Volume 61, Issue 1, Pages 15-26 (January 2016) The Cohesin Complex Prevents the End Joining of Distant DNA Double-Strand Ends  Camille Gelot, Josée Guirouilh-Barbat, Tangui Le Guen, Elodie Dardillac, Catherine Chailleux, Yvan Canitrot, Bernard S. Lopez  Molecular Cell  Volume 61, Issue 1, Pages 15-26 (January 2016) DOI: 10.1016/j.molcel.2015.11.002 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 61, 15-26DOI: (10.1016/j.molcel.2015.11.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Substrates Used (A) Intrachromosomal EJ substrates. The proximal GFP-34 bp (in both the GCS5 and GCS17 cell lines) and distal GFP-3,200 bp (in the GCSH14 cell line) substrates allow GFP expression after the EJ of I-SceI-induced DNA breaks separated by 34 bp or 3,200 bp, respectively. The GFP-34 bp substrate has been described previously under the name sGEJ (Xie et al., 2009). All cell lines also contained the CD4-3200 bp integrated substrate, which has been described previously under the name pCOH-CD4 (Guirouilh-Barbat et al., 2004) and monitors the joining of distant DSEs 3,200 bp apart (see details in Figure S1). (B) Monitoring of EJ (expression of GFP or CD4) via fluorescent microscopy. The cell line used here was GSC5. (C) Monitoring of EJ via flow cytometry through the expression of CD4 or GFP upon I-SceI expression (right). The example shown comes from the GSC17 cell line, which contains both the GFP-34 bp and CD4-3200 bp substrates. Molecular Cell 2016 61, 15-26DOI: (10.1016/j.molcel.2015.11.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Depletion of the Cohesin Complex Specifically Increases the Efficiency of the EJ of Distal Extremities (A) Silencing of RAD21 stimulates the EJ of distant ends to the level of close ends. Protein expression is shown. (B) RAD21 or NIPBL silencing specifically stimulates distal EJ. (Top) Protein expression is shown. (Bottom) The frequency of EJ in depleted cells was normalized to the frequency in cells exposed to the control siRNA (Student’s t test; see Figure S3). (C) EJ of distant DSEs (CD4-3,200 bp) in U2OS cells. (Left) The frequency of EJ (CD4+ cells) was normalized to the frequency in cells exposed to the control siRNA. The values correspond to at least five independent experiments. (Right) Protein expression is shown. Molecular Cell 2016 61, 15-26DOI: (10.1016/j.molcel.2015.11.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Effect of the Cell Cycle on the Repression of EJ of Distal Ends (A) Inhibition of Sororin stimulates the EJ of distal ends. (Top) Expressions of RAD21 and Sororin (western blot) are shown. (Bottom) The frequency of EJ was normalized to the frequency in cells exposed to the control siRNA (Student’s t test; see Figure S3). The values represent the average of at least five independent experiments. SEM is represented. (B) Kinetics of the expression of I-SceI, GFP, and CD4. I-SceI expression peaked 24 hr after transfection, while GFP and CD4 expressions were delayed. (C) Blocking cells in late G1 abolished the repression of the EJ of distal ends. (Left) The experimental protocol was designed based on kinetics (B); one example of a cell-cycle distribution analysis performed 24 and 48 hr after I-SceI transfection and mimosine administration is given. AS, asynchronous cells. (Right) The frequency of close EJ versus distal EJ (in GCS17 cells) upon exposure to mimosine is shown. The results correspond to seven independent experiments. Molecular Cell 2016 61, 15-26DOI: (10.1016/j.molcel.2015.11.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 RAD21 Inhibits Both the C-NHEJ and A-EJ of Distant DSEs and Prevents Deletions and Insertions at Repair Junctions (A) Silencing of LIG4 (C-NHEJ) is shown. (B) Silencing of LIG1 and LIG3 (A-EJ). Normalized frequency of CD4+ cells is shown (Student’s t test: siCTRL (siControl) versus siRAD21, p = 0.0032; siCTRL versus siLigIV, p = 0.001). The values correspond to at least five independent experiments. SEM is represented. (C) Analysis of repair junctions upon RAD21 depletion. The frequency of the different events is shown. (D) Types of insertion events (>45 nt). The frequency and origin of the inserted sequences are shown. Molecular Cell 2016 61, 15-26DOI: (10.1016/j.molcel.2015.11.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 RAD21 Protects against Genome Rearrangements: DES (A) siRad21 versus siControl (siNT) in SV40-transformed cells is shown. (B) siRad21 versus siControl (siNT) in U20S cells is shown. (C) siSororin versus siControl (siNT) in U2OS cells. The circos plots show the location of the genome rearrangements present in cells treated with the siRAD21 or siSororin compared to cells exposed to siControl. The chromosome numbering is shown in the circle. The tables detail the rearrangements shown in the circos plot. Molecular Cell 2016 61, 15-26DOI: (10.1016/j.molcel.2015.11.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 RAD21 Protects against Chromosomal Rearrangements (A) Examples of chromatid breaks, chromosome breaks, and radial structures are shown. (B) Quantification of chromosome aberrations in (A) is shown (Fisher’s test: chromatid breaks under treatment with siCTRL versus siRAD21, p < 0.0001; radial structures under treatment with siCTRL versus siRAD21, p < 0.0001). (C) γ-H2AX foci upon exposure to APH (0.3 μM) in S phase. (Top) Examples of immunofluorescence microscopy are shown. (Bottom) Quantification of foci in BrdU-positive cells is shown. The values correspond to three independent experiments. (D) (Top) Scheme of the experiment. (Middle) Examples of interchromosomal fusions are shown. The centromeres (green) and telomeres (red) are labeled via PNA FISH. (Bottom) The frequency of chromosome fusions is shown. At least 1,000 chromosomes were counted per condition (Student’s t test: siCTRL + APH versus siRAD21 + APH, p = 0.0188). Molecular Cell 2016 61, 15-26DOI: (10.1016/j.molcel.2015.11.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 The Cohesin Complex Represses the Joining of Distal Ends (A) Model of the repression of distal DSEs. In the presence of the cohesin complex (left), sister chromatids are coupled, restricting their mobility. In the absence of the cohesin complex (right), DSEs are more mobile and distant DSEs can be joined (black dotted arrow). In the case of EJ substrates, the internal fragment (in red) might more easily be lost. (B) Multiple roles for the cohesin complex in maintaining genome stability following replication stress. Replication stress generates single-ended DSBs (one example in the upper panels). (1) The cohesin complex stabilizes the arrested replication fork, while another fork is migrating in the opposite direction, reaching the blocked fork. This process leads to the formation of two close ends, which can be ligated. Indeed, the cohesin complex does not impair the EJ of close ends. Moreover, the EJ of close ends is more conservative (J.G.-B., C.G., E.D., and B.S.L., unpublished data). (2) Sister chromatid exchange (SCE) allows the resumption of arrested replication; because the sister chromatids are identical, this process does not generate genetic instability. (3) In the absence of the cohesin complex, one single-ended DSB (red/gray) can interact with another (dark/light blue), which should be distant. Therefore, the joining of replication stress-induced DSEs generates chromosome fusions and genome rearrangements. The cohesin complex not only favors the two former situations, which allow the maintenance of genome stability, but also specifically restrains the joining of distant ends (without affecting the EJ of close DSEs) and, therefore, protects against the associated genetic instability. Molecular Cell 2016 61, 15-26DOI: (10.1016/j.molcel.2015.11.002) Copyright © 2016 Elsevier Inc. Terms and Conditions