Fig 2: USP7 is recruited to replication foci in the absence of DNMT1. Replication foci where identified by staining for the replication factor PCNA. Colocalization of USP7 and PCNA is clearly apparent in both wild type (top three rows) and Dnmt1-null (bottom three rows) ES cell nuclei

Fig 3: Ablation of USP7 does not affect steady-state level of DNMT1 or global DNA methylation. a MEFs that lack USP7 after Cre-mediated excision of a Floxed allele of Usp7 show normal levels of DNMT1 (lane 2) but no detectable USP7 protein. Biological replicates are shown as Experiments 1 and 2. b Normal expression of DNMT1 and UHRF1 in human H1299 lung carcinoma cells containing an inducible shRNA against USP7 mRNA. Ablation of USP7 has no detectable effect on expression of other proteins. c Normal steady-state expression of USP7 in the absence of DNMT1 in ES cells. d Normal DNA methylation in MEFs that lack USP7. McrBC cleaves methylated DNA; HpaII cleaves unmethylated DNA at CCGG sites; MspI cleaves CCGG sites whether methylated or unmethylated. DNA from wild-type and Usp7−/− MEFs show similar patterns of resistance to both McrBC and HpaII. e Removal of USP7 by an inducible shRNA against USP7 from H1299 human lung carcinoma cells does not affect DNA methylation levels as assessed by resistance of DNA to HpaII. f LUMA analysis shows that DNA methylation is not measurably affected by removal of USP7. n = 2 (biological replicates). Error bars show standard deviations, center value is mean, and p values were calculated using the two-tailed t test

Fig 4: USP7 deletion in glutamatergic neurons causes disease-relevant behavioral deficits in mice(A and B) Immunofluorescence of USP7 in the cerebral cortex on embryonic day 14 (E14; A) and post-natal day 42 (P42; B). Dashed lines demarcate the cortical plate (c.p.). Scale bars, 100 μm.(C) Virtual optomotor system (VOS) test: spatial frequency threshold. **p < 0.01 by Bonferroni’s multiple-comparisons test.(D) Motor strength test: time to grip onto the inverted screen. *p < 0.05 by Dunn’s multiple-comparisons test.(E and F) CatWalk: relative mean intensity distributed onto front- and hindpaws (E) and limb swing speed (F). **p < 0.01, ***p < 0.001 by Tukey’s multiple-comparisons test. See also Video S2.(G–I) Fear conditioning: percentage of freezing time in tone/shock pairing (G), contextual conditioning (H), and auditory cued conditioning (I). *p < 0.05, **p < 0.01 by Bonferroni’s multiple-comparisons test (Usp7 WT vs. cKO).(J) Representative biting injury on mice co-caged with Usp7 cKO mice. See also Video S3.(K) Percentage of tail biters among Usp7 cKO mice from 3 to 12 weeks old.(L) Schematic of behavioral phenotypes in Usp7 cKO mice that are relevant to HAFOUS.Data are presented as mean ± SEM. See also Figure S1.

Fig 5: USP7 targets and deubiquitinates Ppil4 to promote dendritic spinogenesis(A) Representative GFP and PSD95 micrographs of dendritic spines (arrowheads) of DIV18 primary cortical neurons with knockdown of Ppil4 or luciferase (Luci). Micrographs of dendritic spines with knockdown of other candidate substrates are shown in Figure S8. Scale bar, 5 μm.(B–G) Density of all (B–D) and PSD95+ (E–G) spines on apical, basal, and all dendrites of cortical neurons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Dunnett’s multiple-comparisons test (compared to Luci).(H) Streptavidin pull-down of HEK293T lysate with overexpression of streptavidin-binding peptide-FLAG (SFB)-tagged USP7 followed by immunoblot analyses.(I) IP of endogenous Ppil4 in HEK293T lysates followed by immunoblot analyses.(J) Reciprocal IP of endogenous USP7 and Ppil4 in mouse brain at age P4, followed by immunoblot analyses. Input lysate with long exposure for clear visualization is shown on the left.(K) TUBE pull-down of MG132-treated neuronal lysate with USP7 loss driven by lentiviral Cre followed by immunoblotting of Ppil4. Densitometric quantification is shown at the bottom. IB, immunoblot; a.u., arbitrary unit.(L and M) In vitro deubiquitination using FLAG-immunoprecipitated Ppil4 from MG132-treated HEK293T cells (L) and densitometric quantification (M) of high-molecular-weight Ppil4 over all Ppil4 signals with 3 biological replicates. **p < 0.01 by 2-tailed unpaired t test.(N and O) Immunoblot and densitometric quantification (n = 5 biological replicates) of endogenous Ppil4 in HEK293T cells expressing patient variants of USP7. USP7 C223A is catalytically dead as a positive control. *p < 0.05, **p < 0.01 by Dunnett’s multiple-comparisons test after repeated-measurement one-way ANOVA.(P) Schematic of the USP7-Ppil4 signaling pathway regulating dendritic spines.Data are presented as mean ± SEM. See also Figure S7.