The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5

  1. Ryota Miyashita
  2. Atsuya Nishiyama  Is a corresponding author
  3. Weihua Qin
  4. Yoshie Chiba
  5. Satomi Kori
  6. Norie Kato
  7. Chieko Konishi
  8. Soichiro Kumamoto
  9. Hiroko Kozuka-Hata
  10. Masaaki Oyama
  11. Yoshitaka Kawasoe
  12. Toshiki Tsurimoto
  13. Tatsuro S Takahashi
  14. Heinrich Leonhardt
  15. Kyohei Arita
  16. Makoto Nakanishi  Is a corresponding author
  1. Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, Japan
  2. Faculty of Biology, Ludwig-Maximilians-Universität München, Germany
  3. Structural Biology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Japan
  4. Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, Japan
  5. Laboratory of Chromosome Biology, Department of Biology, Faculty of Science, Kyushu University, Japan
9 figures, 1 table and 4 additional files

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 …

Figure 1—figure supplement 1
USP7 was identified as a PAF15 binding protein.

(A) Interphase egg extracts supplemented with UbVS or UbVS+Ub used in Figure 1A were analyzed by immunoblotting using the antibodies indicated. (B) Purified GST or GST-rPAF15 mutants used in Figure …

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 …

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) …

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 …

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 …

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 …

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 …

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 …

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 …

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)…

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 …

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 …

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 …

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 …

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 …

Model of the termination of PAF15 ubiquitin signaling post replication.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (insect cells)Sf9This paperCell line maintained in M. Nakanishi Lab
Cell line (mouse cells)mESC J1 lineThis paperCell line maintained in H. LeonhardtLab
AntibodyAnti-Xenopus PAF15 (Rabbit polyclonal)PMID:32145273PMID:32145273WB (1:500)
Nakanishi Lab
AntibodyAnti-Xenopus DNMT1 (Rabbit polyclonal)PMID:24013172PMID:24013172WB (1:500)
Nakanishi Lab
AntibodyAnti-USP7 (Rabbit polyclonal)Thermo Fisher ScientificCat# A300-033A, RRID:AB_203276WB (1:1000)
AntibodyAnti-Xenopus UHRF1 (Rabbit polyclonal)PMID:24013172WB (1:500)
Nakanishi Lab
AntibodyAnti-histone H3 (Rabbit polyclonal)abcamCat# ab1791, RRID:AB_302613WB (1:3000)
AntibodyAnti-PCNA (mouse monoclonall)Santa Cruz BiotechnologyCat# sc-56, RRID:AB_628110WB (1:1000)
AntibodyAnti-Xenopus ATAD5 (rabbit polyclonal)This studyWB (1:1000)
AntibodyAnti-tubulin (mouse monoclonal)Sigma-AldrichCat# T9026, RRID:AB_477593WB (1:2000)
AntibodyAnti-PAF15 (mouse monoclonal)Santa Cruz BiotechnologyCat# sc-390515WB (1:500)
AntibodyHRP-anti-mouse IgG (rabbit polyclonal)Sigma-AldrichCat# A9044, RRID:AB_258431WB (1:5000)
Recombinant DNA reagentpGEX4T-3-xPAF15PMID:32145273PMID:32145273Nakanishi Lab
Recombinant DNA reagentpVL1392-xUSP7-3xFLAGThis studyExpression and purification of xUSP7 in insect cells
Recombinant DNA reagentpVL1392-xDNMT1-3xFLAGPMID:29053958Nakanishi Lab
Recombinant DNA reagentpGEX-4T-3-mDPPA3PMID:33235224Nakanishi Lab
Recombinant DNA reagentpGEX-6P-1-hUSP7 (561–1102)This studyExpression and purification of hUSP7 fragment in bacteria cells
Recombinant DNA reagentpGEX-4T-1-SUMO-hPAF15-FLAGThis studyExpression and purification of full-length hPAF15 in bacteria cells
Recombinant DNA reagentpCSII-EF-mini-AzamiGreen ATAD5This studyExpression and purification of full-length hATAD5 in human 293T cells
Recombinant DNA reagentpCSII-EF-RFC2, 3, 4 and 5This studyExpression and purification of full-length hRFC complex in human 293T cells
Peptide, recombinant proteinUbiquitinR&D systems
(Boston biochem)
U-100H58 μM
Peptide, recombinant proteinRecombinant Human Ubiquitin Vinyl Sulfone ProteinR&D systems
(Boston biochem)
U-20220 μM
Commercial assay or kitEZ DNA Methylation-Gold KitZymoD5005
Chemical compound and drugFT-671MedChem
Express
HY-107985100 μM

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