Fig 2: The FDF motif of CENP-U directly binds to the composite interface between Scc1 and SA2.(A) Structural superposition of Scc1-SA2 (purple and cyan) bound to CENP-U (orange) and CTCF (purple-blue). F44, F46 of CENP-U and Y226, F228 of CTCF are shown in stick. (B) Fo-Fc omit electron-density Fourier map contoured at 2.0 σ. Residues of CENP-U are shown in orange, and SA2 and Scc1 are in green and blue, respectively. (C) HeLa cell lysates were subjected to pull-down with GST or GST-CENP-U (1–60) in the forms of WT, ADA, and FKF, followed by immunoblotting with antibodies for Scc1 and SA2, and CBB staining. (D) Lysates prepared from HEK-293T cells transiently expressing CENP-U-GFP in the forms of WT, ADA, and the indicated fragments were subjected to pull-down with GST or GST-Scc1-SA2, followed by immunoblotting with the antibody for GFP, and CBB staining. (E) U2OS-LacO cells transiently expressing the indicated proteins were stained with antibodies for the Flag-tag, Myc-tag, and DAPI. Example images are shown. (F) U2OS-LacO cells transiently expressing the indicated proteins were stained with the antibody for Plk1, and DAPI. Example images are shown. (G) Lysates prepared from HEK-293T cells transiently expressing SFB-CENP-Q and CENP-U-GFP (WT or ADA) were subjected to pull-down with GST or GST-Scc1-SA2, followed by immunoblotting with antibodies for GFP and the Flag-tag, and CBB staining. Data information: The white arrows point to the LacO repeats (E, F). Scale bars, 10 µm (E, F). Irrelevant lanes were removed (C, D, G).

Fig 3: A genome-wide CRISPR-Cas9 screen reveals STAG2 as a pro-RV host factor. a Schematic flowchart for RV CRISPR-based loss-of-function screening approach. b Bubble plot of host factors essential to RV infection. The top 20 genes were colored and grouped by function. Size of bubbles corresponds to the number of significant sgRNAs scored for each gene. c Wild-type (WT), STAG2−/−, and STAG2−/− HT-29 cells transduced with V5-tagged STAG2 were infected with RV (MOI = 1) and viral NSP5 mRNA level was measured at 24 h.p.i. by RT-qPCR (left panel). Cell lysates were analyzed by western blot with the indicated antibodies (right panel). For c, experiments were repeated at least three times in triplicates. Data are represented as mean ± SEM (***P ≤ 0.001)

Fig 4: Model for the role of the inner kinetochore in protecting sister-chromatid cohesion at mitotic centromeres.The CENP-OPQUR complex anchors a subset of cohesin at the inner kinetochore through direct binding of its CENP-U subunit to the composite interface between Scc1 and SA2, which antagonizes the competitive binding of Wapl to Scc1-SA2. Sgo1-bound cohesin is both distributed on chromosome arms and enriched at centromeres.

Fig 5: 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.