Maternal Smc3 protects the integrity of the zygotic genome through DNA replication and mitosis

Aneuploidy is frequently observed in oocytes and early embryos, begging the question of how genome integrity is monitored and preserved during this critical period. SMC3 is a subunit of the cohesin complex that supports genome integrity, but its role in maintaining the genome in this window of mammalian development is unknown. We discovered that although depletion of Smc3 following meiotic S phase in mouse oocytes allowed accurate meiotic chromosome segregation, adult females were infertile. We provide evidence that DNA lesions accumulated following S phase in SMC3-deficient zygotes, followed by mitosis with lagging chromosomes, elongated spindles, micronuclei, and arrest at the 2-cell stage. Remarkably, although centromeric cohesion was defective, the dosage of SMC3 was sufficient to enable embryogenesis in juvenile mutant females. Our findings suggest that despite previous reports of aneuploidy in early embryos, chromosome missegregation in zygotes halts embryogenesis at the 2-cell stage. Smc3 is a maternal gene with essential functions in repair of spontaneous damage associated with DNA replication and subsequent chromosome segregation in zygotes, making cohesin a key protector of the zygotic genome.

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DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity

The Journal of Cell Biology ◽

10.1083/jcb.201306023 ◽

2014 ◽

Vol 204 (2) ◽

pp. 165-175 ◽

Cited By ~ 22

Author(s):

Maria M. Magiera ◽

Elisabeth Gueydon ◽

Etienne Schwob

Keyword(s):

Dna Replication ◽

Chromosome Segregation ◽

Replication Stress ◽

S Phase ◽

Genome Integrity ◽

Replication Forks ◽

Mitotic Entry ◽

Transient Activation ◽

Spindle Checkpoints ◽

Spindle Assembly Checkpoints

Deoxyribonucleic acid (DNA) replication and chromosome segregation must occur in ordered sequence to maintain genome integrity during cell proliferation. Checkpoint mechanisms delay mitosis when DNA is damaged or upon replication stress, but little is known on the coupling of S and M phases in unperturbed conditions. To address this issue, we postponed replication onset in budding yeast so that DNA synthesis is still underway when cells should enter mitosis. This delayed mitotic entry and progression by transient activation of the S phase, G2/M, and spindle assembly checkpoints. Disabling both Mec1/ATR- and Mad2-dependent controls caused lethality in cells with deferred S phase, accompanied by Rad52 foci and chromosome missegregation. Thus, in contrast to acute replication stress that triggers a sustained Mec1/ATR response, multiple pathways cooperate to restrain mitosis transiently when replication forks progress unhindered. We suggest that these surveillance mechanisms arose when both S and M phases were coincidently set into motion by a unique ancestral cyclin–Cdk1 complex.

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An intrinsic S/G2 checkpoint enforced by ATR

Science ◽

10.1126/science.aap9346 ◽

2018 ◽

Vol 361 (6404) ◽

pp. 806-810 ◽

Cited By ~ 78

Author(s):

Joshua C. Saldivar ◽

Stephan Hamperl ◽

Michael J. Bocek ◽

Mingyu Chung ◽

Thomas E. Bass ◽

...

Keyword(s):

Dna Replication ◽

Chromosome Segregation ◽

Gene Network ◽

S Phase ◽

Genome Integrity ◽

Control Mechanisms ◽

Cyclin Dependent Kinase ◽

G2 Checkpoint ◽

A Cell ◽

Early Mitosis

The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)–directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.

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Retinoic acid signaling is critical during the totipotency window in early mammalian development

Nature Structural & Molecular Biology ◽

10.1038/s41594-021-00590-w ◽

2021 ◽

Author(s):

Ane Iturbide ◽

Mayra L. Ruiz Tejeda Segura ◽

Camille Noll ◽

Kenji Schorpp ◽

Ina Rothenaigner ◽

...

Keyword(s):

Retinoic Acid ◽

Regulatory Networks ◽

Es Cells ◽

Embryonic Stem ◽

Mammalian Development ◽

Cell Stage ◽

Cell Potential ◽

Cellular Models ◽

Early Embryos ◽

Genome Activation

AbstractTotipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

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The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

The Journal of Cell Biology ◽

10.1083/jcb.200412076 ◽

2005 ◽

Vol 168 (7) ◽

pp. 999-1012 ◽

Cited By ~ 25

Author(s):

Jeff Bachant ◽

Shannon R. Jessen ◽

Sarah E. Kavanaugh ◽

Candida S. Fielding

Keyword(s):

Dna Replication ◽

Chromosome Segregation ◽

Replication Fork ◽

S Phase ◽

Origin Of Replication ◽

Independent Manner ◽

Checkpoint Signaling ◽

Hydroxyurea Treatment ◽

Chromatid Cohesion ◽

S Phase Checkpoint

The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore–spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

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Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription

Development ◽

10.1242/dev.121.1.113 ◽

1995 ◽

Vol 121 (1) ◽

pp. 113-122 ◽

Cited By ~ 4

Author(s):

E. Christians ◽

E. Campion ◽

E.M. Thompson ◽

J.P. Renard

Keyword(s):

Dna Replication ◽

Mouse Embryo ◽

Culture Conditions ◽

Hsp 70 ◽

Cell Stage ◽

Embryonic Genome ◽

Genome Activation ◽

Alpha Amanitin ◽

Zygotic Genome

Activation of the mouse embryonic genome at the 2-cell stage is characterized by the synthesis of several alpha-amanitin-sensitive polypeptides, some of which belong to the multigenic hsp 70 family. In the present work we show that a member of this family, the HSP 70.1 gene, is highly transcribed at the onset of zygotic genome activation. Transcription of this gene began as early as the 1-cell stage. Expression of the gene continued through the early 2-cell stage but was repressed before the completion of the second round of DNA replication. During this period we observed that the level of transcription was modulated by in vitro culture conditions. The coincidence of repression of HSP70.1 transcription with the second round of DNA replication was not found for other transcription-dependent polypeptides synthesized at the 2-cell stage.

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The yeast core spliceosome maintains genome integrity through R-loop prevention and α-tubulin expression

10.1101/272807 ◽

2018 ◽

Author(s):

Annie S. Tam ◽

Veena Mathew ◽

Tianna S. Sihota ◽

Anni Zhang ◽

Peter C. Stirling

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Chromosome Segregation ◽

Genome Stability ◽

Spindle Assembly Checkpoint ◽

Genome Instability ◽

Genome Integrity ◽

Genome Maintenance ◽

Maintenance Factors ◽

Spindle Assembly Checkpoint Protein

To achieve genome stability cells must coordinate the action of various DNA transactions including DNA replication, repair, transcription and chromosome segregation. How transcription and RNA processing enable genome stability is only partly understood. Two predominant models have emerged: one involving changes in gene expression that perturb other genome maintenance factors, and another in which genotoxic DNA:RNA hybrids, called R-loops, impair DNA replication. Here we characterize genome instability phenotypes in a panel yeast splicing factor mutants and find that mitotic defects, and in some cases R-loop accumulation, are causes of genome instability. Genome instability in splicing mutants is exacerbated by loss of the spindle-assembly checkpoint protein Mad1. Moreover, removal of the intron from the α-tubulin gene TUB1 restores genome integrity. Thus, while R-loops contribute in some settings, defects in yeast splicing predominantly lead to genome instability through effects on gene expression.

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The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

Macedonian Veterinary Review ◽

10.1515/macvetrev-2016-0085 ◽

2016 ◽

Vol 39 (2) ◽

pp. 209-217 ◽

Cited By ~ 1

Author(s):

Martin Morovic ◽

Matej Murin ◽

Frantisek Strejcek ◽

Michal Benc ◽

Dusan Paál ◽

...

Keyword(s):

Somatic Cell ◽

Coming Out ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Dna Methyltransferase ◽

Dna Methyltransferases ◽

Preimplantation Embryos ◽

Mammalian Development ◽

Cell Stage ◽

Early Embryos

AbstractOne of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

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Maternal UHRF1 Is Essential for Transcription Landscapes and Repression of Repetitive Elements During the Maternal-to-Zygotic Transition

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.610773 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yanqing Wu ◽

Juan Dong ◽

Shenglei Feng ◽

Qiang Zhao ◽

Peng Duan ◽

...

Keyword(s):

Embryonic Development ◽

Critical Role ◽

Conditional Knockout ◽

Developmental Competence ◽

Early Embryonic Development ◽

Transcriptional Level ◽

Cell Stage ◽

Early Embryos ◽

Maternal To Zygotic Transition ◽

Zygotic Genome

Maternal factors that modulate maternal-to-zygotic transition (MZT) are essential for the growth from specialized oocytes to totipotent embryos. Despite several studies, the mechanisms regulating epigenetic reprogramming during MZT remain largely elusive. UHRF1 plays a role in maintaining GC methylation in oocytes and early embryos. However, little is known about its role in mouse MZT. Here, we explored the function of maternal UHRF1 in zygotic genome regulation during early embryonic development in mice. We showed that the conditional knockout (cKO) of UHRF1 in either primordial or growing oocytes causes infertility but differentially affects early embryonic development. UHRF1 deficiency in primordial oocytes led to early embryonic developmental arrest at the two-cell stage, accompanied by significant alterations in global DNA and H3K4me3 methylation patterns. In comparison, UHRF1 ablation in growing oocytes significantly reduced developmental competence from two-cell embryos to blastocysts. At the transcriptional level, the absence of maternal UHRF1 led to aberrant transcriptional regulation of the zygotic genome during MZT at the two-cell stage. Furthermore, we observed that retrotransposable elements in UHRF1-deficient oocytes and embryos were not silenced properly; in particular, the LINE-1 and long terminal repeat (LTR) subfamily were activated abnormally. Collectively, the findings of our study reveal that maternal UHRF1 plays a critical role in establishing the correct epigenetic chromatin reprogramming of early embryos, regulating essential genes during MZT, and preserving genome integrity that drives early embryonic development in mice.

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Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration

Scientific Reports ◽

10.1038/s41598-019-54366-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Marcelo S. da Silva ◽

Gustavo R. Cayres-Silva ◽

Marcela O. Vitarelli ◽

Paula A. Marin ◽

Priscila M. Hiraiwa ◽

...

Keyword(s):

Dna Replication ◽

Mechanistic Model ◽

S Phase ◽

Dna Lesions ◽

Phase Duration ◽

African Trypanosomes ◽

Transcription Activity ◽

Dna And Rna ◽

Polycistronic Transcription ◽

Dna Combing

AbstractThe co-synthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences are still little studied. Here, we showed that the number of constitutive origins mapped in the Trypanosoma brucei genome is less than the minimum required to complete replication within S-phase duration. By the development of a mechanistic model of DNA replication considering replication-transcription conflicts and using immunofluorescence assays and DNA combing approaches, we demonstrated that the activation of non-constitutive (backup) origins are indispensable for replication to be completed within S-phase period. Together, our findings suggest that transcription activity during S phase generates R-loops, which contributes to the emergence of DNA lesions, leading to the firing of backup origins that help maintain robustness in S-phase duration. The usage of this increased pool of origins, contributing to the maintenance of DNA replication, seems to be of paramount importance for the survival of this parasite that affects million people around the world.

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An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903418116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13374-13383 ◽

Cited By ~ 26

Author(s):

Tatiana N. Moiseeva ◽

Yandong Yin ◽

Michael J. Calderon ◽

Chenao Qian ◽

Sandra Schamus-Haynes ◽

...

Keyword(s):

Dna Replication ◽

Kinase Activity ◽

Kinase Inhibitors ◽

S Phase ◽

Dna Lesions ◽

Dependent Manner ◽

Kinase Signaling ◽

Origin Firing ◽

Atr Kinase ◽

Chk1 Kinase

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

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