Fig 2: USP13 is required for release of cohesin from chromatin in mitosis.A, 293T parental cells and USP13 KO derivatives #1 and #2 were treated for 20 h with DMSO vehicle alone (DM); hydroxyurea (HU) to arrest cells in the S phase; RO-3306 (RO) to arrest cells in G2; and nocodazole (NOC) to arrest cells in mitosis. Chromatin lysates and whole cell lysates were prepared, and Western blot was performed with the antibodies to cohesin subunits SMC3, SMC1A, STAG2, RAD21, PDS5A, WAPL, NIPBL, and Sororin, as well as the control antibodies indicated. B, same as for (A) except Western blots were performed with antibodies to Ac-SMC3 and SMC3, as well as the control antibodies indicated. Quantification of Western blot bands is shown in Fig. S3.

Fig 3: GFP–NIPBL is recruited to DNA damage independently of MAU2, and HP1 mediates the recruitment of NIPBL only to DSBs. (A) The expression of GFP fusions for either wild-type NIPBL isoform A (GFP–NIPBLA) or NIPBL isoform A featuring the G15R mutation (GFP–NIPBLG15R) were induced with doxycycline for 48 h. NIPBL was immunoprecipitated (I.P.) on GFP–Sepharose beads and then probed for associated native MAU2 protein by western blotting. (B) The FokI cells were transiently transfected with GFP–NIPBLG15R and fixed 5 h after induction. Cells were imaged via confocal microscopy. (C) Following 48 h of induced GFP–NIPBLG15R expression, cells were laser microirradiated, and labeled as in Fig. 1D. (D) A HEK293 stable cell line was generated for GFP–NIPBL incorporating the PxVxL to PxAxA double mutation within the HP1-binding motif (GFP–NIPBLPxAxA). (E) A representative confocal microscopy image of the FokI U2OS cell line transiently transfected with the GFP–NIPBLPxAxA plasmid. (F) The GFP–NIPBLPxAxA stable cell line was induced for 48 h, and then laser microirradiated, fixed after 30 min and immunolabeled for ?H2AX. Arrows in B and E highlight the LacO array. Scale bars: 3 µm.

Fig 4: NIPBL accumulates at laser-induced DNA damage throughout the cell cycle. (A) FACS analysis of the cell cycle distribution and GFP expression of the NIPBLN and NIPBLC stable cell lines in the absence or presence of doxycycline for 48 h. The fraction of cells residing in each cell cycle phase based on propidium iodide incorporation is indicated. (B) The NIPBLN and NIPBLC stable cell-lines were induced with doxycycline for 48 h and then laser microirradiated as standard. Cells were immunolabeled for both ?H2AX (Cy3), and cyclin B1 (Alexa Fluor 647). Cells positive for cyclin B1 were considered to be in either S or G2 phase. Cells lacking cyclin B1 commonly showed recruitment of either NIPBLN or NIPBLC to DNA damage tracks. Note the ?H2AX signal from the red channel bleeds through into the weaker signal of cyclin B1 in the far-red channel. (C) Quantification of the number of cells displaying GFP-NIPBL recruitment to ?H2AX lines in each group irrespective of cell cycle phase. Results are mean±s.d., n=2. Scale bars: 3 µm.

Fig 5: Genome-wide distribution of SMC1A in CdLS-derived cells.a Heatmap showing the signal around all SMC1A peaks in control and CdLS patient 3-derived cells and its colocalization with CTCF and RAD21. b (left) Venn diagrams showing the overlap of genomic positions of SMC1A (dark blue) with CTCF (light red) and RAD21 (green) (up), SMC1A (dark blue) with NIPBL (light red) (bottom) or NIPBL (dark blue) with CTCF (light blue) and RAD21 (green) (right). b (right) Bam graph showing the mean signal of NIPBL (Control, black; Patient, red), SMC1A (Control, blue; Patient, orange), CTCF (green), and MNase (gray) around NIPBL peaks in control and CdLS patient 3-derived cells. c Heatmap comparing the signal in control and CdLS patient 3-derived cells around differential SMC1A peaks. Signals of SMC1A, NIPBL, CTCF, DNase, and MNase genomic distribution are depicted. The heatmap is divided into two categories, SMC1A peaks gained (pink) or lost (light blue) in the patient compared with the control. d Bam graphs showing mean signal of the indicated proteins around gained (left) and lost (right) SMC1A genomic positions. NIPBL (Control, black; Patient, red), SMC1A (Control, blue; Patient, orange), CTCF (green), DNase (brown), and MNase (gray). e Pie charts showing the overlap of gained (left) and lost (right) SMC1A peaks with NIPBL and CpG islands (CGI). NIPBL, green; NIPBL&CGI, red; no NIPBL, blue; no NIPBL&CGI, violet. f Plots of percentages of SMC1A gained and lost peaks overlapping with CpG islands (CGI, black), promoters (blue), enhancers (pink), and CTCF (green).