Noncannonical functions of Ku may underlie essentiality in human cells
Notice bibliographique
Résumé
Abstract The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but has also been involved in other cellular functions like telomere regulation and maintenance, in which Ku’s role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells to study the essentiality of Ku70 function. Endogenous Ku70 knockout was achieved using CRISPR/Cas9 editing in cells where Ku70 expression was maintained through integration of an HA-tagged Ku70 cDNA under the control of a doxycycline-inducible promoter. Ku70 conditional knockout cell lines were identified via western blotting, and edits were validated by Sanger sequencing. We visually observed cell death in Ku70 knockout cells 8-10 days post Ku70-HA depletion, and loss of viability following Ku depletion was quantified using crystal violet assays. Interestingly, assessment of telomere length in Ku70 knockout cells using telomere restriction fragment analyses did not reveal any changes in average telomere length following Ku70-HA depletion. Immunofluorescence analysis used to assess γH2AX foci accumulation as a measure of double-stranded DNA breaks following Ku70-HA depletion allowed us to conclude that increased DNA damage is not the driving cause of loss of cell viability. Finally, quantitative proteome analysis of Ku70 knockout cells following Ku70-HA depletion identified a number of pathways and proteins that are significantly dysregulated following the loss of Ku70, including processes which Ku function has been previously associated with such as cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Overall, this conditional Ku70 knockout system reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in other cellular processes beyond DNA repair and telomere maintenance to maintain cell viability. Author Summary The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair, where it acts as a scaffold for other repair factors needed to process double-stranded DNA breaks. Ku has also been involved in other cellular functions like telomere regulation and maintenance, in which Ku’s role is not fully characterized. Previous data suggest that while loss of Ku70/80 can be tolerated in other species, Ku is essential to humans. We have established a conditional Ku70 knockout in HEK293 cells to evaluate the basis of Ku essentiality in human cells. While we observed loss of cell viability upon Ku depletion, we did not observe significant changes in telomere length nor did we record lethal levels of DNA damage upon loss of Ku, suggesting that the reasons for the loss of viability is not linked to the functions of Ku in DNA repair or at telomeres. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,001 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».