Fig 2: CHAMP1 overexpression is common in tumors with underlying HR deficiency and correlates with poor cancer patient prognosis(A and B) Kaplan-Meier curves depicting overall survival of patients from TCGA with CHAMP1 expression and wild-type BRCA1 (A), and mutated BRCA1 (B). This analysis combines tumors from these TCGA studies: BLCA (bladder), BRCA (breast), COAD/READ (colorectal), LUAD (lung), LUSC (lung squamous), OV (ovarian), and SKCM (skin). N represents number of patients.(C) CHAMP1 expression positively correlates with cyclin E expression. N represents number of cancer cell lines.(D) Breast cancer cells with high expression of CCNE1 are more dependent on CHAMP1 for survival. N represents number of cancer cell lines.(E) Schematic of PARPi-resistant RPE1p53-/-BRCA1-/- cells generation. RPE1p53-/-BRCA1-/- cells were treated with increasing concentrations of the PARPis niraparib/olaparib over 3 months, and then isolated by single-cell clones from the niraparib- and olaparib-resistant pools.(F) A 14-day clonogenic survival of RPE1p53-/-, RPE1p53-/-BRCA1-/-, and niraparib/olaparib-resistant RPE1p53-/-BRCA1-/- NA5 cell clone treated with various doses of olaparib after siControl or siCHAMP1 treatment. Error bars indicate SD, n = 3 independent experiments. ***p < 0.001. Statistical analysis was performed using two-way ANOVA.

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: DNA damage activates CHAMP1 binding to REV7(A) Representative images of CHAMP1 and REV7 foci 1 h after IR (5Gy) treatment in RPE1 cells. Colocalizations of CHAMP1 and REV7 are shown as indicated. DAPI was used to stain the nuclei. Scale bar, 5 µm.(B) Quantification of CHAMP1 foci shown in (A). Error bars indicate SEM, and p values were calculated using two-tailed Student’s t test. ****p < 0.0001.(C) Quantification of CHAMP1-REV7 colocalized foci shown in (A). Error bars indicate SD (n = 3), and p values were calculated using two-tailed Student’s t test. ***p < 0.001.(D) Quantification of CHAMP1-REV7 colocalized foci with or without IR (5Gy) treatment as indicated. Error bars indicate SD.(E) (Left) Western blot showing GFP-immunoprecipitation of GFP-tagged CHAMP1 in 293T cells after IR (5Gy) treatment as indicated and the co-immunoprecipitation of endogenous REV7. VC, vector control. (Right) Quantification of the relative CHAMP1-REV7 binding activity.(F) A cartoon showing that CHAMP1 interacts with the closed REV7, which is regulated by TRIP1-p31 complex and DNA damage.(G) Western blot showing GFP-immunoprecipitation of GFP-CHAMP1 in wild-type (WT), TRIP13-/-, and p31-/- U2OS cells, and the co-immunoprecipitation of endogenous REV7.(H) Western blot showing GFP-immunoprecipitation of GFP-CHAMP1 in U2OS-vector control (VC) and TRIP13 overexpressed U2OS cells, and the co-immunoprecipitation of endogenous TRIP13 and REV7. All of the immunoblots are representative of at least two independent experiments.

Fig 5: CHAMP1 increases HR activity by binding to REV7 and reducing the level of the REV7/SHLD3 complex(A) (Top) Schematic of CHAMP1 protein showing its various domains and REV7 binding region. (Bottom) Schematic of CHAMP1-full-length (FL) and W334A/K335A (2A) mutants. SBM, REV7 seatbelt-binding motif.(B) Structure of the REV7-CHAMP1 complex. REV7 is shown in cyan and blue (seatbelt domain), and the CHAMP1 fragment (residues 331–343) is shown in red.(C) Western blot showing GFP-immunoprecipitation of GFP-empty vector, GFP-CHAMP1 wild-type, and GFP-CHAMP1-2A mutant, and the co-immunoprecipitation of endogenous REV7 in 293T cells.(D) RPE1P53-/-CHAMP1-/- cells were transfected with GFP-empty vector, GFP-CHAMP1 wild-type, or GFP-CHAMP1-2A mutant. The GFP-positive cells were sorted for clonogenic assay. A 14-day clonogenic assay of indicated cell lines treated with various doses of olaparib; error bars indicate SD, n = 3 independent experiments, **p < 0.01. Statistical analysis was performed using two-way ANOVA.(E) (Top) Western blot showing GFP-immunoprecipitation of GFP-SHLD3 in 293T cells, treated with or without siCHAMP1, and the co-immunoprecipitation of endogenous CHAMP1 and REV7. (Bottom) Quantification of the relative SHLD3-REV7 binding activity from three independent immunoprecipitation western blots. Error bars indicate SD, n = 3. **p < 0.01.(F) Representative images of GFP-SHLD3 foci formation in GFP-SHLD3 stable expressed RPE1 cells treated with siRNA negative control or siCHAMP1, and 1 h after 5Gy IR treatment. Scale bar, 5 µm.(G) Quantification of RAD51 foci in (F). More than 60 cells were counted per treatment. ***p < 0.001. Statistical analysis was performed using two-tailed Student’s t test. Error bar means SEM.