Fig 2: ASD-related de novo mutations in POGZ impair the nuclear localization of the POGZ protein.a De novo mutations in POGZ identified in patients with NDDs (blue, ASD; green, ID; red, both ASD and ID; purple, unclassified NDDs). b De novo mutations in POGZ identified in unaffected controls. c The number of missense, nonsense, and frameshift mutations identified in patients with NDDs and unaffected controls. d Distribution of POGZ mutations identified in patients with NDDs and unaffected controls (blue, missense mutations; red, nonsense mutations; green, frameshift mutations). e, g ASD-related nonsense e or missense g mutations disrupted the nuclear localization of Myc-tagged overexpressed mouse (m) POGZ in Neuro2a cells. C cytosolic fraction; N nuclear fraction; WT wild-type. Note that Q1038R mutation in mouse POGZ corresponds to Q1042R mutation in human POGZ. f, h, j Quantification of POGZ in cytosolic and nuclear fractions (each n = 4). i Control mutations identified in unaffected healthy controls did not affect the nuclear localization of Myc-tagged overexpressed mouse POGZ in Neuro2a cells. C cytosolic fraction; N nuclear fraction; WT wild-type. Note that the amino acid numbers are based on the human protein (NP_055915.2) a, b and mouse protein (NP_766271.2) e, g, i. f, h, j One-way ANOVA with Bonferroni–Dunn post hoc tests, f F3, 12 = 23.12; h F2, 9 = 67.45; j F3, 12 = 0.491. ***P < 0.001. Data are presented as the mean ± s.e.m. The source data underlying figures e, g and i are provided as a Source Data file.

Fig 3: POGZ is epistatic with CHAMP1 in the regulation of homologous recombination(A) Graph showing the percentage of GFP-positive cells after DR-GFP analysis. U2OS cells were infected with I-SceI adenovirus and knocked down for BRCA1 or POGZ using siRNA. N = 3 biologically independent experiments. Error bars indicate SD, and p values were calculated using two-tailed Student’s t test, ****p < 0.0001.(B) Quantification of resected ssDNA measured by SMART assay in U2OS cells treated by siControl or siRNAs targeting CHAMP1 for 48 h. Approximately 50 fibers were counted per experiment. Error bars indicate SD, and p values were calculated using Student’s t test, ***p < 0.001.(C) Quantification of >10 RAD51 foci in wild-type and two CHAMP1 knockout U2OS cell lines 6 h after 5Gy IR treatment. Error bars indicate SD, n = 3 biologically independent experiments. ****p < 0.0001. Statistical analysis was performed using two-tailed Student’s t test.(D) Western blot showing GFP-immunoprecipitation of GFP-REV7 in HEK293T cells treated with or without irradiation (5Gy), and the co-immunoprecipitation of endogenous CHAMP1 and POGZ.(E) Western blot showing GFP-immunoprecipitation of GFP-CHAMP1 in HEK293T cells treated with or without siPOGZ, and the co-immunoprecipitation of endogenous CHAMP1 and POGZ.(F) 3-day cytotoxicity analysis of RPE1p53−/− and RPE1p53−/−CHAMP1−/− cells treated with various doses of olaparib after 48 h siControl or siPOGZ treatment. Cell viability was detected by CellTiter-Glo (Promega). Error bars indicate SD, n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA.(G) 3-day cytotoxicity analysis of RPE1p53−/− and RPE1p53−/−POGZ−/− cells treated with various doses of olaparib after 48 h siControl or siCHAMP1 treatment. Cell viability was detected by CellTiter-Glo (Promega). Error bars indicate SD, n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA. All of the immunoblots are representative of at least two independent experiments.

Fig 4: POGZ regulates the neuronal differentiation of mouse cortical neural stem cells.a Slight, non-significant migration defects caused by shRNA-mediated knockdown of Pogz in E16.5 mouse cortices electroporated at E14.5. CP cortical plate; IZ intermediate zone; SVZ subventricular zone; VZ ventricular zone; E embryonic day. Scale bars, 50 µm. b Quantification of GFP+ cells in each layer (each n = 4). CP cortical plate; IZ intermediate zone; SVZ subventricular zone; VZ ventricular zone. c Increased number of PAX6+ NSCs caused by Pogz knockdown in E16.5 mouse cortices electroporated at E14.5. d Quantification of PAX6+ cells (each n = 4). e Decreased number of TBR2+ differentiated IPs caused by Pogz knockdown in E16.5 mouse cortices electroporated at E14.5. f Quantification of TBR2+ cells (each n = 4). g Decreased number of SATB2+ differentiated neurons caused by Pogz knockdown in E16.5 mouse cortices electroporated at E14.5. h Quantification of SATB2+ cells (each n = 4). c, e, g Right panels, magnifications of the areas outlined with orange boxes. Arrowheads indicate co-labeled cells. White scale bars, 50 µm; orange scale bars, 10 µm. b Two-way repeated-measures ANOVA with Bonferroni–Dunn post hoc tests, F4, 27 = 1.861. d, f, h One-way ANOVA with Bonferroni–Dunn post hoc tests; d F2, 9 = 18.37; f F2, 9 = 5.710; h F2, 9 = 11.91. *P < 0.05, **P < 0.01. Data are presented as the mean ± s.e.m.

Fig 5: ASD-related POGZ mutations impair neuronal development.a Forced expression of the ASD-related POGZ mutants failed to rescue the Pogz-knockdown-mediated migration defect in the developing mouse cortex. E embryonic day. Scale bars, 50 µm. b, d Quantification of GFP+ cells in each layer (each n=4). CP cortical plate; IZ intermediate zone; SVZ subventricular zone. c Forced expression of the control POGZ mutants rescued the Pogz-knockdown-mediated migration defect. Scale bars, 50 µm. e Low number of MAP2+ differentiated neurons in the patient-derived NSCs harboring the Q1042R POGZ mutation. Scale bars, 100 µm. f Quantification of MAP2+ neurons (each n = 9). g Increased BrdU incorporation in the patient-derived NSCs (each n = 9). h Impaired radial migration of the patient-derived neurons. Scale bars, 50 µm. i Quantification of the migration distance (control, n = 25; patient, n = 20). b, d Two-way repeated-measures ANOVA with Bonferroni–Dunn post hoc tests; b F12, 63 = 54.55; d F10, 54 = 9.426. f, g, i Student’s t-test. **P < 0.01, ***P < 0.001, ###P < 0.001 (vs. shPOGZ1 + WT mPOGZ in b). Data are presented as the mean ± s.e.m.