Fig 2: Validation of the differential SMC1A peaks by ChIP-qPCR experiments.a Cells from a control (white) and two CdLS patients (P2, blue; P3 purple) were used in ChIP-qPCR experiments to validate the SMC1A ChIP-Seq data using primers at the indicated genes. An intergenic region was used as a negative control (Neg -). Means and SEMs were calculated from biological triplicates (left). Two-sided unpaired student’s t-test (**p < 0.01; *p < 0.05). Snapshots of SMC1A and NIPBL peak distributions in control (C) and CdLS patient cells (P3–4) at three validated genome regions (right). The red arrows indicate the validated SMC1A peaks. b Gene ontology (GO) analysis reveals enriched biological processes in the SMC1A differential genomic positions in CdLS-derived fibroblasts. c ChIP-qPCR experiments were performed to validate the SMC1A ChIP-Seq data at three regions of HOXB, two of HOXC, two of HOXD, and four regions of the PCDHB clusters using cells from a control (white) and a CdLS patient (blue). Means and SEMs were calculated from biological duplicates (HOX) and triplicates (PCDHB). Two-sided unpaired student’s t-test (****p < 0.0001; *p < 0.05) (left). Snapshots of SMC1A peak distributions in control (C) and CdLS patient cells (P3–4) for the HOXB cluster (right). Red arrows indicate three validated SMC1A peaks within the HOXB cluster.

Fig 3: Chromatin-bound cohesin is less stable in CdLS-derived cells.a Representatives images of an iFRAP experiment of control (C) and two CdLS patients (P2–3) cells before (0 min) and 5, 30, and 60 min after photobleaching (green channel). The entire cell, except for a small nuclear region, was photobleached (yellow line) and the fluorescence of EGFP-RAD21 redistribution in the bleached and unbleached regions was followed by time-lapse microscopy. b Fluorescence recovery after bleaching was quantified as the difference between bleached and unbleached nuclear regions over 1 h in one control (C, blue) and two CdLS patients (P2, red; P3, green) and normalized to the first post-bleach frame after 2 min. Means and SEMs are shown. Control, n = 10; P2, n = 8; P3, n = 7 cells examined over more than 3 biologically independent experiments. Two-sided unpaired student’s t-test. c The drop in mean fluorescence intensity in the unbleached region at the first post-bleach frame was used to calculate the chromatin-bound fraction. Same cells than in (b) were used. Means and SEMs are shown. Two-sided unpaired student’s t-test. d Chromatin fractions were prepared from cells derived from control (C) and CdLS patient (P) derived cells and washed with 0.25 and 0.5 M of NaCl containing buffer for 30 min. The amount of RAD21 bound to the chromatin was analyzed by immunoblotting. A representative experiment is shown (left panel), in which histone3 (H3) was used as a loading control. Plots of RAD21 levels in controls (white) and 3 biologically independent samples (CdLS patients P1, P2, and P3, blue) are depicted (right panel). Means and SEMs are shown. Two-sided unpaired student’s t-test.

Fig 4: Genes involved in development and differentiation are deregulated in CdLS patient-derived cells.a Volcano plot representation of gene expression changes between control and CdLS-derived fibroblasts. Significantly downregulated and upregulated genes are indicated in red. Green, blue, and gray show non-significant differential genes. b Gene ontology (GO) enrichment analyses reveal biological processes that are downregulated and upregulated in CdLS-derived fibroblasts (blue bars). c Representation of the log-fold changes in expression of genes related to developmental processes (black bars). Genes involved in embryonic system development and differentiation are marked in red, those involved in nervous system development are marked in blue. Two-sided unpaired student’s t-test (**p < 0.01; *p < 0.05). d, e The expression levels of some nervous system development genes (d) and embryonic development genes (e) were analyzed in a control (C, white) and three CdLS patient-derived fibroblasts (P1, blue; P2, purple; P3, green) by reverse transcription-qPCR. The graphs show the amount of transcript of each gene relative to that in the control. Means and SEMs were calculated from biological triplicates. Two-sided unpaired student’s t-test (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05).

Fig 5: Cohesin Establishment and Loading Factors Are Important for Transcriptional Repression at DNA DSBs in Both G1 and G2 Phase Cells(A, D, and F) Quantification of transcription in asynchronous U2OS reporter cells with or without induction of the FokI endonuclease (DSB) treated with siRNA targeting Sororin, NIPBL, SA2, or BRG1 (A), Esco2 (D), BRG1 or WAPL (F), and/or with 10 µM ATM inhibitor (NTC, non-targeting control). 150 cells were analyzed per condition, per repeat. Data are presented as mean ± SD; n = 3 (A), n = 4 (D), n = 4 (F) biological repeats.(B, E, and G) Western blot analysis of whole-cell extracts prepared from cells treated with siRNA targeting NIPBL (B), Esco2 (E), or BRG1 or WAPL (G). NTC, non-targeting control. a-Tubulin was used as a loading control.(C) qRT-PCR analysis of Sororin mRNA levels following siNTC or siSororin treatment to provide an indication of depletion efficiency.(H) Quantification of transcription in cyclin-D1-positive (G1 phase) U2OS reporter cells treated with the indicated siRNAs. Data are presented as the mean ± SD; n = 3 biological repeats.(I) Quantification of transcription in CENPF-positive (G2 phase) U2OS reporter cells treated with the indicated siRNA with or without induction of the FokI endonuclease (DSB). More than 110 cells were analyzed per condition, per repeat. Data are presented as mean ± SD, n = 3 biological repeats.*p < 0.05, **p < 0.01 using paired Student’s t test. See also Figure S3.