Replication Stress Causes Mitotic Nuclear Envelope Reassembly Defect
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PROVIDER: PRJNA1134251 | ENA |
REPOSITORIES: ENA
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Project description:Replication stress, if not effectively and timely addressed, could result in DNA damage in mitosis. However, it remains unknown the relationship between mitotic DNA damage and other mitotic events such as nuclear envelope (NE) breakdown and reassembly. Here we report that replication stress could generate NE rupture. Rather than de novo formation, the rupture per se is a result of nuclear envelope reassembly defect (NERD) in mitosis. Repair of mitotic DNA damage by DNA polymerase theta (Polθ), a key microhomology-mediated end joining (MMEJ) factor, suppresses NERD. Furthermore, exacerbated NERD is observed in multiple conditions of synthetic lethality, suggesting NERD might be a general consequence of synthetic lethality. In addition, genomic mapping of LADs identifies a population of RESS-LADs (replication stress-sensitive LADs). Replication stress causes the loss of CFSs at RESS-LADs, likely due to the sustained phosphorylation of Lamin A/C at the NE rupture sites. Altogether, our findings establish a novel link between replication stress-induced genome instability and nuclear vulnerability.
2025-08-16 | GSE271990 | GEO
Project description:In animals, mitosis involves the breakdown of the nucleus. The reassembly of a nucleus after mitosis requires the reformation of the nuclear envelope around a single mass of chromosomes. This process requires Ankle2 (also known as LEM4 in humans) which interacts with PP2A and promotes the function of Barrier-to-Autointegration Factor (BAF). Upon dephosphorylation, BAF dimers cross-bridge chromosomes and bind lamins and transmembrane proteins of the reassembling nuclear envelope. How Ankle2 functions in mitosis is incompletely understood. Using a combination of approaches in Drosophila, along with structural modeling, we provide several lines of evidence that suggest that Ankle2 is a regulatory subunit of PP2A, explaining how it promotes BAF dephosphorylation. In addition, we discovered that Ankle2 interacts with the endoplasmic reticulum protein Vap33, which is required for Ankle2 localization at the reassembling nuclear envelope during telophase. We identified the interaction sites of PP2A and Vap33 on Ankle2. Through genetic rescue experiments, we show that the Ankle2/PP2A interaction is essential for the function of Ankle2 in nuclear reassembly and that the Ankle2/Vap33 interaction also promotes this process. Our study sheds light on the molecular mechanisms of post-mitotic nuclear reassembly and suggests that the endoplasmic reticulum is not merely a source of membranes in the process, but also provides localized enzymatic activity.
2025-01-31 | PXD059738 | Pride
Project description:Entry into mitosis is triggered by mitotic kinases that phosphorylate multiple proteins to induce profound cellular changes including nuclear envelope breakdown, chromosome condensation and spindle assembly. Conversely, mitotic exit requires protein dephosphorylation as cells return to their interphase organization. Protein Phosphatase 2A in complex with its B55/Tws regulatory subunit (PP2A-B55) is known to play a crucial role in this transition. However, the precise events and substrates that it regulates are incompletely understood. We used proteomic approaches in Drosophila to identify proteins that interact with PP2A-B55 and are dephosphorylated in a PP2A-B55 dependent manner. Among several candidates, we identified Otefin (also known as Emerin) as a target of PP2A-B55 in the process of nuclear envelope reformation after mitosis. Emerin is a protein of the inner nuclear membrane that interacts with the DNA-binding protein Barrier-to-Autointegration Factor (BAF) via a LEM domain. Our results indicate that phosphorylation of Emerin at Ser50 and Ser54 near its LEM domain negatively regulates its association with BAF, Lamin and additional Emerin. Using live imaging, we show that dephosphorylation of Emerin at the identified sites determines the timing of nuclear envelope reformation. Genetic rescue experiments indicate that this regulation is required in vivo during embryonic development. Phosphoregulation of the Emerin-BAF complex formation by PP2A-B55 appears as a key event of mitotic exit that is likely to be conserved across species.
2023-08-10 | PXD041091 | Pride
Project description:Entry into mitosis is triggered by mitotic kinases that phosphorylate multiple proteins to induce profound cellular changes including nuclear envelope breakdown, chromosome condensation and spindle assembly. Conversely, mitotic exit requires protein dephosphorylation as cells return to their interphase organization. Protein Phosphatase 2A in complex with its B55/Tws regulatory subunit (PP2A-B55) is known to play a crucial role in this transition. However, the precise events and substrates that it regulates are incompletely understood. We used proteomic approaches in Drosophila to identify proteins that interact with PP2A-B55 and are dephosphorylated in a PP2A-B55 dependent manner. Among several candidates, we identified Otefin (also known as Emerin) as a target of PP2A-B55 in the process of nuclear envelope reformation after mitosis. Emerin is a protein of the inner nuclear membrane that interacts with the DNA-binding protein Barrier-to-Autointegration Factor (BAF) via a LEM domain. Our results indicate that phosphorylation of Emerin at Ser50 and Ser54 near its LEM domain negatively regulates its association with BAF, Lamin and additional Emerin. Using live imaging, we show that dephosphorylation of Emerin at the identified sites determines the timing of nuclear envelope reformation. Genetic rescue experiments indicate that this regulation is required in vivo during embryonic development. Phosphoregulation of the Emerin-BAF complex formation by PP2A-B55 appears as a key event of mitotic exit that is likely to be conserved across species.
2023-08-10 | PXD041032 | Pride
Project description:Entry into mitosis is triggered by mitotic kinases that phosphorylate multiple proteins to induce profound cellular changes including nuclear envelope breakdown, chromosome condensation and spindle assembly. Conversely, mitotic exit requires protein dephosphorylation as cells return to their interphase organization. Protein Phosphatase 2A in complex with its B55/Tws regulatory subunit (PP2A-B55) is known to play a crucial role in this transition. However, the precise events and substrates that it regulates are incompletely understood. We used proteomic approaches in Drosophila to identify proteins that interact with PP2A-B55 and are dephosphorylated in a PP2A-B55 dependent manner. Among several candidates, we identified Otefin (also known as Emerin) as a target of PP2A-B55 in the process of nuclear envelope reformation after mitosis. Emerin is a protein of the inner nuclear membrane that interacts with the DNA-binding protein Barrier-to-Autointegration Factor (BAF) via a LEM domain. Our results indicate that phosphorylation of Emerin at Ser50 and Ser54 near its LEM domain negatively regulates its association with BAF, Lamin and additional Emerin. Using live imaging, we show that dephosphorylation of Emerin at the identified sites determines the timing of nuclear envelope reformation. Genetic rescue experiments indicate that this regulation is required in vivo during embryonic development. Phosphoregulation of the Emerin-BAF complex formation by PP2A-B55 appears as a key event of mitotic exit that is likely to be conserved across species.
2023-08-10 | PXD041030 | Pride
Project description:Repo-Man targets protein phosphatase 1 γ (PP1γ) to chromatin at anaphase onset and regulates chromosome structure during mitotic exit. Here, we show that a Repo-Man:PP1 complex forms in anaphase following dephosphorylation of Repo-Man. Upon activation, the complex localizes to chromosomes and causes the dephosphorylation of histone H3 (Thr3, Ser10, and Ser28). In anaphase, Repo-Man has both catalytic and structural functions that are mediated by two separate domains. A C-terminal domain localizes Repo-Man to bulk chromatin in early anaphase. There, it targets PP1 for the dephosphorylation of histone H3 and possibly other chromosomal substrates. An N-terminal domain localizes Repo-Man to the chromosome periphery later in anaphase. There, it is responsible for the recruitment of nuclear components such as Importin β and Nup153 in a PP1-independent manner. These observations identify Repo-Man as a key factor that coordinates chromatin remodeling and early events of nuclear envelope reformation during mitotic exit.
| S-EPMC3480639 | biostudies-literature
Project description:DNA replication is a major contributor to genomic instability, and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S phase of the cell cycle. If these stalled forks persist into the M phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5'-triphosphate-induced stalled forks were able to restart with the addition of excess dCTP during early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after the NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of the NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase activities because of which DNA replication and mitosis occur in a mutually exclusive and sequential manner.
| S-EPMC7948514 | biostudies-literature
Project description:Guanylate Binding Proteins (GBPs) are prominent regulators of immunity unknown to be required for nuclear envelope formation and function. The Arabidopsis GBP orthologue AtGBPL3 was identified as a novel component of the plant lamina, with essential functions in mitotic nuclear envelope reformation. AtGBPL3 is preferentially expressed in mitotically active root tips and accumulates at the nuclear envelope. Furthermore, fluorescent lifetime imaging experiments in the presence or absence of the chromatin-labelling dye Sytox-Orange suggested that GBPL3 might be able to interact with chromatin. Chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) revealed only few GBPL3-associated chromatin domains, which were concentrated particularly within or near centromeric regions.
2022-12-23 | GSE221668 | GEO
Project description:Guanylate Binding Proteins (GBPs) are prominent regulators of immunity unknown to be required for nuclear envelope formation and function. The Arabidopsis GBP orthologue AtGBPL3 was identified as a novel component of the plant lamina, with essential functions in mitotic nuclear envelope reformation. AtGBPL3 is preferentially expressed in mitotically active root tips, accumulates at the nuclear envelope and interacts with further components of the nuclear lamina. To investigate effects of defective lamina components on gene expression, we analysed the transcriptomes of 4 day old wild-type, gbpl3-5, crwn4-1, crwn1-1crwn4-1, as well as gbpl3-5 mutants expressing GBPL3-GFP or the GTPase-dead GBPL3(K83A)-GFP under control of the AtGBPL3-promoter.
2022-12-23 | GSE221661 | GEO
Project description:Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, which primarily affect the heart and skeletal muscle. A mutation in the NEP LEMD2 causes severe cardiomyopathy in humans. However, the roles of LEMD2 in the heart and the pathological mechanisms responsible for its association with cardiac disease are unknown. To explore the mechanistic basis of this pathology, we generated mice carrying the human c.T38>G LEMD2 mutation. These mice phenocopied the human disease and developed severe dilated cardiomyopathy with extensive cardiac fibrosis leading to premature death. At the cellular level, LEMD2 mutant cardiomyocytes exhibited disorganization of the transcriptionally silent heterochromatin associated with the nuclear envelope. Moreover, mice with cardiac-specific deletion of LEMD2 also died shortly after birth due to heart abnormalities. LEMD2 mutant mice displayed aberrant cardiac gene expression and extensive DNA damage. The development of cardiac disease in both mouse models is linked to p53 activation. Together, our results reveal the essentiality of the nuclear envelope protein LEMD2 for genome stability and normal cardiac function.
2022-10-13 | GSE194218 | GEO