Anti-silencing factor 1A is associated with genome stability maintenance of mouse preimplantation embryos†

2020 ◽  
Vol 102 (4) ◽  
pp. 817-827
Author(s):  
Kai Deng ◽  
Wanyou Feng ◽  
Xiaohua Liu ◽  
Xiaoping Su ◽  
Erwei Zuo ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Genome Stability ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
End Joining ◽  
Dna Damages ◽  
Developmental Potential ◽  
Damage Repair ◽  
Non Homologous End Joining ◽  
Early Mouse

Abstract Genome stability is critical for the normal development of preimplantation embryos, as DNA damages may result in mutation and even embryo lethality. Anti-silencing factor 1A (ASF1A) is a histone chaperone and enriched in the MII oocytes as a maternal factor, which may be associated with the maintenance of genome stability. Thus, this study was undertaken to explore the role of ASF1A in maintaining the genome stability of early mouse embryos. The ASF1A expressed in the preimplantation embryos and displayed a dynamic pattern throughout the early embryonic development. Inhibition of ASF1A expression decreased embryonic development and increased DNA damages. Overexpression of ASF1A improved the developmental potential and decreased DNA damages. When 293T cells that had been integrated with RGS-NHEJ were co-transfected with plasmids of pcDNA3.1-ASF1A, gRNA-NHEJ, and hCas9, less cells expressed eGFP, indicating that non-homologous end joining was reduced by ASF1A. When 293T cells were co-transfected with plasmids of HR-donor, gRNA-HR, hCas9, and pcDNA3.1-ASF1A, more cells expressed eGFP, indicating that homologous recombination (HR) was enhanced by ASF1A. These results indicate that ASF1A may be associated with the genome stability maintenance of early mouse embryos and this action may be mediated by promoting DNA damage repair through HR pathway.

scholarly journals Pak2 reduction induces a failure of early embryonic development in mice

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Juan Zeng ◽  
Nengqing Liu ◽  
Yinghong Yang ◽  
Yi Cheng ◽  
Yuanshuai Li ◽  
...  
Keyword(s):  
Dna Repair ◽  
Embryonic Development ◽  
Cell Apoptosis ◽  
Spindle Assembly ◽  
Dna Lesions ◽  
Mouse Embryos ◽  
Early Embryonic Development ◽  
Preimplantation Embryos ◽  
Early Mouse

Abstract Background The quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. In mice, Pak2 knock out and endothelial depletion of Pak2 showed embryonic lethality. However, the role of Pak2 in preimplantation embryos remains unelucidated. Methods In the present work, Pak2 was reduced using a specific small interfering RNA in early mouse embryos, validating the unique roles of Pak2 in spindle assembly and DNA repair during mice early embryonic development. We also employed immunoblotting, immunostaining, in vitro fertilization (IVF) and image quantification analyses to test the Pak2 knockdown on the embryonic development progression, spindle assembly, chromosome alignment, oxidative stress, DNA lesions and blastocyst cell apoptosis. Areas in chromatin with γH2AX were detected by immunofluorescence microscopy and serve as a biomarker of DNA damages. Results We found that Pak2 knockdown significantly reduced blastocyst formation of early embryos. In addition, Pak2 reduction led to dramatically increased abnormal spindle assembly and chromosomal aberrations in the embryos. We noted the overproduction of reactive oxygen species (ROS) with Pak2 knockdown in embryos. In response to DNA double strand breaks (DSBs), the histone protein H2AX is specifically phosphorylated at serine139 to generate γH2AX, which is used to quantitative DSBs. In this research, Pak2 knockdown also resulted in the accumulation of phosphorylated γH2AX, indicative of increased embryonic DNA damage. Commensurate with this, a significantly augmented rate of blastocyst cell apoptosis was detected in Pak2-KD embryos compared to their controls. Conclusions Collectively, our data suggest that Pak2 may serve as an important regulator of spindle assembly and DNA repair, and thus participate in the development of early mouse embryos.

scholarly journals Pak2 Depletion Induces a Failure of Early Embryonic Development in Mice

2021 ◽  
Author(s):  
Juan Zeng ◽  
Nengqing Liu ◽  
Yinghong Yang ◽  
Yi Cheng ◽  
Yuanshuai Li ◽  
...  
Keyword(s):  
Dna Repair ◽  
Embryonic Development ◽  
Cell Apoptosis ◽  
Spindle Assembly ◽  
Dna Lesions ◽  
Mouse Embryos ◽  
Early Embryonic Development ◽  
Preimplantation Embryos ◽  
Early Mouse

Abstract Background: The quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. However, the role of Pak2 in preimplantation embryos remains unelucidated. Methods: In the present work, Pak2 was depleted using a specific small interfering RNA in early mouse embryos, validating the unique roles of Pak2 in spindle assembly and DNA repair during mice early embryonic development. We also employed immunoblotting, immunostaining, in vitro fertilization (IVF) and image quantification analyses to test the Pak2 knockdown on the embryonic development progression, spindle assembly, chromosome alignment, oxidative stress, DNA lesions and blastocyst cell apoptosis.Results: We found that Pak2 deletion significantly reduced blastocyst formation of early embryos. In addition, Pak2 depletion led to dramatically increased abnormal spindle assembly and chromosomal aberrations in the embryos. We noted the overproduction of reactive oxygen species (ROS) with Pak2 knockdown in embryos. Pak2 knockdown also resulted in the accumulation of phosphorylated γH2AX, indicative of increased embryonic DNA damage. Commensurate with this, a significantly augmented rate of blastocyst cell apoptosis was detected in Pak2-KD embryos compared to their controls. Conclusions: Collectively, our data suggest that Pak2 may serve as an important regulator of spindle assembly and DNA repair, and thus participate in the development of early mouse embryos.

scholarly journals cGAS guards against chromosome end-to-end fusions during mitosis and facilitates replicative senescence

2021 ◽  
Author(s):  
Xiaocui Li ◽  
Xiaojuan Li ◽  
Chen Xie ◽  
Sihui Cai ◽  
Mengqiu Li ◽  
...  
Keyword(s):  
Genome Stability ◽  
Mitotic Chromosome ◽  
Replicative Senescence ◽  
Genome Instability ◽  
Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
End To End ◽  
Non Homologous End Joining ◽  
Sting Pathway

AbstractAs a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.

scholarly journals Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21- Mediated Early Senescence Signalling

2015 ◽  
Vol 11 (5) ◽  
pp. e1004246 ◽  
Author(s):  
David W. P. Dolan ◽  
Anze Zupanic ◽  
Glyn Nelson ◽  
Philip Hall ◽  
Satomi Miwa ◽  
...  
Keyword(s):  
Dna Damage ◽  
Stochastic Model ◽  
Dna Damage Repair ◽  
End Joining ◽  
Damage Repair ◽  
Non Homologous End Joining

scholarly journals Loss of Cdc13 causes genome instability by a deficiency in replication-dependent telomere capping

2019 ◽  
Author(s):  
Rachel E Langston ◽  
Dominic Palazzola ◽  
Erin Bonnell ◽  
Raymund J. Wellinger ◽  
Ted Weinert
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Genome Instability ◽  
Chromosome Instability ◽  
S Phase ◽  
Temperature Sensitive ◽  
End Joining ◽  
Telomere Capping ◽  
Non Homologous End Joining

AbstractIn budding yeast, Cdc13, Stn1, and Ten1 form a telomere binding heterotrimer dubbed CST. Here we investigate the role of Cdc13/CST in maintaining genome stability, using a Chr VII disome system that can generate recombinants, loss, and enigmatic unstable chromosomes. In cells expressing a temperature sensitive CDC13 allele, cdc13F684S, unstable chromosomes frequently arise due to problems in or near a telomere. Hence, when Cdc13 is defective, passage through S phase causes Exo1-dependent ssDNA and unstable chromosomes, which then are the source for whole chromosome instability events (e.g. recombinants, chromosome truncations, dicentrics, and/or loss). Specifically, genome instability arises from a defect in Cdc13’s replication-dependent telomere capping function, not Cdc13s putative post-replication telomere capping function. Furthermore, the unstable chromosomes form without involvement of homologous recombination nor non-homologous end joining. Our data suggest that a Cdc13/CST defect in semi-conservative replication near the telomere leads to ssDNA and unstable chromosomes, which then are lost or subject to complex rearrangements. This system defines a links between replication-dependent chromosome capping and genome stability in the form of unstable chromosomes.

scholarly journals Ku-dependent non-homologous end-joining as the major pathway contributes to sublethal damage repair in mammalian cells

2015 ◽  
Vol 91 (11) ◽  
pp. 867-871 ◽  
Author(s):  
Min Liu ◽  
Solah Lee ◽  
Bailong Liu ◽  
Hongyan Wang ◽  
Lihua Dong ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
End Joining ◽  
Major Pathway ◽  
Damage Repair ◽  
Non Homologous End Joining

scholarly journals KU70 Inhibition Impairs Both Non-Homologous End Joining and Homologous Recombination DNA Damage Repair Through SHP-1 Induced Dephosphorylation of SIRT1 in Adult T-Cell Leukemia-Lymphoma Cells

2018 ◽  
Vol 49 (6) ◽  
pp. 2111-2123 ◽  
Author(s):  
Wenlei Yu ◽  
Liang Li ◽  
Guangming Wang ◽  
Wenjun Zhang ◽  
Jun Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Jurkat Cells ◽  
Cell Leukemia ◽  
End Joining ◽  
Adult T Cell Leukemia ◽  
Damage Repair ◽  
Repair Efficiency ◽  
Promising Target ◽  
Non Homologous End Joining

Background/Aims: Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease which is highly resistant to chemotherapy. Studies show that enhanced ability of DNA damage repair (DDR) in cancer cells plays a key role in chemotherapy resistance. Here, we suggest that defect in DDR related genes might be a promising target to destroy the genome stability of tumor cells. Methods: Since KU70 is highly expressed in Jurkat cells, one of the most representative cell lines of ATL, we knocked down KU70 by shRNA and analyzed the impact of KU70 deficiency in Jurkat cells as well as in NOD-SCID animal models by western blot, immunofluorescence, flow cytometry and measuring DNA repair efficiency. Results: It is observed that silencing of KU70 resulted in accumulated DNA damage and impaired DDR in Jurkat cells, resulting in more apoptosis, decreased cell proliferation and cell cycle arrest. DNA damage leads to DNA double-strand breaks (DSBs), which are processed by either non-homologous end joining(NHEJ) or homologous recombination(HR). In our study, both NHEJ and HR are impaired because of KU70 defect, accompanied with increased protein level of SHP-1, a dephosphorylation enzyme. In turn, SHP-1 led to dephosphorylation of SIRT1, which further impaired HR repair efficiency. Moreover, KU70 deficiency prolonged survival of Jurkat-xenografted mice. Conclusion: These findings suggest that targeting KU70 is a promising target for ATL and might overcome the existing difficulties in chemotherapy.

Ku80 Gene is Related to Non-Homologous End-Joining and Genome Stability in Aspergillus niger

2011 ◽  
Vol 62 (4) ◽  
pp. 1342-1346 ◽  
Author(s):  
Jinxiang Zhang ◽  
Zhihui Mao ◽  
Wei Xue ◽  
Ying Li ◽  
Guomin Tang ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Genome Stability ◽  
End Joining ◽  
Non Homologous End Joining

Sodium Fluoride Affects DNA Methylation of Imprinted Genes in Mouse Early Embryos

2015 ◽  
Vol 147 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Lei Zhao ◽  
Sheng Zhang ◽  
Xinglan An ◽  
Wentao Tan ◽  
Bo Tang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Embryonic Development ◽  
Mrna Level ◽  
Mouse Embryos ◽  
Imprinted Genes ◽  
Male Mice ◽  
Early Embryos ◽  
Pregnant Mice ◽  
H19 Gene ◽  
Early Mouse

Fluorine is reported to affect embryonic development, but the underlining mechanism is unclear. The modification of DNA methylation of the H19 and Peg3 genes is important in embryonic development. Therefore, the effect of fluorine on methylation of H19 and Peg3 during early mouse embryos was studied. It was shown that the H19 gene was significantly downmethylated in E2.5, E3.5, and E4.5 embryos from pregnant mice treated with 120 mg/l NaF in drinking water for 48 h. But methylation of both H19 and Peg3 genes was disrupted when the parent male mice were treated with NaF for 35 days. H19 DNA methylation decreased significantly, while Peg3 was almost completely methylated. However, when pregnant mice, mated with NaF-treated male mice, were again treated with NaF for 48 h, either H19 or Peg3 methylation in the embryos decreased significantly. In addition, the mRNA level of H19 considerably increased in E3.5 and E4.5 embryos from NaF-treated pregnant mice. Further, the expression of DNMT1 decreased significantly after NaF treatment. Conclusively, we demonstrated that fluorine may adversely affect early embryonic development by disrupting the methylation of H19 and Peg3 through downregulation of DNMT1.

scholarly journals Non-Homologous end Joining Factors XLF, PAXX and DNA-PKcs Maintain the Neural Stem and Progenitor Cell Population

2020 ◽  
Author(s):  
Raquel Gago-Fuentes ◽  
Valentyn Oksenych
Keyword(s):  
Mammalian Cells ◽  
Progenitor Cell ◽  
Neuronal Apoptosis ◽  
Embryonic Lethality ◽  
End Joining ◽  
Double Knockout ◽  
Dna Damages ◽  
Stem And Progenitor Cells ◽  
Non Homologous End Joining ◽  
The Impact

Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damages throughout the cell cycle, and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking NHEJ factors Ku70 and Ku80. Inactivation of other NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of either Paxx, Mri or Dna-pkcs NHEJ gene results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of NHEJ factors on early stages of neurodevelopment, we isolated neural stem and progenitor cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf, Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that XLF, DNA-PKcs and PAXX maintain the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.

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