A non-transcriptional function of Yap regulates the DNA replication program in Xenopus laevis
- Paris-Saclay Institute of Neuroscience, CNRS, Université Paris-Saclay, CERTOFrance, France
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), France
Figures
Figure 1 with 3 supplements
Yap is recruited to chromatin during DNA replication and the absence of Yap accelerates DNA synthesis in Xenopus egg extracts.
(A) Sperm nuclei were incubated in Xenopus egg extracts in the absence (Control) or presence of geminin (+Gem). Chromatin was isolated for immunoblotting at indicated times points before and during …
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Figure 1—source data 1
Related to Figure 1.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig1-data1-v2.zip
Figure 1—figure supplement 1
Yap protein concentration in Xenopus egg extracts.
(A) Western blot showing different amounts of recombinant Yap (rYap) used to estimate endogenous Yap in egg extracts (LSS for low-speed supernatant). (B, C) The optical densities (OD) of the protein …
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Figure 1—figure supplement 1—source data 1
Related to Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
Yap depletion increases nascent strand synthesis in egg extracts.
(A) Immunodepleted extracts with either a rabbit anti-Yap antibody (ΔYap) or a rabbit IgG as a control (ΔMock) were supplemented with sperm nuclei and incubated with [α32P]dCTP for the indicated …
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Figure 1—figure supplement 2—source data 1
Related to Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig1-figsupp2-data1-v2.zip
Figure 1—figure supplement 3
Yap depletion does not affect entry into S-phase.
(A) Nascent DNA strands synthesized during early S-phase were analysed at the indicated times by alkaline gel electrophoresis, as described in Figure 1—figure supplement 2A. (B) The amount of …
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Figure 1—figure supplement 3—source data 1
Related to Figure 1—figure supplement 3.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig1-figsupp3-data1-v2.zip
Figure 2
Egg extracts lacking Yap exhibit more active replication origins.
Sperm nuclei were incubated in egg extracts in the presence of biotin-dUTP and DNA combing was performed. (A) Three representative combed DNA fibers from one combing experiment (replicate 1) after …
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Figure 2—source data 1
Related to Figure 2.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig2-data1-v2.zip
Figure 3
Rif1 interacts with Yap.
(A) Chord plot representation related to GO annotations belonging to biological processes of proteins enriched by at least threefold in Yap versus control co-immunoprecipitations performed in …
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Figure 3—source data 1
Related to Figure 3.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig3-data1-v2.zip
Figure 4
Rif1 depletion increases global fork density in egg extracts.
(A) Western blot of egg extracts after Mock- or Rif1-depletion using the indicated antibodies. Tubulin is used as a loading control. (B) Mock- or Rif1-depleted extracts were incubated with sperm …
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Figure 4—source data 1
Related to Figure 4.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig4-data1-v2.zip
Figure 5 with 1 supplement
Yap and Rif1 depletions accelerate cell cycles in early Xenopus embryos.
(A) Time course analysis of Yap expression throughout development by western blot. Tubulin is used as a loading control. (B) Diagram of the experimental procedure used in (C). (C) Evaluation of the …
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Figure 5—source data 1
Related to Figure 5.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig5-data1-v2.zip
Figure 5—figure supplement 1
Yap knockdown using the Trim-Away strategy is effective at very early stages of development.
(A) Western blot performed on whole protein extracts from a pool of 8 embryos injected with either control morpholinos (-) or yap morpholinos (yap-MO, +), and/or pre-immune serum +TRIM21 (-) or …
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Figure 5—figure supplement 1—source data 1
Related to Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig5-figsupp1-data1-v2.zip
Figure 6 with 2 supplements
rif1 is expressed in retinal stem cells and its knockdown leads to small eye phenotype.
(A) Schematic transversal section of a Xenopus tadpole retina (RPE: retinal pigmented epithelium; NR: neural retina; ON: optic nerve). Within the ciliary marginal zone (CMZ; lower diagram), retinal …
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Figure 6—source data 1
Related to Figure 6.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig6-data1-v2.zip
Figure 6—figure supplement 1
Validation of the specificity of YAP and Rif1 antibodies.
Retinal sections from stage 41 Xenopus tadpoles microinjected at the one-cell stage with either control-MO, yap-MO or rif1-MO as indicated and immunostained for either Yap or Rif1. The staining area …
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Figure 6—figure supplement 1—source data 1
Related to Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig6-figsupp1-data1-v2.zip
Figure 6—figure supplement 2
The rif1-MO-induced small eye phenotype is rescued by co-injection with rif1 mRNA.
(A) Western blot analysis showing the levels of Rif1 proteins in tadpoles at stage 41 following microinjection at one-cell stage as indicated in the table of either Control-MO or rif1-MO together …
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Figure 6—figure supplement 2—source data 1
Related to Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig6-figsupp2-data1-v2.zip
Figure 7
rif1 loss of function affects DNA replication timing in retinal stem/early progenitor cells.
(A) One-cell stage embryos were microinjected with either the control-MO (Control), yap-MO or rif1-MO and analysed for EdU-labelling (1 hr-pulse) at stage 41. Quantifications of EdU+ cell number in …
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Figure 7—source data 1
Related to Figure 7.
- https://cdn.elifesciences.org/articles/75741/elife-75741-fig7-data1-v2.zip
Figure 8
Diagram illustrating Yap function in the control of DNA replication dynamics.
We found that Yap and Rif1 can interact (A) and are co-expressed in Xenopus early embryos as well as in retinal stem (RSC)/progenitor cells (P1) (B). (C) We found that yap and rif1 knockdowns in …
Author response image 1
Rif1 depletion (A) or Yap depletion (B) does not inhibit Yap or Rif1 recruitment, respectively, onto chromatin in egg extracts.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-human MCM2 (rabbit polyclonal) | Bethyl lab, Euromedex, Souffelweyersheim, France | Cat# A300-191 RRID: AB_162709 | WB (1:2000) |
| Antibody | Anti-Xenopus MCM7 (rabbit polyclonal) | Gift from R. A. Laskey | doi: 10.1073/pnas.93.19.10189 | WB (1:1000) |
| Antibody | Anti-α Tubulin (mouse monoclonal) | Sigma, Saint-Quentin-Fallavier, France | Cat# T5168 RRID: AB_477579 | WB (1:10,000) |
| Antibody | Anti-rat PCNA (mouse monoclonal) | ThermoFisher, Illkirch, France | Cat# MA5-11358 RRID: AB_10982348 | WB (1:500) |
| Antibody | Anti-human Yap (mouse monoclonal) | Abcam Cambridge, UK | Cat# Ab56701 RRID: AB_2219140 | WB (1:1000), IHC (1:50) |
| Antibody | Anti-human Yap (rabbit polyclonal) | Abcam Cambridge, UK | Cat# Ab62752 RRID: AB_956477 | Immunodepletion (1 µl per 20 µl extract), IP (1 µl per 20 µl extract) |
| Antibody | Anti-human H3 (rabbit polyclonal) | Abcam Cambridge, UK | Cat# Ab1791 RRID: AB_302613 | WB (1:10000) |
| Antibody | Anti-Flag (rabbit polyclonal) | Cell Signaling, OZYME, Saint-Cyr-l'École, France | Cat# F7425 RRID: AB_439687 | IP (1 µl per test) |
| Antibody | Anti-human ssDNA (mouse monoclonal) | Merck Millipore, Guyancourt, France | Cat# MAB3034 RRID: AB_11212688 | DNA combing (1:50) |
| Antibody | Anti-Xenopus Yap (rabbit polyclonal) | This paper Covalab, Villeurbanne, France | IHC (1:100), WB (1:2000), IP (1 µl per 2,5 µl extract) Immunodepletion (1 µl per 2,5 µl extract), Trim away (50 nl per injection) | |
| Antibody | Anti-Xenopus Rif1(rabbit polyclonal) | This paper Covalab, Villeurbanne, France | doi: 10.1093/n10.1093/nar/gkab756 | IHC (1:100), WB (1:2000), IP (1 µl per 2 µl extract) Immunodepletion(1 µl per 2 µl extract), Trim away (50 nl per injection) |
| Antibody | Anti-Mouse IgG (rabbit polyclonal) | Sigma, Saint-Quentin-Fallavier, France | Cat# M7023 RRID: AB_260634 | Immunodepletion (1 µl per 2 µl extract) |
| Antibody | Anti-mouse Alexa 488 (rabbit polyclonal) | ThermoFisher, Illkirch, France | Cat# A11059 RRID: AB_2534106 | DNA combing (1:50) IHC (1:50) |
| Antibody | Anti-rabbit Alexa 448 (goat polyclonal) | ThermoFisher, Illkirch, France | Cat# A11008 RRID: AB_143165 | DNA combing (1:50) |
| Antibody | Anti-mouse Alexa 594 (goat polyclonal) | ThermoFisher, Illkirch, France | Cat# A11005 RRID: AB_2534073 | DNA combing (1:50), IHC (1:1000) |
| Antibody | Anti-mouse Alexa 488 (rabbit polyclonal) | ThermoFisher, Illkirch, France | Cat# A11001 RRID: AB_2534069 | IHC (1:1000) |
| Antibody | Anti-streptavidin biotinylated (goat polyclonal) | Eurobio, Les Ulis, France | Cat# BA-0500 RRID: AB_2336221 | DNA combing (1:50), IHC (1:50) |
| Antibody | Anti-mouse IgG HRP (goat polyclonal) | Sigma, Saint-Quentin-Fallavier, France | Cat# A4416 RRID: AB_258167 | WB (1:10000) |
| Antibody | Anti-rabbit IgG HRP (donkey polyclonal) | GE Healthcare, France | Cat# NA934 RRID: AB_772206 | WB (1:10000) |
| Peptide, recombinant protein | Streptavidin Alexa 594 | ThermoFisher, Illkirch, France | Cat# S11227 | (1:50) DNA combing |
| Sequence-based reagent | yap-MO | This paper | Morpholinos Gene Tools, LLC | 5’TAGGAGACTGTG PGTCACTTCACC 3’ |
| Sequence-based reagent | rif1-MO | This paper | Morpholinos Gene Tools, LLC | 5’AATCCACAGAA CAGACGACAGCCAT 3’ |
| Sequence-based reagent | Control-MO | This paper | Morpholinos control (Gene Tools Standard Control) Gene Tools, LLC | 5'CCTCTTACCTCA GTTACAATTTATA 3' |
| Recombinant DNA reagent | HLTV-hTRIM21 | Gift from Leo James | RRID: Addgene_104973 doi:10.1038/s41596-018-0028-3 | Protein expression |
| Recombinant DNA reagent | Xenopus rif1 C-Terminal cloned in pET30a vector | Gift from A. Kumagai and W. Dunphy | doi:10.4161/cc.11.6.19636 | Protein expression |
| Recombinant DNA reagent | His-tagged Xenopus Yap cloned in pFastBac1vector | Invitrogen | baculovirus Bac-to-Bac expression system Cat# 10359016 | Protein expression |
Additional files
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Supplementary file 1
Depletion of Yap increases replication origin firing in Xenopus egg extracts.
Extended DNA combing data to Figure 2 of 2 independent experiments, Replicate 1 and Replicate 2.
- https://cdn.elifesciences.org/articles/75741/elife-75741-supp1-v2.docx
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Supplementary file 2
Depletion of Rif1 increases replication origin firing in Xenopus egg extracts.
Extended DNA combing data to Figure 4 of 2 independent experiments, Replicate 1 and Replicate 2.
- https://cdn.elifesciences.org/articles/75741/elife-75741-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/75741/elife-75741-transrepform1-v2.docx
