The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5
- Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, Japan
- Faculty of Biology, Ludwig-Maximilians-Universität München, Germany
- Structural Biology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Japan
- Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, Japan
- Laboratory of Chromosome Biology, Department of Biology, Faculty of Science, Kyushu University, Japan
Figures
Figure 1 with 1 supplement
USP7 was identified as a PAF15 binding protein.
(A) Sperm chromatin was added to interphase egg extracts supplemented with either buffer (+Buffer), 20 µM UbVS (+UbVS), or 20 µM UbVS and 58 µM ubiquitin (+UbVS + Ub). Samples were analyzed by …
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Figure 1—source data 1
Figure 1 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig1-data1-v2.zip
Figure 1—figure supplement 1
USP7 was identified as a PAF15 binding protein.
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Figure 1—figure supplement 1—source data 1
Figure 1—figure supplement 1 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig1-figsupp1-data1-v2.zip
Figure 2 with 1 supplement
PAF15 associates with the TRAF and UBL1-2 domains of USP7.
(A) Schematic illustration of PAF15-USP7 binding experiment. USP7 recognizes P/A/ExxS or KxxxK motifs in its substrates via TRAF or ubiquitin-like (UBL) domains, respectively. PAF15 has three …
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Figure 2—source data 1
Figure 2 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig2-data1-v2.zip
Figure 2—figure supplement 1
hUSP7561-1102 interacts with hPAF15 dependently on KxxxK motif.
(A) Superimposition of plots of enthalpy changes in the interaction between hUSP7561-1102 and full-length hPAF15 wild-type (WT) or K93A/K96A mutant by isothermal titration calorimetry (ITC) …
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Figure 2—figure supplement 1—source data 1
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig2-figsupp1-data1-v2.zip
Figure 3 with 1 supplement
USP7 promotes PAF15 dissociation from chromatin.
(A) Sperm chromatin was added to Mock- or PAF15-depleted interphase extracts supplemented with either buffer (+Buffer), wild-type rPAF15-3xFLAG (+WT) or rPAF15 SAKA-3xFLAG (+SAKA). Chromatin …
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Figure 3—source data 1
Figure 3 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig3-data1-v2.zip
Figure 3—figure supplement 1
USP7 directly deubiquitylates PAF15 to promote PAF15 chromatin dissociation.
(A) rPAF15 WT-3xFLAG or SAKA-3xFLAG were added to PAF15-depleted extracts. The extracts used in Figure 3A were analyzed by immunoblotting using the antibodies indicated. (B) rUSP7 WT-3xFLAG or …
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Figure 3—figure supplement 1—source data 1
Figure 3—figure supplement 1 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig3-figsupp1-data1-v2.zip
Figure 4 with 1 supplement
USP7 is recruited to chromatin through the interaction with DNMT1 for PAF15 deubiquitylation.
(A) Sperm chromatin was added to interphase extracts. Replicating chromatin was digested by micrococcal nuclease (MNase). Immunoprecipitation was performed by PAF15, USP7, and DNMT1 antibodies from …
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Figure 4—source data 1
Figure 4 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig4-data1-v2.zip
Figure 4—figure supplement 1
USP7 is recruited to chromatin through the interaction with DNMT1.
(A) rDNMT1WT-3xFLAG or 4KA-3xFLAG were added to DNMT1-depleted extracts. The extracts used in Figure 4C were analyzed by immunoblotting using the antibodies indicated. (B) Mock-, PAF15-, DNMT1-, or …
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Figure 4—figure supplement 1—source data 1
Figure 4—figure supplement 1 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig4-figsupp1-data1-v2.zip
Figure 5 with 1 supplement
Unloading of PAF15 requires DNMT1-dependent DNA methylation.
(A) Sperm chromatin was added to Mock- or DNMT1-depleted interphase extracts supplemented with either buffer (+Buffer), wild-type rDNMT1-3xFLAG (+WT), or catalytic mutant rDNMT1 C1101S-3xFLAG …
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Figure 5—source data 1
Figure 5 Orignal blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig5-data1-v2.zip
Figure 5—figure supplement 1
Unloading of PAF15 requires DNMT1-dependent DNA methylation.
(A) rDNMT1 WT-3xFLAG or C1101S-3xFLAG were added to DNMT1-depleted extracts. The extracts used in Figure 5A were analyzed by immunoblotting using the antibodies indicated. (B) rDNMT1 WT-3xFLAG or …
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Figure 5—figure supplement 1—source data 1
Figure 5—figure supplement 1 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig5-figsupp1-data1-v2.zip
Figure 6 with 1 supplement
ATAD5 unloads PAF15Ub0 from chromatin.
(A) Sperm chromatin was added to Mock-, USP7-, ATAD5-, and USP7/ATAD5-depleted interphase extracts and isolated at indicated time points. Chromatin bound proteins were analyzed by immunoblotting. (B)…
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Figure 6—source data 1
Figure 6 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig6-data1-v2.zip
Figure 6—figure supplement 1
ATAD5 unloads PAF15Ub0 from chromatin.
(A) Mock-, USP7-, ATAD5-, or USP7/ATAD5 co-depleted extracts were used in Figure 6A and analyzed by immunoblotting using the antibodies indicated. (B) rhATAD5-RFCs was added to ATAD5-depleted …
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Figure 6—figure supplement 1—source data 1
Figure 6—figure supplement 1 Original blots.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig6-figsupp1-data1-v2.zip
Figure 7 with 1 supplement
PAF15 dissociation negatively regulates aberrant increase of DNA methylation.
Sperm chromatin and radiolabeled S-(methyl-3H)-adenosyl-L-methionine were added to either Mock- and USP7-, ATAD5-, or USP7/ATAD5 co-depleted extracts. Purified DNA samples were analyzed to determine …
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Figure 7—source data 1
Figure 7 DNA methylation.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig7-data1-v2.zip
Figure 7—figure supplement 1
DNA replication and methylation analysis in USP7 or ATAD5-depleted extracts.
(A) Mock-, USP7-, ATAD5-, or USP7/ATAD5 co-depleted extracts used in Figure 7 were analyzed by immunoblotting using the antibodies indicated. (B) Sperm chromatin and radiolabeled (α-32P) dCTP were …
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Figure 7—figure supplement 1—source data 1
Figure 7—figure supplement 1 Original blots, DNA replication, and methylation data.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig7-figsupp1-data1-v2.zip
Figure 8 with 1 supplement
mUSP7 interacts with mPAF15 to assure a complete DNA methylation maintenance.
(A) Immunoprecipitation (IP) of endogenous mPAF15 from whole cell lysates of wt and mPAF15 SAKA mouse embryonic stem cells (mESCs) using an anti-PAF15 antibody. Bound fractions were subjected to …
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Figure 8—source data 1
Figure 8 Original blots and DNA methylation data.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig8-data1-v2.zip
Figure 8—figure supplement 1
Generation and characterization of mouse embryonic stem cell (mESC) lines carrying the SAKA mutants by genome editing.
(A) Schematic representation of the CRISPR/Cas9 gene editing strategy. The restriction enzyme recognition site for restriction fragment length polymorphism (RFLP) screening is shown. (B) Genotyping …
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Figure 8—figure supplement 1—source data 1
Figure 8—figure supplement 1 Original gel and sequencing data.
- https://cdn.elifesciences.org/articles/79013/elife-79013-fig8-figsupp1-data1-v2.zip
Figure 9
Model of the termination of PAF15 ubiquitin signaling post replication.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (insect cells) | Sf9 | This paper | Cell line maintained in M. Nakanishi Lab | |
| Cell line (mouse cells) | mESC J1 line | This paper | Cell line maintained in H. LeonhardtLab | |
| Antibody | Anti-Xenopus PAF15 (Rabbit polyclonal) | PMID:32145273 | PMID:32145273 | WB (1:500) Nakanishi Lab |
| Antibody | Anti-Xenopus DNMT1 (Rabbit polyclonal) | PMID:24013172 | PMID:24013172 | WB (1:500) Nakanishi Lab |
| Antibody | Anti-USP7 (Rabbit polyclonal) | Thermo Fisher Scientific | Cat# A300-033A, RRID:AB_203276 | WB (1:1000) |
| Antibody | Anti-Xenopus UHRF1 (Rabbit polyclonal) | PMID:24013172 | WB (1:500) Nakanishi Lab | |
| Antibody | Anti-histone H3 (Rabbit polyclonal) | abcam | Cat# ab1791, RRID:AB_302613 | WB (1:3000) |
| Antibody | Anti-PCNA (mouse monoclonall) | Santa Cruz Biotechnology | Cat# sc-56, RRID:AB_628110 | WB (1:1000) |
| Antibody | Anti-Xenopus ATAD5 (rabbit polyclonal) | This study | WB (1:1000) | |
| Antibody | Anti-tubulin (mouse monoclonal) | Sigma-Aldrich | Cat# T9026, RRID:AB_477593 | WB (1:2000) |
| Antibody | Anti-PAF15 (mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-390515 | WB (1:500) |
| Antibody | HRP-anti-mouse IgG (rabbit polyclonal) | Sigma-Aldrich | Cat# A9044, RRID:AB_258431 | WB (1:5000) |
| Recombinant DNA reagent | pGEX4T-3-xPAF15 | PMID:32145273 | PMID:32145273 | Nakanishi Lab |
| Recombinant DNA reagent | pVL1392-xUSP7-3xFLAG | This study | Expression and purification of xUSP7 in insect cells | |
| Recombinant DNA reagent | pVL1392-xDNMT1-3xFLAG | PMID:29053958 | Nakanishi Lab | |
| Recombinant DNA reagent | pGEX-4T-3-mDPPA3 | PMID:33235224 | Nakanishi Lab | |
| Recombinant DNA reagent | pGEX-6P-1-hUSP7 (561–1102) | This study | Expression and purification of hUSP7 fragment in bacteria cells | |
| Recombinant DNA reagent | pGEX-4T-1-SUMO-hPAF15-FLAG | This study | Expression and purification of full-length hPAF15 in bacteria cells | |
| Recombinant DNA reagent | pCSII-EF-mini-AzamiGreen ATAD5 | This study | Expression and purification of full-length hATAD5 in human 293T cells | |
| Recombinant DNA reagent | pCSII-EF-RFC2, 3, 4 and 5 | This study | Expression and purification of full-length hRFC complex in human 293T cells | |
| Peptide, recombinant protein | Ubiquitin | R&D systems (Boston biochem) | U-100H | 58 μM |
| Peptide, recombinant protein | Recombinant Human Ubiquitin Vinyl Sulfone Protein | R&D systems (Boston biochem) | U-202 | 20 μM |
| Commercial assay or kit | EZ DNA Methylation-Gold Kit | Zymo | D5005 | |
| Chemical compound and drug | FT-671 | MedChem Express | HY-107985 | 100 μM |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/79013/elife-79013-mdarchecklist1-v2.docx
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Supplementary file 1
MS-based quantification of GST interacting protein.
- https://cdn.elifesciences.org/articles/79013/elife-79013-supp1-v2.xlsx
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Supplementary file 2
MS-based quantification of xPAF15 interacting proteins.
- https://cdn.elifesciences.org/articles/79013/elife-79013-supp2-v2.xlsx
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Supplementary file 3
Oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/79013/elife-79013-supp3-v2.xlsx
