Fig 2: miR-638-mediated regulation of BRCA1 in breast cancer cell lines. (A) The top part shows the predicted location of the miR-638 binding site of the 3’-UTR of BRCA1. The bottom part indicates the binding site in the CDS of BRCA1 (BRCA1 CDS) as well as the mutant BRCA1 sequence (BRCA1 CDS mut) corresponding to miR-638 sequence, along with the pGL-3 vector. (B) Relative luciferase activity was measured in breast cell lines co-transfected with either 200 ng of miR-638 mimic (dark bars) or a scrambled control (white bar), 100 ng of either pGL3-BRCA1-CDS (BRCA1-wt) or pGL3-BRCA1-CDS mut (BRCA1-mu) by Lipofectamine 2000 (Life Technologies) for 48 h. The data was reported as mean ± SE for three independent experiments. The luciferase activity was significantly decreased in MDA-MB-231 and Hs578T cells when co-transfected with miR-638 mimic and BRCA1-wt (*P <0.05), while either increased in MCF-7 or not changed in T47D and MCF-10A cells. (C) The expression of BRCA1 protein in breast cancer cells when transfected with miR-638 mimic (OV) compared to the mock (Mock). BRCA1 expression was significantly downregulated by miR-638 in TNBC cells, MDA-MB-231 and Hs578T compared to non-TNBC cells, MCF-7 and T47D. The experiments were repeated three times. Band intensities were quantified using the ImageJ software, and the relative expression level was shown in a bar graph. BRCA1, breast cancer susceptibility gene 1; CDS, coding sequence; TNBC, triple-negative breast cancer; UTR, untranslated region.

Fig 3: Knockout of STAG2 decreases homologous recombination repair. A) Left: Western blot of STAG2 and BRCA1 expression in the U2OS‐DR‐GFP cells after the treatment with si‐Control (NC), si‐BRCA1, and si‐STAG2. Right: The homologous recombination efficiency was measured using the U2OS‐DR‐GFP cell line. Cells were transfected with si‐NC, si‐STAG2, or si‐BRCA1, exposed to I‐Scel adenovirus or not (Scel‐free) for 6 h, and cultured with fresh medium. GFP‐positive cells were quantified after 72 h using FACS. B) Left: Western blot of STAG2 and 53BP1 expression in the U2OS‐EJ5‐GFP cells after the treatment with si‐NC, si‐53BP1, and si‐STAG2. Right: The non‐homologous end joining efficiency was measured using the U2OS‐EJ5‐GFP cell line. Cells were transfected with si‐NC, si‐53BP1, or si‐STAG2, exposed to I‐Scel adenovirus or not (Scel‐free) for 6 h, and cultured with fresh medium. GFP‐positive cells were quantified after 72 h using FACS. C–E) Immunofluorescence images and quantification of RPA32, RAD51, and 53BP1 foci 4 h after treatment without (W/o) or with irradiation (W/IR, 5 Gy) in STAG2WT, STAG2KO, and STAG2KO+STAG2 U2OS cells. At least 50 nuclei were counted in each condition. Data are shown as mean ± SEM and were analyzed by one‐way ANOVA. ns, not significant, * p <0.05, ** p <0.01, ***p <0.001.

Fig 4: STAG2 influences the recruitment of BRCA1 and BARD1 to DNA damage site by regulating H4K20 methylation mediated by KMT5A. A) Western blot of the indicated proteins in subcellular fractions of STAG2WT and STAG2KO U2OS cells after treatment with 2 mM ATMi (KU‐60019) for 24 h. GAPDH was used as the control for the total cell lysates. Histone H3 was used as the control for the nucleus chromatin proteins. Lamin B1 was used as the control for the nucleus‐soluble proteins. Arrows indicate the target bands. B) Left: Immunofluorescence images of BRCA1 foci 4 h after treatment without (W/o) or with irradiation (W/IR, 5 Gy) in STAG2WT and STAG2KO U2OS cells. Right: Quantification analysis of BRCA1 foci 4 h after treatment without (W/o) or with irradiation (W/IR, 5 Gy) in STAG2WT and STAG2KO U2OS cells, related to left panel. At least 50 nuclei were randomly counted in each group. C) Western blot of the indicated proteins from STAG2WT and STAG2KO U2OS cell lines. D) Western blot showing FLAG‐immunoprecipitation of FLAG‐BARD1 in U2OS cells treated with siRNA negative control and si‐STAG2. E) CUT & Tag‐seq for FLAG or FLAG‐STAG2 at the KMT5A gene locus (indicated by the black arrow) in U2OS cells. The bed files showed the detected signal of vector or FLAG‐STAG2 peaks. F) A schematic diagram of the luciferase reporter plasmids constructed with three different fragments of the KMT5A promoter. STAG2‐Peak indicates the specific sequence of STAG2 binding to KMT5A which is obtained by CUT‐Tag sequencing. G) Relative values of luciferase (LUC) activity measured after transfection of different reporter plasmids in wild‐type (STAG2WT) or STAG2 knockout (STAG2KO) U2OS cells (Relative to STAG2WT each group). H) Relative values of luciferase (LUC) activity measured after transfection of different reporter plasmids in U2OS cells followed by si‐NC or si‐STAG2 (Relative to si‐NC each group). I) A cartoon showing that BRCA1‐BARD1 complex and 53BP1 interact with different methylated H4 histones. J) Western blot of the indicated proteins from STAG2WT and STAG2KO U2OS cells treated with siRNA negative control and si‐KMT5A. K) Left: Immunofluorescence images and quantification of BRCA1 foci 4 h after treatment with irradiation (W/IR, 5 Gy) in STAG2WT and STAG2KO U2OS cells treated with siRNA negative control and si‐KMT5A. Right: Quantification of BRCA1 foci 4 h after treatment with irradiation (W/IR, 5 Gy) in STAG2WT and STAG2KO U2OS cells with siRNA negative control and si‐KMT5A, related to the left panel. At least 50 nuclei were randomly counted in each group. L) Quantification of HR efficiency using direct repeat U2OS DR‐GFP reporter assay. Cells were treated with si‐NC, si‐STAG2, si‐BRCA1, si‐KMT5A, si‐BRCA1+si‐STAG2, si‐BRCA1+si‐KMT5A, and si‐STAG2+si‐KMT5A. M) Left: Representative images of colony formation by STAG2WT or STAG2KO U2OS cells treated with si‐NC and si‐KMT5A, and treated with DMSO or ATMi (KU‐60019, 1 µM). Right: Clonogenic survival assays of STAG2WT and STAG2KO U2OS cells treated with siRNA negative control and si‐KMT5A and treated with DMSO or ATMi (KU‐60019). Data are shown as mean ± SEM from three independent experiments and were analyzed using the one‐way ANOVA test (B, G, H, K, and L), and two‐way ANOVA (M). ns, not significant, *p<0.05, **p<0.01, ***p < 0.001.