Fig 2: ZMYM2 interacts with ZMYM3 and regulates its association with DNA damage. (A) Co-Immunoprecipitation (co-IP) analysis identifies an interaction between ZMYM2 and ZMYM3. Differentially SFB and GFP-tagged ZMYM2 and ZMYM3 were co-expressed in HEK293T cells and IPed using anti-GFP beads followed by western blotting analysis with the indicated antibodies. GFP alone expressing cells were analyzed as controls. (B) Interactions between ZMYM2 and ZMYM3 are not altered by IR treatment. Samples from untreated or IR-treated HEK293T cells were obtained and analyzed by co-IP as in (A) with either SFB alone or SFB-ZMYM2 expressing cells. Endogenous ZMYM3 was detected in co-IP samples with a specific antibody. (C, D) ZMYM2 promotes retention of ZMYM3 at damage sites. U2OS ZMYM2 and ZMYM3 KO cells expressing GFP-tagged ZMYM3 or ZMYM3 were damaged using a UV-C laser and analyzed over time post-damage by live-cell fluorescence confocal microscopy. Right panel: quantification of GFP protein signal at the damage site over time (0–90 min. Data represent mean ± S.E.M., n ≥ 6 cells analyzed per condition (ns; not significant, *P < 0.05, **P < 0.01, ****P < 0.0001 by multiple unpaired t-test). Dotted white lines mark cell nuclei.

Fig 3: ZMYM2-deficiency results in defective responses and survival to DNA damage and PARP inhibitors. (A, B) ZMYM2-deficient cells are sensitive to ionizing radiation (IR; top panel) and PARP inhibitors (PARPi; bottom panel). U2OS cells either treated with control or ZMYM2 siRNAs or depleted of ZMYM2 by CRISPR-Cas9 gene editing were challenged with IR or PARP inhibitors as indicated and analyzed by colony formation assays. Data represent mean ± S.E.M., n ≥ 3 (**P < 0.01, ***P < 0.001; multiple unpaired t-test for (A), *P < 0.05; two-tailed paired t-test for (B)). (C) Complementation analysis of ZMYM2 KO cells. ZMYM2 KO cells with empty vector or ZMYM2 WT were analyzed as in (B), BRCA1 siRNA transfected cells were used as positive control. Data represent mean ± S.E.M., n = 3, (*P < 0.05; two-tailed paired t-test). (D) ZMYM2 or ZMYM3 depletion in U2OS cells results in sensitivity to DNA crosslink agents and PARP inhibitor. ZMYM2 and ZMYM3 KO cells were treated with Cisplatin, Mitomycin C and Olaparib with the indicated dosages. Cell survival was measured by MTT assay. Data represent mean ± S.E.M., n ≥ 3 (*P < 0.05, **P < 0.002; two-tailed paired t-test). (E, F) ZMYM2-deficient cells exhibit defects in DNA damage signaling and checkpoint activation following IR-treatment. siCtrl and siZMYM2 U2OS cells were treated with IR and analyzed by western blotting with the indicated antibodies. (G) Parental WT and ZMYM2 KO U2OS cells were damaged with IR (5Gy, 6hr) followed by FACS analysis with anti-H3 pS10 and propidium iodide to identify mitotic cells. Data represent mean ± S.E.M., n = 3 (*P < 0.05, ***P < 0.001, one-way ANOVA test).

Fig 4: ZMYM2 functions in concert with ZMYM3 to promote HR repair. (A) Efficiency of HR repair by DR-GFP cell-based assay. U2OS DR-GFP cells were treated with either control or gene-specific siRNAs (ZMYM2 and/or ZMYM3). GFP + cells (HR-events) were identified by FACS analysis. HR efficiencies were normalized to siCtrl cells. Data are represented as mean ± S.E.M., n = 3, (ns; not significant, **P < 0.01, ****P < 0.0001 one-way ANOVA test). (B, C) IR-induced RAD51 foci are reduced in ZMYM2 KO U2OS cells. WT and ZMYM2 KO cells were treated with IR (10 Gy) and analyzed by IF 3 h post-treatment. Cells were stained with cyclin A to identify S and G2 cells. Quantification of RAD51 foci in cyclin A-positive cells in (C). Data represented as mean ± S.E.M. from >120 cells, n = 3 (****P < 0.0001, one-way ANOVA test). Scale bar = 20 μm.

Fig 5: ZMYM2-deficiency increases 53BP1 engagement with DNA breaks. (A) Loss of ZMYM2 or ZMYM3 increases 53BP1 foci following IR-treatment. Foci formation of 53BP1 and γH2AX was analyzed following IR treatment (10 Gy, 3h) by IF in parental U2OS and ZMYM2 and ZMYM3 KO cells. (B) Quantification of (A). Data represented as mean ± S.E.M. from > 300 cells, n = 3, Scale bar = 20 μm, (****P < 0.0001, one-way ANOVA test). (C) 53BP1 is hyper-accumulated at FokI-mediated DSBs. U2OS mCherry-LacI-FokI DSB reporter cells transfected with indicated siRNAs were treated with 4-OHT and Shield-1 for >4 h to induce FokI-mediated DSBs. 53BP1 foci number and size were analyzed. (D) Quantification of (C). Data represent mean ± S.E.M., n = 3, (ns; not significant, two-tailed unpaired t-test). (E) 53BP1 foci sizes (area) were analyzed using ImageJ. Data represent mean ± S.E.M., from >120 cells, n = 3 (*P < 0.05 two-tailed unpaired t-test). (F) Defective BRCA1 foci formation in ZMYM2 KO U2OS cells is rescued by depletion of 53BP1. BRCA1 foci were analyzed by IF following IR-treatment (10 Gy. 3h) in WT and ZMYM2 KO cells (two independent KO clones; Cl#1 and Cl#2) treated with control siRNAs or siRNAs targeting 53BP1. (G) Quantification of (F). Data represented as mean ± S.E.M., n = 3, Scale bar = 10 μm (ns; not significant, ****P < 0.0001, one-way ANOVA test). (H) Depletion of 53BP1 rescues the HR deficiency in ZMYM2-depleted cells. HR levels were obtained in DR-GFP U2OS cells transfected with siRNAs targeting the indicated genes. Two-independent siRNAs targeting 53BP1 were analyzed. Data represent mean ± S.E.M., n = 3, (ns; not significant, ****P < 0.0001, one-way ANOVA test).