating-Dual Oxidase. Duox is required for high ROS levels in the trachea cells and suppression of high ROS levels by overexpression of super oxide dismutase causes precocious proliferation. Duox regulation of ATR and cell cycle arrest is independent of DNA-damage or activation of ATRIP, TOPBP1 or Claspin. Exogenous H2O2 can block excessive proliferation and restore ATR phosphorylation deficits resulting from knockdown of Duox. The experiments are well done and rigorous, and the paper is well written. No major concerns are noted.
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Reviewer #3 (Public Review):
Kizhedathu et al. propose that Duox generated ROS activate ATR/Chk1 in the tracheoblasts in the Drosophila Tr2 metamere, and that this ROS-mediated ATR/Chk1 activation is DNA damage independent. This is a novel, unusual mechanism of activation of ATR and Chk1 that should be of rather broad interest, though it is not without precedent. They present data supporting the conclusion that ROS-dependent activation of ATR & Chk1 plays an instructive role in arresting tracheoblasts in G2, and that the developmentally controlled loss of ROS during the late L3 larva stage deactivates ATR/Chk1 and thereby activates tracheoblast cell division. The authors have performed extensive experiments to support these claims and the data are, overall, consistent with the model they present. The limitations of this study are that …
Reviewer #3 (Public Review):
Kizhedathu et al. propose that Duox generated ROS activate ATR/Chk1 in the tracheoblasts in the Drosophila Tr2 metamere, and that this ROS-mediated ATR/Chk1 activation is DNA damage independent. This is a novel, unusual mechanism of activation of ATR and Chk1 that should be of rather broad interest, though it is not without precedent. They present data supporting the conclusion that ROS-dependent activation of ATR & Chk1 plays an instructive role in arresting tracheoblasts in G2, and that the developmentally controlled loss of ROS during the late L3 larva stage deactivates ATR/Chk1 and thereby activates tracheoblast cell division. The authors have performed extensive experiments to support these claims and the data are, overall, consistent with the model they present. The limitations of this study are that the authors have not elucidated how, molecularly, ROS activates ATR, and that they do not provided data or discussion relevant to how ROS levels might change according to developmental stage.
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