Yamaguchi L , Nishiyama A , Misaki T , Johmura Y , Ueda J , Arita K , Nagao K , Obuse C , Nakanishi M .

	Figure 1. DUBs activity is important for maintenance of DNA methylation, but not for DNA replication, in Xenopus egg extracts. (A,B) Sperm chromatin was incubated with interphase Xenopus egg extracts supplemented with buffer (+buffer), 20 μM Ub-VS (+Ub-VS), or 20 μM Ub-VS and 0.2 mg/ml ubiquitin (+Ub-VS +Ub). Prior to addition of sperm chromatin, radiolabeled [α-32P]dCTP (A) or S-[methyl-3H]-adenosyl-L-methionine (B) was added to extracts. Purified DNA samples were analyzed to determine the efficiency of DNA replication (A) and DNA methylation at 90 min after addition of sperm DNA (B), as described in Experimental Procedures. (C) Interphase egg extracts were treated as in (A) and chromatin fractions were isolated and subjected to immunoblotting using the antibodies indicated. (D) Mock- or Uhrf1-depleted interphase egg extracts were treated with Ub-VS and ubiquitin as in (A). ∆ Denotes immunodepletion of the protein indicated. The resultant chromatin fractions were subjected to immunoblotting using the antibodies indicated. Source data are available online for this figure.
	Figure 2. Usp7 forms a stable complex with Dnmt1 in Xenopus egg extracts. (A) Dnmt1 was immunoprecipitated from interphase HSS using specific antibodies. The associated polypeptides were identified by mass spectrometry. The resultant immunoprecipitates were subjected to SDS-PAGE and stained with a SyproRuby; the peptides corresponding to Dnmt1 and Usp7 are indicated. The numbers of unique spectra identified for each protein are shown in the table. (B) Immunoprecipitates using anti-Dnmt1 (lane 3), anti-Usp7 (lane 4), anti-Uhrf1 (lane 5) antibodies, or control IgG (lane 2) as well as egg extracts (lane 1) were subjected to immunoblotting using the antibodies indicated. (C) Sequence alignment of the KG linker of Dnmt1 across different species. Red denotes residues mutated in a 4KA mutant used in this study. (D) FLAG-tagged wild-type or a 4KA mutant of recombinant Xenopus Dnmt1 was introduced into Xenopus egg extracts. Recombinant or endogenous Usp7 was immunoprecipitated using either anti-FLAG-M2 agarose beads or protein A agarose beads conjugated with anti-Usp7 antibodies, respectively. Immunoprecipitates were then subjected to immunoblotting using the antibodies indicated. Source data are available online for this figure.
	Figure 3. Usp7 binds to replicating chromatin in a Dnmt1-dependent manner. (A) CSF extracts (M-phase) were released into interphase by addition of CaCl2. Sperm nuclei were added to either CSF or interphase extracts. Aphidicolin (APD, 150 μM) was added to extracts 10 min before sperm addition. At the indicated times after incubation, chromatin fractions were isolated and subjected to immunoblotting using the antibodies indicated. (B) Sperm chromatin was added to mock- or xDnmt1-depleted interphase extracts. xDnmt1-depleted extracts were supplemented with either buffer alone (lanes 9–12), purified wild-type xDnmt1-FLAGx3 or its 4KA mutant-FLAGx3 (10 ng/μl final concentration, lanes 13–16 or 17–20, respectively). At the indicated time points, chromatin fractions as well as extracts were subjected to immunoblotting using the antibodies indicated. ∆ Denotes immunodepletion of the protein indicated. (C) Sperm chromatin was added to mock-depleted or xUhrf1-depleted interphase extracts. xUhrf1-depleted extracts were supplemented with either buffer alone (lanes 7–9) or purified hUhrf1 (30 ng/μl final concentration, lanes 10–12). At the indicated time points, chromatin fractions were isolated and subjected to immunoblotting using the antibodies indicated. Note that xUhrf1 antibodies recognized hUhrf1. ∆ Denotes immunodepletion of the protein indicated. (D) Either mock- or Dnmt1-depleted HSS were incubated with chromatin fractions from mock- (M) or Dnmt1- (D) depleted extracts. The resultant mixtures were immunoprecipitated with anti-histone H3 antibodies and subjected to immunoblotting using the antibodies indicated. ∆ Denotes immunodepletion of the protein indicated. Source data are available online for this figure.
	Figure 4. Usp7 depletion and inhibition result in accumulation of ubiquitylated histone H3 and enhancement of Dnmt1 chromatin loading. (A) Sperm chromatin was added to either mock-, Usp7-, or Usp7/Uhrf1-double depleted extracts for 90 min. Isolated chromatin fractions as well as extracts were subjected to immunoblotting using the antibodies indicated. ∆ Denotes immunodepletion of the protein indicated. (B) Sperm chromatin was added to either mock- or Usp7-depleted extracts for 150 min in the presence or absence of 0.1 mg/ml His6-ubiquitin. Chromatin fractions were isolated and subjected to immunoblotting using anti-histone H3 antibodies. ∆ Denotes immunodepletion of the protein indicated. (C,D) HeLa cells were infected with lentiviruses expressing tet-on shRNAs targeting Luciferase (Control), Dnmt1, Usp7, and Uhrf1, in the combinations indicated. Cells were then harvested after treatment with doxycycline (1 μg/ml) for 3 days (C) and whole cell extracts were subjected to immunoblotting using the antibodies indicated. Histones were acid-extracted from cells depleted of the indicated proteins and subjected to immunobloting using anti-histone H3 antibodies (D). xDnmt1-depleted chromatin was used as a positive control. (E) USP7 deubiquitylates histone H3 in vitro. Dnmt1/Usp7 complex deubiquitylates histone H3 either in the absence or presence of SAM. The indicated immunoprecipitates (IP) were incubated with MNase-digested chromatin containing unmodified (lane1) or ubiquitylated histone H3 in the presence or absence of S-adenosylmetionine (SAM). (F) Purified recombinant Usp7 was incubated with ubiquitylated histone H3 for 2 hrs. The reaction mixtures were analyzed by immunoblotting using the antibodies indicated. (G) Sperm chromatin was added to mock- or xUsp7-depleted egg extracts. At the time points indicated, chromatin fractions were isolated and subjected to immunoblotting using the antibodies indicated. ∆ Denotes immunodepletion of the protein indicated. Source data are available online for this figure.
	Figure 5. Usp7 is involved in recruitment of Dnmt1 to DNA replication sites in mammals. HeLa cells expressing shRNA-resistant wild-type Usp7(Wt) or its mutant (C223S) were infected with lentiviruses expressing tet-on shRNAs targeting Luciferase (Control) or Usp7 and treated with doxycycline (1 μg/ml) for 72 hrs. The resultant cells were subjected to immunostaining using anti-Dnmt1 (green) and anti-PCNA (red) antibodies.
	Figure 6. Usp7 plays a key role in efficient DNA methylation. (A) Sperm chromatin was added to either mock- or Usp7-depleted extracts in the presence of radiolabelled S-[methyl-3H]-adenosyl-L-methionine. The efficiency of DNA methylation was measured at the time points indicated. ∆ Denotes immunodepletion of the protein indicated. (B) Interphase egg extracts were treated with 100 μM P22077. The efficiency of DNA methylation was measured at the indicated time points as in (A).
	Figure 7. A schematic model showing the role of Usp7 in the maintenance of DNA methylation. During S-phase, Uhrf1 binds to hemi-methylated DNA and ubiquitylates histone H3. Ubiquitylation of histone H3 facilitates the recruitment of Dnmt1/Usp7 complex to DNA methylation sites. Usp7 then deubiquitylates ubiquitylated histone H3, promoting DNA methylation.

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