Splicing function of mitotic regulators links R-loop–mediated DNA damage to tumor cell killing
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ABSTRACT:
SUBMITTER: Yihan Wan
PROVIDER: S-JCBD-201409073 | bioimages |
REPOSITORIES: bioimages
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Project description:Although studies suggest that perturbing mitotic progression leads to DNA damage and p53 activation, which in turn lead to either cell apoptosis or senescence, it remains unclear how mitotic defects trigger p53 activation. We show that BuGZ and Bub3, which are two mitotic regulators localized in the interphase nucleus, interact with the splicing machinery and are required for pre-mRNA splicing. Similar to inhibition of RNA splicing by pladienolide B, depletion of either BuGZ or Bub3 led to increased formation of RNA-DNA hybrids (R-loops), which led to DNA damage and p53 activation in both human tumor cells and primary cells. Thus, R-loop-mediated DNA damage and p53 activation offer a mechanistic explanation for apoptosis of cancer cells and senescence of primary cells upon disruption of the dual-function mitotic regulators. This demonstrates the importance of understanding the full range of functions of mitotic regulators to develop antitumor drugs.
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Project description:We discovered that two mitotic regulators, BuGZ and Bub3, involved in splicing regulation during interphase 8 samples from primary Human foreskin fibroblast cells (HFFs) , 12 samples from TOV21G cells. Control siRNA. BuGZ siRNA or Bub3 siRNA were transfected for 48 h before sample collection. Cells treated with pladienolide B served as positive controls. For each RNAi experiment, we had two biological replicates.
2015-04-11 | E-GEOD-67770 | biostudies-arrayexpress
Project description:We discovered that two mitotic regulators, BuGZ and Bub3, involved in splicing regulation during interphase
2015-04-11 | GSE67770 | GEO
Project description:Mitotic catastrophe is the response of mammalian cells to mitotic DNA damage. It produces tetraploid cells with a range of different nuclear morphologies from binucleated to multimicronucleated. In response to DNA damage, checkpoints are activated to delay cell cycle progression and to coordinate repair. Cells in different cell cycle phases use different mechanisms to arrest their cell cycle progression. It has remained unclear whether the termination of mitosis in a mitotic catastrophe is regulated by DNA damage checkpoints. Here, we report the presence of a mitotic exit DNA damage checkpoint in mammalian cells. This checkpoint delays mitotic exit and prevents cytokinesis and, thereby, is responsible for mitotic catastrophe. The DNA damage-induced mitotic exit delay correlates with the inhibition of Cdh1 activation and the attenuated degradation of cyclin B1. We demonstrate that the checkpoint is Chk1-dependent.
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Project description:The H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its noncatalytic FLOS domain-mediated recruitment of cyclin K and regulation of DNA damage response genes. In this study, we identify a functional nuclear localization signal in and interaction partners of the FLOS domain. Our screen for FLOS domain-binding partners reveals that the SETD1A FLOS domain binds mitosis-associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3-binding motifs in SETD1A shows synergistic antileukemic effects. BuGZ/BUB3 localize to SETD1A-bound promoter-TSS regions and SETD1A-negative H3K4me1-positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required for both SETD1A-binding and leukemia cell proliferation. Cell-cycle-specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells.
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Project description:The H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its non-catalytic FLOS domain-mediated recruitment of cyclin K and regulation of DNA damage response genes. In this study, we identify a functional nuclear localization signal in and interaction partners of the FLOS domain. Our screen for FLOS domain-binding partners reveals that the SETD1A FLOS domain binds mitosis-associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3 binding motifs in SETD1A shows synergistic anti-leukemic effects. BuGZ/BUB3 localize to SETD1A-bound promoter-TSS regions and SETD1A-negative H3K4me1-positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required for both SETD1A binding and leukemia cell proliferation. Cell-cycle specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells.
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Project description:PurposeSpecificity protein 1 (SP1) is involved in the transcription of several genes implicated in tumor maintenance. We investigated the effects of mithramycin A (MTA), an inhibitor of SP1 DNA binding, on radiation response.Methods and materialsClonogenic survival after irradiation was assessed in 2 tumor cell lines (A549, UM-UC-3) and 1 human fibroblast line (BJ) after SP1 knockdown or MTA treatment. DNA damage repair was evaluated using γH2AX foci formation, and mitotic catastrophe was assessed using nuclear morphology. Gene expression was evaluated using polymerase chain reaction arrays. In vivo tumor growth delay was used to evaluate the effects of MTA on radiosensitivity.ResultsTargeting of SP1 with small interfering RNA or MTA sensitized A549 and UM-UC-3 to irradiation, with no effect on the BJ radiation response. MTA did not alter γH2AX foci formation after irradiation in tumor cells but did enhance mitotic catastrophe. Treatment with MTA suppressed transcription of genes involved in cell death. MTA administration to mice bearing A549 and UM-UC-3 xenografts enhanced radiation-induced tumor growth delay.ConclusionsThese results support SP1 as a target for radiation sensitization and confirm MTA as a radiation sensitizer in human tumor models.
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Project description:Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.
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Project description:DNA damage response (DDR) during interphase involves active signaling and repair to ensure genomic stability. However, how mitotic cells respond to DNA damage remains poorly understood. Supported by correlative live-/fixed-cell microscopy, it was found that mitotic cells exposed to several cancer chemotherapy compounds acquire and signal DNA damage, regardless of how they interact with DNA. In-depth analysis upon DNA damage during mitosis revealed a spindle assembly checkpoint (SAC)-dependent, but ataxia telangiectasia mutated-independent, mitotic delay. This delay was due to the presence of misaligned chromosomes that ultimately satisfy the SAC and missegregate, leading to micronuclei formation. Mechanistically, it is shown that mitotic DNA damage causes missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesins. Importantly, with the exception of DNA damage induced by etoposide-a topoisomerase II inhibitor-this outcome was independent of a general effect on kinetochore microtubule stability. Colony formation assays in pan-cancer cell line models revealed that mitotic DNA damage causes distinct cytotoxic effects, depending on the nature and extent of the damage. Overall, these findings unveil and raise awareness that therapeutic DNA damage regimens may contribute to genomic instability through a surprising link with chromokinesin-mediated missegregation of polar chromosomes in cancer cells.
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Project description:Double-strand breaks (DSBs) can initiate mitotic catastrophe, a complex oncosuppressive phenomenon characterized by cell death during or after cell division. Here we unveil how cell cycle-regulated DSB repair guides disparate cell death outcomes through single-cell analysis of extended live imaging. Following DSB induction in S or G2, passage of unresolved homologous recombination intermediates into mitosis promotes non-immunogenic intrinsic apoptosis in the immediate attempt at cell division. Conversely, non-homologous end joining, microhomology-mediated end joining and single-strand annealing cooperate to enable damaged G1 cells to complete the first cell cycle with an aberrant cell division at the cost of delayed extrinsic lethality and interferon production. Targeting non-homologous end joining, microhomology-mediated end joining or single-strand annealing promotes mitotic death, while suppressing mitotic death enhances interferon production. Together the data indicate that a temporal repair hierarchy, coupled with cumulative DSB load, serves as a reliable predictor of mitotic catastrophe outcomes following genome damage. In this pathway, homologous recombination suppresses interferon production by promoting mitotic lethality.
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