Sororin locks the DNA-exit gate of cohesin to preserve sister-chromatid cohesion

Domain: biology
Published: 2026-03-10
Editorial outcome: major_revision

Abstract
The cohesin complex mediates sister-chromatid cohesion by topologically entrapping DNA within an SMC1-SMC3-RAD21 ring, yet how Sororin preserves cohesion beyond its known role of antagonizing Pds5 binding to the release factor Wapl has remained unclear. Here, we show that the extreme C-terminal region (CTR) of Sororin functions as a direct structural lock for cohesin’s DNA-exit gate by engaging the RAD21-SMC3 interface. Centromere-tethered Sororin-CTR fully restores cohesion after Sororin depletion, whereas constitutive chromatin tethering prevents cohesin removal, recapitulating Wapl-loss phenotypes, including impaired mitotic chromosome condensation, decatenation and segregation. Through biochemical reconstitution, AlphaFold3-guided modeling, and targeted mutagenesis, we define conserved hydrophobic and electrostatic contacts between Sororin-CTR and the RAD21-SMC3 gate, the disruption of which abolishes cohesion in a Wapl-dependent manner. Furthermore, mitotic phosphorylation of Sororin selectively disrupts Pds5 binding while leaving gate engagement intact, providing a regulated molecular switch for cohesin release. Together, these findings redefine Sororin as a dual-function regulator that both antagonizes Wapl-Pds5 and directly locks the RAD21-SMC3 exit gate to stabilize sister-chromatid cohesion while permitting its timely dissolution.

Introduction
Accurate chromosome segregation is fundamental to genomic stability and cellular viability1. This process relies on the cohesin complex, a conserved ring-shaped molecular machine that topologically entraps DNA to perform two essential functions: mediating sister-chromatid cohesion in trans and organizing chromatin architecture through DNA loop extrusion in cis2. Cohesive cohesin maintains the physical connection between sister chromatids from S-phase until anaphase to ensure their faithful separation3–6, whereas extrusive cohesin shapes the three-dimensional genome organization7–12. Given these central roles, dysregulation of cohesin contributes to developmental disorders and tumorigenesis, underscoring the need for a detailed mechanistic understanding of cohesin regulation13–17.

Structurally, the cohesin ring is formed by two SMC proteins, SMC1 and SMC3, bridged by the kleisin subunit RAD21 (also called Scc1/Mcd1); RAD21 further associates with Pds5 and SA1/SA2 to recruit regulatory factors (Fig. 1A)18. SMC1 and SMC3 dimerize at their hinge domains, while their ATPase head domains are connected by RAD21 at the opposite ends19. DNA is thought to enter the ring through the SMC1-SMC3 hinge and exit via a transiently opened interface between RAD21 and SMC320–23. This DNA-exit gate is regulated by the release factor Wapl, which, together with Pds5, promotes gate opening and cohesin turnover24–34.

During S-phase, acetylation of SMC3 by Esco1 and Esco2 promotes recruitment of Sororin, which stabilizes cohesion primarily by competing with Wapl for binding to Pds526,27,35–45. In early mitosis, phosphorylation of Sororin disrupts its association with Pds5, allowing Wapl to rebind and trigger cohesin release from chromosome arms24–27,46–50. In contrast, centromeric cohesion is protected by Sgo1, which associates with PP2A to counteract Sororin phosphorylation, until Separase-mediated cleavage of RAD21 initiates anaphase51–56.

Despite its essential role in cohesion maintenance27,36,44,57,58, Sororin’s mechanistic contribution beyond Wapl displacement remains unresolved26,27,37,49,54,59–63. A central unanswered question is whether Sororin stabilizes cohesion solely through indirect competition with Wapl for Pds5 binding or whether it also directly engages cohesin to reinforce closure of the DNA-exit gate58,64. Although the C-terminal region of Sororin has been implicated in cohesion and proposed to interact with the SA2 subunit65,66, the molecular basis and functional relevance of this interaction remain unclear.

Here, we show that the extreme C-terminal region (CTR) of Sororin directly binds the RAD21-SMC3 interface, functioning as a molecular lock for cohesin’s DNA-exit gate. Through integrated cell-biological, biochemical, and structural analyses, we demonstrate that this gate-locking activity is both necessary and sufficient for cohesion maintenance, operates independently of Pds5 competition, and is tightly regulated during the cell cycle. Our findings redefine Sororin as a dual-function regulator that both antagonizes Wapl binding to Pds5 and directly stabilizes cohesin ring closure to ensure faithful chromosome segregation.

Results
The extreme C-terminus of Sororin is necessary and sufficient to promote cohesion
To define the minimal Sororin domain required for sister-chromatid cohesion, we generated GFP-tagged Sororin truncation mutants (Fig. 1B), and assessed their ability to promote cohesion in mitotic HeLa cells arrested with the spindle destabilizer nocodazole (Fig. 1C–E). Chromosome spreads from control cells displayed the characteristic X-shaped morphology, reflecting normal chromosome-arm resolution. As expected, expression of full-length Sororin-GFP, but not H2B-GFP, markedly impaired arm resolution, consistent with Sororin’s established role. Strikingly, the C-terminal fragments Sororin (150-252)-GFP and Sororin (223–252)-GFP also strongly inhibited arm resolution, whereas the N-terminal fragments Sororin (1-149)-GFP and Sororin (1-222)-GFP did not.

Consistent with previous reports27,44,67, all Sororin-GFP variants localized to interphase nuclei but were largely excluded from mitotic chromosomes (Fig. 1E and Supplementary Fig. 1). To exclude the possibility that the inactivity of the N-terminal fragments resulted from defective chromatin association, we forcibly tethered them to chromatin by fusion to histone H2B (Fig. 1F–H). While H2B-Sororin-GFP potently blocked arm resolution, H2B-Sororin (1-149)-GFP and H2B-Sororin (1-222)-GFP remained ineffective.

Together, these results demonstrate that the extreme C-terminal region (residues 223–252), hereafter termed the CTR, is both necessary and sufficient for Sororin to promote sister-chromatid cohesion.

Centromere-tethered Sororin-CTR bypasses the requirement for Sororin but not Esco1/2 in cohesion protection
Given that Sororin accumulates at mitotic centromeres27,54,59,62, we asked whether targeted localization of the CTR to centromeres could functionally replace full-length Sororin. To this end, we generated HeLa cell lines stably expressing either Sororin-CTR-GFP fused to the centromere-targeting domain of CENP-B (CB-Sororin-CTR-GFP) or a CB-GFP control (Fig. 2A), then depleted endogenous Sororin by siRNA and examined its effect on mitotic index (Fig. 2B–D). As expected, Sororin knockdown in parental HeLa cells induced profound mitotic arrest, increasing the mitotic index from 4.5% to 39.8%, consistent with spindle-assembly-checkpoint activation following cohesion loss44. Comparable mitotic arrest was observed upon Sororin depletion in CB-GFP-expressing cells. In contrast, Sororin depletion had little effect on the mitotic index of CB-Sororin-CTR-GFP cells, indicating that centromeric Sororin-CTR effectively rescued this defect. Consistent with this, chromosome-spread analysis revealed severe cohesion loss in ~85% of Sororin-depleted parental or CB-GFP cells, compared with only 5.9% of CB-Sororin-CTR-GFP cells (Fig. 2E, F). Thus, centromeric tethering of Sororin-CTR is sufficient to maintain sister-chromatid cohesion in the absence of full-length Sororin.

We next examined whether centromeric Sororin-CTR could compensate for the loss of Sgo1, which promotes Sororin-dependent cohesion protection at mitotic centromeres54,62. Depletion of Sgo1 caused cohesion loss in 84% of CB-GFP cells, a phenotype comparable to Sororin knockdown (Supplementary Fig. 2A–C). This defect was partially but significantly rescued in CB-Sororin-CTR-GFP cells, where only 32.9% showed cohesion loss. This partial rescue suggests that while centromeric Sororin-CTR can mitigate Sgo1 depletion, it cannot fully substitute for Sgo1, which also protects cohesin through PP2A-mediated dephosphorylation54.

During S phase, Esco1 and Esco2 acetylate two conserved lysine residues (K105 and K106) in the SMC3 ATPase head, thereby promoting Sororin recruitment27,43,68–70. We therefore asked if these two acetyltransferases remain essential when Sororin-CTR is constitutively localized to centromeres. Co-depletion of Esco1/2 by siRNA induced similar levels of cohesion loss in CB-GFP and CB-Sororin-CTR-GFP cells (~23.3% and ~20.5%, respectively) (Fig. 2G–J). Thus, enforced centromeric localization of Sororin-CTR cannot bypass the requirement for Esco1/2.

In summary, constitutive centromeric targeting of Sororin-CTR is sufficient to replace full-length Sororin, but not Sgo1 or Esco1/2, in protecting sister-chromatid cohesion.

Chromatin-tethered Sororin-CTR impairs mitotic chromosome condensation and decatenation
The cohesin release factor Wapl regulates cohesin’s dynamic association with chromatin25,47. Depletion of Wapl prevents cohesin release from mitotic chromosomes, leading to defective sister-chromatid resolution, impaired chromosome condensation, and segregation errors48,50. Because Sororin antagonizes Wapl, we asked whether constitutive retention of Sororin-protected cohesin on chromatin could phenocopy Wapl depletion.

To address this, we generated HeLa cell lines stably expressing H2B-Sororin-CTR-GFP, which tethers the CTR constitutively to chromatin, or an H2B-GFP control (Fig. 3A). In these cells, we first examined the release of cohesin from chromosome arms in nocodazole-arrested mitotic cells. Following endogenous Sororin depletion, the cohesin subunit SMC1 remained largely chromosome-associated in H2B-Sororin-CTR-GFP cells, but was efficiently removed in H2B-GFP cells (Fig. 3B), confirming that chromatin-tethered Sororin-CTR blocks cohesin release. Consistently, expression of H2B-Sororin-CTR-GFP but not H2B-GFP potently inhibited chromosome arm resolution (Fig. 3C). Moreover, mitotic chromosomes in H2B-Sororin-CTR-GFP cells were significantly longer than those in control H2B-GFP cells (2.65 μm vs. 1.77 μm) (Fig. 3D), indicating defective chromosome condensation.

We next assessed the requirement for Sgo1 and Esco1/2 under these conditions. Sgo1 knockdown caused severe cohesion loss in H2B-GFP cells, but this defect was largely suppressed in H2B-Sororin-CTR-GFP cells (Supplementary Fig. 3A, B). This indicates that chromatin-tethered Sororin-CTR can largely bypass the requirement for Sgo1, supporting the model that Sgo1 protects cohesin both at centromeres and on chromosome arms71–75. In contrast, co-depletion of Esco1/2 resulted in comparable cohesion defects in both H2B-GFP and H2B-Sororin-CTR-GFP cells (Supplementary Fig. 3C–F), demonstrating that Esco1/2 perform non-redundant functions that cannot be bypassed by Sororin.

To determine the consequences of impaired cohesin removal, we analyzed mitosis by live-cell and fixed-cell imaging. Live-cell imaging revealed comparable mitotic durations in cells expressing H2B-GFP and H2B-Sororin-CTR-GFP (38.5 min vs. 36.7 min; Fig. 3E, F and Supplementary Movie 1, 2). However, anaphase chromosome-segregation errors increased dramatically, from 4.4% in control cells to 38.3% in H2B-Sororin-CTR-GFP cells (Fig. 3G). Analysis of paraformaldehyde (PFA)-fixed cells showed a specific increase in anaphase chromosome bridges from 6.7% to 32.9%, whereas lagging-chromosome rates remained similar (3.8% vs. 4.5%) (Fig. 3H). Consistently, centromeric accumulation of Aurora B, which is important for correcting erroneous kinetochore-microtubule attachments76–81, was comparable in cells expressing H2B-GFP and H2B-Sororin-CTR-GFP (Supplementary Fig. 3G, H), indicating that the increased chromosome bridges do not result from Aurora B mislocalization. Furthermore, H2B-Sororin-CTR-GFP cells exhibited a ~3-fold increase in ultrafine DNA bridges marked by PICH (37.9% vs. 12.4%; Fig. 3I), indicative of defective decatenation. A similar increase in anaphase bridges was observed upon overexpression of untethered Sororin-CTR-GFP (Supplementary Fig. 3I–K), linking these defects directly to the CTR’s cohesion-stabilizing activity.

In summary, constitutive chromatin tethering of Sororin-CTR prevents mitotic cohesin removal and recapitulates key features of Wapl loss, including defective chromosome condensation, impaired decatenation, and erroneous chromosome segregation.

Sororin-CTR directly binds the closed RAD21-SMC3 interface
To elucidate the molecular basis of Sororin-CTR-mediated cohesion stabilization, we examined its interaction with the cohesin complex. Bacterially expressed GST-Sororin fragments were used in pull-down assays with HeLa cell lysates. Full-length GST-Sororin and GST-Sororin-CTR, but not GST-Sororin (1-222), bound endogenous SMC3, SMC1, RAD21, and SA2 (Fig. 4A, B). While full-length Sororin and the N-terminal fragment interacted with Pds5B, GST-Sororin-CTR did not, indicating that the CTR associates with the cohesin core independently of Pds5.

A previous study suggested that the C-terminus of Sororin interacts with the SA2 subunit65. To test whether SA2 is required for cohesin’s interaction with Sororin-CTR, we performed pull-down assays in SA2-knockout HeLa cells generated by CRISPR/Cas9. GST-Sororin-CTR retained binding to SMC3, SMC1, RAD21, and SA1 in the absence of SA2 (Fig. 4C, D and Supplementary Fig. 4A, B). In contrast, siRNA-mediated depletion of RAD21 abolished the interaction between GST-Sororin-CTR and the SMC subunits (Fig. 4E), identifying RAD21 as the critical anchor. Consistent with the previous observation that the D793K mutation prevented SA2 from binding RAD2182, this mutation eliminated the co-immunoprecipitation of Myc-tagged SA2 with GFP-fused RAD21 in HEK-293T cells (Supplementary Fig. 4C). Notably, this mutation also prevented Myc-SA2 from binding GST-Sororin-CTR (Supplementary Fig. 4D), indicating that SA2 binding to Sororin-CTR depends on its interaction with RAD21.

Within the cohesin ring, RAD21 connects to the head domains of SMC3 and SMC1 via its N-terminal helical domain (NHD) and C-terminal winged-helix domain (WHD), respectively (Fig. 4F)34,83. To determine which protein is recognized by Sororin-CTR, we co-expressed Myc-tagged RAD21 with GFP-tagged SMC3 or SMC1 in HEK-293T cells. GST-Sororin-CTR selectively pulled down RAD21-Myc together with SMC3-GFP, but not with SMC1-GFP (Fig. 4G), demonstrating preferential binding to the RAD21-SMC3 interface.

Because the RAD21-SMC3 interaction involves RAD21’s NHD and the head-proximal coiled-coil of SMC3 (referred to as SMC3-HD)34,83, we tested whether these regions were sufficient for Sororin-CTR binding. Pull-down assays showed that GST-Sororin-CTR bound Flag-tagged SMC3-HD only when co-expressed with Myc-tagged RAD21 fragments comprising residues 1-281 (referred to as N281) or residues 1-103 (referred to as N103), and that deletion of RAD21’s Pds5-interaction region (residues 102-122) did not affect this interaction (Fig. 4H and Supplementary Fig. 4E).

We further generated a recombinant protein complex formed between 2xStrep-tagged RAD21-N103 and SMC3-HD-Flag, which were co-expressed in E. coli and purified with the streptavidin beads. Pull-down assays showed that this minimal RAD21-N103/SMC3-HD complex directly bound GST-Sororin-CTR and GST-Sororin (Fig. 4I, J), establishing a direct, specific interaction.

We next examined the cell-cycle regulation of this interaction. We synchronized HeLa cells in early-S phase and G2 phase by a double-thymidine block-and-release protocol and performed pull-down assays. GST-Sororin-CTR bound endogenous SMC3, SMC1, and RAD21 more efficiently in G2-phase cell lysates than in early S-phase lysates (Fig. 4K). Furthermore, this interaction persisted in late-G2 phases (arrested by the Cdk1 inhibitor RO-3306) but was markedly reduced in mitotic cells (arrested by nocodazole) (Fig. 4L). Thus, Sororin-CTR binding to cohesin peaks during the cohesion-maintenance phase of the cell cycle.

Finally, to distinguish chromatin-bound from soluble cohesin pools, we performed chromatin fractionation followed by co-immunoprecipitation. Sororin-GFP stably expressed in HeLa cells co-precipitated RAD21, SMC3, and Pds5B specifically from the chromatin-bound fraction of mitotic cells, but not from the soluble fraction (Fig. 4M, N), confirming that Sororin interacts with functional, chromatin-associated cohesin.

Collectively, these results demonstrate that Sororin’s CTR directly and specifically binds the RAD21-SMC3 interface in a cell-cycle-regulated manner on chromatin, consistent with a model in which Sororin stabilizes cohesion by locking the closed DNA exit gate of cohesin.

Mitotic phosphorylation of Sororin selectively disrupts Pds5B binding while preserving interaction with the RAD21-SMC3 interface
Sororin phosphorylation in mitosis promotes cohesin release from chromosome arms27,46,54,62. To examine whether this modification also affects Sororin’s interaction with the RAD21-SMC3 interface, we arrested HeLa cells co-expressing Sororin-GFP, RAD21-N103-Myc, and SMC3-HD-Flag either in G2 (using RO-3306) or in mitosis (with nocodazole), followed by immunoprecipitation of Sororin-GFP. In G2-arrested cells, Sororin-GFP efficiently co-immunoprecipitated RAD21-N103-Myc, SMC3-HD-Flag, and endogenous Pds5B (Fig. 5A). In mitotic cells, however, the Sororin-Pds5B interaction was abolished, whereas Sororin’s binding to RAD21-N103 and SMC3-HD was retained.

Moreover, λ-phosphatase treatment of lysates from nocodazole-treated HEK-293T cells markedly enhanced the Sororin-Pds5B co-immunoprecipitation, without affecting interactions with RAD21-N103 or SMC3-HD (Fig. 5B). Similarly, inhibition of CDK1 (Roscovitine), Aurora B (Hesperadin), or Plk1 (BI2536) in nocodazole-arrested mitotic HeLa cells restored Sororin-Pds5B interaction, but had no effect on Sororin association with RAD21-N103 or SMC3-HD (Fig. 5C). These results indicate that mitotic phosphorylation selectively disrupts Sororin’s interaction with Pds5B while leaving its binding to the RAD21-SMC3 interface intact.

Sororin contains a conserved YSR motif that is required for Pds5 binding26. Consistently, co-immunoprecipitation assays showed that endogenous Pds5B associated with Sororin-GFP, but not with the Sororin-ASE mutant in which the YSR motif (residues 146-148) was replaced by ASE (Fig. 5D). GST pull-down assays further demonstrated that GST-Sororin, but not GST-Sororin-ASE, bound endogenous Pds5B (but not its paralog Pds5A) from HeLa cell lysates (Fig. 5E). Notably, the Sororin-ASE mutant exhibited reduced binding to cohesin core subunits, indicating that Pds5B contributes to the overall association of Sororin with cohesin.

To identify the phosphorylation site responsible for disrupting Pds5B binding, Sororin-GFP immunoprecipitated from HeLa cells was analyzed by mass spectrometry, revealing 18 phosphorylation sites (Supplementary Fig. 5A and Supplementary Data 1). Among these, serine 145 (S145), which lies adjacent to the YSR motif and is conserved across vertebrates, emerged as a strong candidate (Fig. 5F). Using a phospho-specific antibody raised against Sororin phosphorylated at S145 (Sororin-pS145), we found that S145 phosphorylation was detected exclusively in mitotic cells (Fig. 5G). Antibody specificity was confirmed using Flag-tagged Sororin purified from mitotic cells, as the signal was absent in the S145A mutant and abolished by λ-phosphatase treatment (Supplementary Fig. 5B, C).

S145 resides within a consensus Aurora kinase phosphorylation motif (R/K-x-S/T-Φ). Accordingly, treatment of nocodazole-arrested mitotic cells with Hesperadin eliminated the pS145 signal on Flag-Sororin (Fig. 5H), indicating that Sororin-S145 phosphorylation in cells requires Aurora B kinase activity. To determine whether Aurora B can phosphorylate Sororin at S145, we performed in vitro kinase reactions using bacterially produced recombinant GST-Aurora B in complex with the IN-box of INCENP (GST-Aurora B/IN-box)84. In the presence of ATP, this complex phosphorylated Flag-Sororin at S145 (Fig. 5I). These results indicate that Aurora B is the kinase responsible for Sororin-S145 phosphorylation.

We next assessed the effect of S145 phosphorylation on the Sororin-Pds5B interaction. Pull-down assays using a synthesized Sororin peptide (residues 136-158) and a recombinant MBP-tagged Pds5B fragment (residues 1-300) showed that the non-phosphorylated peptide bound Pds5B, whereas the S145-phosphorylated peptide did not (Fig. 5J). Moreover, in vitro phosphorylation of the Sororin peptide by GST-Aurora B/IN-box similarly abolished Pds5B binding (Fig. 5K), demonstrating that S145 phosphorylation directly disrupts this interaction.

Consistent with these in vitro findings, co-immunoprecipitation assays in G2-synchronized HeLa cells revealed that Myc-tagged Pds5B associated with wild-type Sororin-GFP, but not with the phospho-mimetic Sororin-S145E-GFP mutant (Supplementary Fig. 5D). Moreover, Aurora B inhibition in mitotic cells enhanced Pds5B binding to wild-type Sororin-GFP, but not to Sororin-S145E-GFP (Supplementary Fig. 5E), confirming that Aurora B-dependent phosphorylation at S145 disrupts the Sororin-Pds5B interaction in vivo.

We further tested the effect of a phospho-deficient mutation, S145N, on Sororin interactions. In mitotic cells, Sororin-S145N exhibited enhanced association with Pds5B, SMC3, and RAD21 compared with wild-type Sororin, whereas no difference was observed in G2-phase cells (Supplementary Fig. 5F, G).

Finally, because Sgo1-associated PP2A protects centromeric cohesion by dephosphorylating Sororin51–54, we examined whether the S145N mutant could bypass Sgo1 depletion. Although the S145N mutant partially rescued the cohesion defect caused by Sgo1 knockdown, it did not fully restore cohesion (Supplementary Fig. 5H–J). This indicates that preservation of the Sororin-Pds5 interaction via S145 dephosphorylation represents a major, but not exclusive, function of Sgo1-PP2A in cohesion protection.

In summary, Aurora B-mediated phosphorylation of Sororin at S145 acts as a mitotic switch that selectively disrupts its interaction with Pds5B, thereby promoting cohesin release from chromosome arms, while leaving its gate-locking interaction with the RAD21-SMC3 interface intact.

Identification of key residues mediating the interaction between Sororin and the RAD21-SMC3 interface
The 30-residue CTR of Sororin (residues 223-252) is enriched in hydrophobic (17) and acidic (9) residues. AlphaFold predictions indicate that the segment spanning residues K227 to E245 likely adopts an α-helical conformation (AlphaFoldDB: Q96FF9). To elucidate how Sororin interacts with the RAD21-SMC3 interface, we used AlphaFold3 to model the binding of a Sororin-CTR peptide to the interface formed by RAD21-NHD and SMC3-HD (Fig. 6A). The model predicts multiple specific contacts: Sororin W233 with RAD21 Y3; Sororin F241 with RAD21 K50 and SMC3 L1031; Sororin A243 and F247 with SMC3 K1038; Sororin E245 with SMC3 K1034; and Sororin E252 with SMC3 R1099. Sequence alignment revealed that these residues are evolutionarily conserved among vertebrates, with the exception of Sororin W233, which is replaced by a similarly hydrophobic leucine in zebrafish (Fig. 6B and Supplementary Fig. 6A, B).

To experimentally test these predictions, we performed binding assays using bacterially expressed recombinant proteins. A complex consisting of RAD21-N103-2xStrep and SMC3-HD-Flag was efficiently pulled down by GST-Sororin-CTR, but not by the double mutant GST-Sororin-CTR-W233A/E252K (hereafter WA/EK), in which W233 and E252 were mutated to alanine and lysine, respectively (Fig. 6C). A comparable loss of binding was observed for the GST-Sororin-CTR-F241A/F247A mutant (hereafter FA/FA), where both F241 and F247 were replaced by alanine. Consistently, pull-down assays in HeLa cells showed that endogenous SMC3, SMC1, RAD21, and SA2 interacted with GST-Sororin-CTR, but not with the WA/EK or FA/FA mutants (Fig. 6D). Moreover, a cancer-associated Sororin-E252Q mutation reduced co-immunoprecipitation of SMC3 and RAD21 with Sororin-Flag in HEK-293T cells (Supplementary Fig. 6C), suggesting that this mutation partially compromises Sororin’s gate-locking function and may contribute to genomic instability.

We next examined the contribution of residues within RAD21-NHD and SMC3-HD to Sororin binding. In HEK-293T cells, simultaneous mutation of RAD21 Y3 to alanine and K50 to glutamic acid (hereafter YA/KE) abolished the ability of co-expressed RAD21-N103-Myc and SMC3-HD-Flag to bind GST-Sororin-CTR (Fig. 6E). Co-immunoprecipitation assays indicated that the YA/KE mutations did not affect the interaction between RAD21-N103-Myc and SMC3-HD-Flag (Supplementary Fig. 6D). Similarly, mutation of SMC3 K1034 and K1038 to glutamic acid (hereafter KE/KE) strongly reduced pull-down of co-expressed SMC3-HD-Flag and RAD21-N103-Myc by GST-Sororin-CTR (Fig. 6F), without affecting RAD21-SMC3 association (Supplementary Fig. 6E). Additional binding assays performed in HEK-293T cells showed that GST-Sororin-CTR failed to pull down the co-expressed mutants RAD21-N103-Y3A and SMC3-HD-R1099E-Flag, in which residues RAD21 Y3 and SMC3 R1099 were mutated to alanine and glutamic acid, respectively (Fig. 6G). We confirmed that these two mutations did not perturb the binding between RAD21 and SMC3 (Supplementary Fig. 6F).

To further validate these interactions using a fully recombinant system, we co-expressed RAD21-N103-2xStrep and SMC3-HD-Flag in E. coli and immobilized this complex on streptavidin beads. The wild-type RAD21-N103-2xStrep/SMC3-HD-Flag complex efficiently pulled down GST-Sororin-CTR, whereas complexes containing either RAD21-N103-YA/KE or SMC3-HD-KE/KE failed to do so (Fig. 6H). Consistent results were obtained when these complexes were used to pull down Myc-Sororin expressed in HEK-293T cells (Fig. 6I).

Esco1/2-mediated acetylation of SMC3 at K105/K106 promotes Sororin recruitment to chromatin in S phase26,27,35,39–45. Notably, neither acetylation-deficient (K105A/K106A) nor acetylation-mimetic (K105Q/K106Q) mutations in SMC3 affected co-immunoprecipitation of Sororin-GFP with the SMC3-HD-Flag/RAD21-N103-Myc complex (Supplementary Fig. 6G). Consistently, AlphaFold3 modeling indicates that K105 and K106 are spatially distant from the Sororin-binding residues (K1034, K1038, R1099) on SMC3 (Supplementary Fig. 6H; AlphaFoldDB: Q9UQE7). Thus, SMC3 acetylation licenses Sororin recruitment without directly regulating its binding to the RAD21-SMC3 interface.

Collectively, these data identify critical residues in Sororin-CTR, RAD21-NHD, and SMC3-HD that together mediate Sororin binding to the RAD21-SMC3 interface.

Sororin-CTR residues that bind the RAD21-SMC3 interface are required for sister-chromatid cohesion
We next assessed whether the Sororin-CTR residues that mediate binding to the RAD21-SMC3 interface are required for sister-chromatid cohesion. To this end, we analyzed chromosome-arm resolution in HeLa cells transiently expressing H2B-fused Sororin-CTR-GFP, either wild type or carrying the WA/EK or FA/FA mutations (Fig. 7A). Chromosome spreads from nocodazole-arrested mitotic cells showed that expression of wild-type H2B-Sororin-CTR-GFP, but not the WA/EK or FA/FA mutants, prevented arm resolution and markedly impaired chromosome condensation (Fig. 7B, C and Supplementary Fig. 7A). Similar phenotypes were observed in non-transformed RPE-1 cells expressing these H2B fusion proteins (Supplementary Fig. 7B-D). Consistently, expression of H2B-fused full-length Sororin-GFP, but not the corresponding WA/EK or FA/FA mutants, inhibited mitotic arm resolution in both HeLa and RPE-1 cells (Supplementary Fig. 7E–J).

We next tested whether the WA/EK and FA/FA mutants could support Sororin’s function in protecting sister-chromatid cohesion. To this end, we generated HeLa cell lines stably expressing wild-type Sororin-GFP or the WA/EK and FA/FA mutants (Fig. 7D). Co-immunoprecipitation assays confirmed that endogenous RAD21, SMC3, and SMC1 associated with wild-type Sororin-GFP, but not with the WA/EK or FA/FA mutants (Fig. 7E). Following siRNA-mediated depletion of endogenous Sororin, cells expressing the WA/EK or FA/FA mutants displayed a pronounced increase in mitotic index, whereas cells expressing wild-type Sororin-GFP did not (Fig. 7F–H). Consistently, chromosome-spread analyses showed that the premature cohesion loss induced by Sororin depletion was rescued by exogenous expression of wild-type Sororin-GFP or Sororin-SFB, but not by the WA/EK or FA/FA mutants, in both HeLa and RPE-1 cells (Fig. 7I, J and Supplementary Fig. 7K–M).

Collectively, these results demonstrate that Sororin-CTR residues required for binding to the RAD21-SMC3 interface are essential for Sororin-mediated protection of sister-chromatid cohesion.

RAD21 and SMC3 residues that bind Sororin-CTR are essential for sister-chromatid cohesion
We next investigated the functional significance of the RAD21 and SMC3 residues that mediate binding to Sororin-CTR. We first examined sister-chromatid cohesion in cells in which endogenous RAD21 was replaced with the exogenous RAD21-YA/KE mutant defective in Sororin binding. To this end, we established HeLa-derived cell lines stably expressing siRNA-resistant RAD21 (wild-type or the YA/KE mutant) fused to SFB, which is a triple-tag consisting of an S-tag, a Flag tag, and a biotin-binding peptide (Fig. 8A). Co-immunoprecipitation assays confirmed that wild-type RAD21-SFB interacted with endogenous Sororin, whereas the RAD21-YA/KE mutant did not; importantly, the YA/KE mutant retained its interaction with endogenous SMC3 (Fig. 8B). Mitotic chromosome-spread analysis showed that, following depletion of endogenous RAD21, 44.2% of HeLa cells exhibited premature loss of cohesion (Fig. 8C–E). This defect was efficiently rescued by expression of wild-type RAD21-SFB, but not by the YA/KE mutant, demonstrating that RAD21 residues required for Sororin-CTR binding are critical for sister-chromatid cohesion.

We next determined the contribution of SMC3 residues involved in Sororin binding. We generated HeLa cell lines stably expressing siRNA-resistant SMC3-GFP, either wild type or carrying the Sororin-binding-defective KE/KE mutations (Fig. 8F). Co-immunoprecipitation assays showed that the SMC3-KE/KE mutant failed to interact with Sororin but retained binding to RAD21 (Fig. 8G). Depletion of endogenous SMC3 in these cells caused a cohesion defect in control cells (Fig. 8H–J), albeit milder than that observed following RAD21 depletion, likely due to reduced cell viability upon SMC3 loss85,86. Notably, this defect was rescued by expression of wild-type SMC3-GFP but not by the SMC3-KE/KE-GFP mutant, indicating that SMC3 residues mediating Sororin-CTR binding are required for proper cohesion.

Collectively, these results establish that specific residues in both RAD21 and SMC3 that mediate interaction with Sororin-CTR are essential for maintaining sister-chromatid cohesion.

Interaction between Sororin and the RAD21-SMC3 interface is dispensable for cohesion in the absence of Wapl
Previous studies have shown that Sororin is required for sister-chromatid cohesion only in the presence of Wapl27,60, suggesting that Sororin preserves cohesion primarily by antagonizing Wapl activity. We therefore asked whether depletion of Wapl could suppress the cohesion defects caused by Sororin mutants defective in binding the RAD21-SMC3 interface. HeLa cells stably expressing Sororin-GFP mutants were subjected to siRNA-mediated depletion of Sororin and Wapl, either individually or in combination (Fig. 9A). Mitotic chromosome-spread analysis revealed that the premature cohesion loss observed upon replacement of endogenous Sororin with Sororin-WA/EK-GFP or Sororin-FA/FA-GFP was effectively suppressed by Wapl depletion (Fig. 9B, C).

We next examined whether Wapl depletion could similarly rescue cohesion defects caused by Sororin-binding-deficient mutations in cohesin subunits RAD21 and SMC3. Notably, Wapl knockdown prevented premature sister-chromatid separation in cells in which endogenous RAD21 and SMC3 were replaced by exogenous RAD21-YA/KE-SFB (Fig. 9D–F) and SMC3-KE/KE-GFP (Fig. 9G–I), respectively.

Together, these results demonstrate that the cohesion defects resulting from disruption of Sororin binding to the RAD21-SMC3 interface are dependent on Wapl activity, and that this interaction becomes dispensable for cohesion when Wapl is absent.

Methods
Cell lines, plasmids, siRNA, transfection, and drug treatments
HEK-293T cells, HeLa cells, and their derivatives were cultured in Dulbecco’s Modified Eagle Medium (DMEM), while RPE-1 cells and their derivatives were cultured in DMEM/F12. Both media were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. HeLa cells stably expressing Sororin-GFP (WT, WA/EK, or FA/FA), CENP-B-GFP, CENP-B-Sororin-CTR-GFP, H2B-GFP, H2B-Sororin-CTR-GFP, and SMC3-GFP (WT or KE/KE) were cultured in medium supplemented with 1.5 μg/ml blasticidin (Sigma). HeLa cells stably expressing Tet-on inducible RAD21-SFB (WT or YA/KE) were maintained in medium containing 0.5 mg/ml G418 (Calbiochem). Pools of RPE-1 cells expressing Tet-on inducible Sororin-SFB (WT, WA/EK, or FA/FA) were generated by lentiviral infection and selected in medium containing 0.5 mg/ml G418.

The construct for pGEX-6P-Aurora B (60-361)-rbs-INCENP (790-856) was a gift from Dr. Andrea Musacchio (Max Planck Institute of Molecular Physiology). The Myc-Pds5B construct was kindly provided by Dr. Hongtao Yu (Westlake University). To generate the Flag-Sororin, Lenti-Sororin-SFB, and Lenti-RAD21-SFB constructs, the corresponding cDNAs in the entry vectors were transferred into Gateway-compatible destination vectors harboring an N-terminal Flag-tag and a C-terminal SFB-tag, respectively. Myc-SA2 was constructed similarly using a vector harboring an N-terminal Myc-tag. To generate Myc-Sororin, the cDNA encoding Sororin was amplified by PCR and subcloned into an expression vector containing a Myc epitope tag. The pBos-H2B-Sororin-GFP plasmid series was constructed by subcloning PCR-amplified fragments encoding full-length Sororin or truncated variants (1-149, 1-222, 223-252) into pBos-H2B-GFP (BD Pharmingen) using the BamHI restriction site. The pBos-CENP-B-GFP plasmid was constructed by replacing the H2B coding sequence in pBos-H2B-GFP with KpnI/BamHI-digested PCR fragments encoding the centromere-targeting domain of CENP-B (residues 1-163). The pBos-CB-Sororin (223-252)-GFP construct was generated by subcloning the corresponding Sororin fragment into pBos-CENP-B-GFP. The pBos-Sororin-GFP plasmid series was constructed by replacing the H2B coding sequence in pBos-H2B-GFP with PCR-amplified sequences encoding full-length Sororin or its truncated variants (1-149, 150-252, 1-222, 223-252). The pBos-SMC1-GFP, pBos-SMC3-GFP, and pBos-RAD21-GFP constructs were generated by replacing the H2B coding sequence with PCR-amplified coding sequences of SMC1, SMC3, and RAD21, respectively. Plasmids encoding GST-fused Sororin (Full-length or truncations) were generated by subcloning the corresponding PCR fragments into pGEX-4T1 (GE Healthcare). The MBP-Pds5B (1-300) construct was generated by cloning the PCR products into pMAL-C2E (New England Biolabs). For the pEF6-RAD21-Myc construct series, PCR-amplified fragments encoding full-length RAD21 or its variants (1-281, 1-101+123-281, 1-103) were cloned into the pEF6-Myc vector. The pEF-Sororin-Flag and pEF-SMC3-HD-Flag constructs were generated by cloning PCR-amplified Sororin and SMC3-HD fragments (residues 1-253 and 959-1217 connected by a GGGGS linker) into pEF-Flag, respectively. To generate the pET45-RAD21-N103-2xStrep-rbs-SMC3-HD-Flag construct, the PCR-amplified RAD21-N103-2xStrep fragment, rbs fragment, and SMC3-HD-Flag fragment were subcloned into pET45 using the MultiF Seamless Assembly Mix Kit (ABclonal Biotechnology).

All point mutations were introduced using either the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) or the MultiF Seamless Assembly Mix Kit (ABclonal Biotechnology). All constructs were verified by DNA sequencing to confirm the presence of the desired mutations and the absence of unwanted sequence alterations.

SiRNA duplexes targeting Sororin, Esco1, Esco2, Sgo1, Wapl, RAD21, and SMC3 were purchased from Integrated DNA Technologies (IDT) or RiboBio: siSororin targeting the 3’UTR (5’-CUCGGAAAGUUUCCUCGCGUAdTdT-3’), siEsco1 (5’-AGAAGGUCGUUACGCA-GUAdTdT-3’), siEsco2 (5’-GAAAGAACGUGUAGUAGCAdTdT-3’), siSgo1 (5’-GAGGGGACCCUUUUACAGAdTdT-3’), siWapl (5’-CGGACUACCCUUAGCACAAdTdT-3’), siRAD21 (5’-AUACCUUCUUGCAGACUGUdTdT-3’), siRAD21#2 (5’-GCACUACUACUUCUAACCUdTdT-3’), siSMC3 (5’-GGAGGGCAGUCAGUCUCAAGAUGAAdTdT-3’). siRNA transfections were carried out twice at 24-h intervals using Oligofectamine (Invitrogen) or Lipofectamine RNAiMAX (Thermo Fisher Scientific). For SMC3 siRNA transfection, cells at approximately 40% confluence were transfected once using Lipofectamine RNAiMAX. Plasmid transfections were performed using FuGENE 6 (Promega) or Lipofectamine 2000 (Thermo Fisher Scientific).

Cell-cycle synchronization was performed as follows: cells were synchronized at the G1/S boundary using a double-thymidine (2 mM, Sigma) block; in G2 phase using either a single-thymidine block or a double-thymidine block followed by an 8-h release; in late G2 phase using RO-3306 (9 μM; Selleckchem) for 18 h; and in prometaphase using nocodazole (100~300 ng/ml; Selleckchem) for 15–18 h or STLC (5 μM, Tocris Bioscience) for 3 h. Mitotic cells were collected by mechanical detachment (“shake-off”). The kinase inhibitors used in this study were as follows: CDK1 (Roscovitine, Selleckchem; 100 μM), Aurora B kinase (Hesperadin, Selleckchem; 25–100 nM), and Plk1 (BI 2536, Selleckchem; 100 nM). The proteasome inhibitor MG132 (10 μM; Selleckchem) was also used.

Antibodies
The following rabbit polyclonal antibodies were used: GFP (A11122, Invitrogen), GAPDH (14C10, Cell Signaling Technology/CST), RAD21 (ab992, Abcam), SMC1 (A300-055A, Bethyl Laboratories), SMC3 (A300-060A, Bethyl Laboratories), Pds5A (A300-089A, Bethyl Laboratories), Pds5B (A300-537A and A300-538A, Bethyl Laboratories), SA1 (HPA035015, Sigma), SA2 (#5882, CST), Wapl (A300-268A, Bethyl Laboratories), Esco1 (29821-1-AP, Proteintech), Sororin (ab192237, Abcam), Sororin-pS145 (generated by immunization with the synthetic peptide LEMSKKVRRpSYSRLETLGSASTS, where pS indicates phosphoserine), and Flag-tag (A01868, GenScript). Mouse monoclonal antibodies included: Aurora B (611083, BD Transduction Laboratories), α-Tubulin (AC012, ABclonal), Myc-tag (clone 4A6, 05-724, Millipore), Flag-tag (clone M2, F-3165, Sigma), SA2 (sc-81852, Santa Cruz Biotechnology), GFP (M20004, Abmart), PICH (H00054821-B01P, Abnova), Wapl (M221-3, MBL), Strep II-Tag (AE066, ABclonal), MBP (AE016, ABclonal), and GST-tag (M20007, Abmart). The anti-centromere antibody (ACA) was obtained from ImmunoVision (HCT-0100). Guinea pig polyclonal antibodies against CENP-C were purchased from MBL (PD030).

Secondary antibodies for immunoblotting included Streptavidin-HRP (434323, Invitrogen), goat anti-rabbit IgG-HRP (7074, CST), and horse anti-mouse IgG-HRP (7076, CST). Secondary antibodies for immunofluorescence staining included: donkey anti-rabbit IgG conjugated to Alexa Fluor 488 or Cy3 (711-545-152 or 711-166-152, Jackson ImmunoResearch); anti-mouse IgG conjugated to Alexa Fluor 488, 546 (A-21202 or A-11030, Invitrogen) or Cy5 (715-175-150, Jackson ImmunoResearch); goat anti-guinea pig IgG conjugated to Alexa Fluor 647 (A-21450, Invitrogen); and anti-human IgG conjugated to Alexa Fluor 647 (709-605-149, Jackson ImmunoResearch).

Lentivirus packaging and infection
The Tet-On inducible SFB-tagged lentiviral vector and packaging plasmids (pMD2G and pSPAX2) were provided by Dr. Jun Huang (Life Sciences Institute, Zhejiang University). Virus-containing supernatant was collected 48 h after the co-transfection of the lentiviral vector and packaging plasmids (pMD2G and pSPAX2) into HEK-293T cells. HeLa cells were infected with the viral supernatants in the presence of 8 μg/ml polybrene (Sigma), and stable pools were selected using medium containing 0.5 mg/ml G418. For the experiments described in this study, expression of the indicated genes in the stable pools was induced by the addition of 1 μg/ml doxycycline (Selleckchem) for 24 h.

CRISPR/Cas9-mediated editing of the SA2 gene STAG2 in human cells
Single-guide RNA (sgRNA) for human STAG2 (encoding SA2) was designed, ordered as complementary oligonucleotides, annealed, and cloned into the dual Cas9 and sgRNA expression vector pX330 (Addgene, #42230) using BbsI restriction sites. The resulting plasmid was transfected into HeLa cells using FuGENE 6 according to the manufacturer’s protocol. After 48 h incubation, cells were seeded at low density to establish clonal cell lines following brief selection with 1 μg/ml puromycin for 2–3 days. To obtain SA2-KO cells, clones showing undetectable SA2, as confirmed by immunostaining and immunoblotting, were isolated. SA2 KO cells were generated using an sgRNA targeting the sequence 5’-GGCCCAGCAGAAAAGGGCAA-3’.

Fluorescence microscopy, chromosome spreads, and chromosome length measurement
HeLa cells were typically fixed in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min at room temperature, followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min. Cells were subsequently blocked with 3% bovine serum albumin (BSA) in PBS and incubated with primary antibodies for 1–2 h, followed by secondary antibody incubation for 1 h, all incubations were performed at room temperature in 3% BSA/PBS. Nuclear DNA was counterstained with DAPI for 5 min.

For chromosome spread preparation, HeLa cells were treated with 100-300 ng/ml nocodazole for 3 h. Mitotic cells were collected by selective detachment and incubated in hypotonic buffer (75 mM KCl or 0.25× PBS) for 10–15 min at room temperature. Cells were then cytocentrifuged onto glass coverslips using a Cytospin at 260×g for 5 min. Following attachment, chromosome spreads were fixed in 2% PFA in PBS for 15–20 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min, blocked with 3% BSA in PBS, and incubated with primary antibodies for 2 h at room temperature. For SMC1 immunofluorescence staining, cells were pre-extracted with 0.5% Triton in PHEM buffer (60 mM Pipes, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.9) for 5 min, then fixed with 4% PFA in PHEM for 20 minutes.

Fluorescence microscopy was performed at room temperature using a Nikon ECLIPSE Ni microscope equipped with a Plan Apo Fluor 60× oil immersion objective (NA 1.4) and a Clara CCD camera (Andor Technology). To measure the length of mitotic chromosomes, images were visualized using NIS-Elements BR software (Nikon). Chromosome lengths were measured along the outline of the center of the chromosomes using a point-by-point tracing tool. Cells were randomly selected for each condition, and all chromosomes within the selected cell were measured.

Time-lapse live cell imaging
Time-lapse live cell imaging was carried out using the GE DV Elite Applied Precision DeltaVision system (GE Healthcare) equipped with Olympus oil objectives of 60× (NA 1.42) Plan Apo N or 40× (NA 1.35) UApo/340 oil objective, an API Custom Scientific complementary metal-oxide semiconductor camera, and Resolve 3D softWoRx imaging software. Cells were plated in four-chamber glass-bottomed 35-mm dishes (Cellvis) coated with poly-D-lysine, and imaged in a climate-controlled and humidified environment (37°C and 5% CO2). Chromosomes were labeled with H2B-GFP. Images were captured every 3 minutes and processed using Adobe Photoshop and Adobe Illustrator.

Protein expression and purification
Plasmids encoding GST-, Strep-, or MBP-fused proteins were transformed into E. coli BL21 (DE3) competent cells. Transformed cells were cultured in LB medium supplemented with the appropriate antibiotic at 37°C until the optical density at 600 nm (OD600) reached 0.6–0.8. Protein expression was induced by adding 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubating at 16°C for 18 h.

For GST fusion proteins, cells were lysed by sonication in Buffer A (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride/PMSF)). For MBP fusion proteins, lysis was performed in Buffer B (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM EDTA, 0.5% Triton X-100). Lysates were clarified by centrifugation and incubated with Glutathione Sepharose 4B beads (GE Healthcare) for GST fusion proteins or Amylose Resin (New England Biolabs) for MBP fusion proteins. After washing with the respective lysis buffer, proteins were eluted using 100 mM reduced glutathione for GST fusions or 10 mM maltose for MBP fusions.

For Strep-fused protein purification, cells were resuspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM DTT, 1% Triton X-100, 1 mM EDTA, and 1 mM PMSF). Following sonication and a freeze-thaw cycle, the lysate was clarified by centrifugation, and the supernatant was applied to a Strep-Tactin Superflow affinity column (IBA Lifesciences). The resin was washed four times with lysis buffer, and bound proteins were eluted with lysis buffer supplemented with 10 mM desthiobiotin.

In vitro kinase reaction
For the phosphorylation of Flag-Sororin (Fig. 5I), asynchronous HEK-293T cells transiently expressing Flag-Sororin (WT or S145A) were lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% NP-40, and 10 mM MgCl2, supplemented with protease and phosphatase inhibitors as in P150 buffer. The anti-Flag immunoprecipitates were then subjected to phosphorylation by 0.2 μg GST-Aurora B/IN-box. For the phosphorylation of Sororin peptide (Fig. 5K), 0.5 μg of substrates was incubated with 0.2 μg of recombinant GST-Aurora B/IN-box. The kinase reactions were conducted in 50 μl kinase reaction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM MgCl2) with 0.2 mM ATP for 20 min at 30°C. The reactions were terminated by adding SDS sample buffer, boiled, and analyzed by immunoblotting.

Chromatin fractionation
Mitotic cells collected after 18 h of nocodazole treatment were lysed in fractionation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.2% NP-40) supplemented with 1 mM DTT, protease inhibitor cocktail (P8340, Sigma), 10 mM NaF, and 20 mM β-glycerophosphate. After centrifugation at 2000g for 5 min, the supernatant was collected and used for immunoblotting and immunoprecipitation. The chromatin pellets were washed three times with the same buffer and subsequently analyzed by immunoblotting and immunoprecipitation.

Pull-down, immunoprecipitation, and immunoblotting
For GST- or Strep-fused protein pull-down assays with cell lysates, HeLa cells were lysed in P150 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 5 mM EDTA) supplemented with 1 mM dithiothreitol (DTT), protease inhibitor cocktail, 1 mM PMSF, 0.1 μM okadaic acid (Calbiochem), 10 mM sodium fluoride, 20 mM β-glycerophosphate, and Benzonase (GenScript). Insoluble material was removed by high-speed centrifugation, and the lysates were pre-cleared with either glutathione Sepharose 4B beads (GE Healthcare) or StrepTactin 4FF beads (Smart Life Sciences). The precleared lysates were then incubated with bead-immobilized GST- or Strep-fused proteins for 4 h at 4°C. Beads were washed 4 times with lysis buffer, boiled in SDS sample buffer, and subjected to immunoblotting.

For Sororin (136-158) peptide pull-down of recombinant MBP-Pds5B-N300, 1 μg of peptides were incubated with 4 μg of MBP-Pds5B-N300 in 1 ml of binding buffer (50 mM Tris, pH 7.5, 300 mM NaCl, 0.5% Triton X-100) for 3 h, followed by 1-h incubation with NeutrAvidin Plus Ultralink Resin (Thermo Fisher Scientific). The resin was then washed 4 times with binding buffer. The Sororin peptide of residues, corresponding to residues 136-158 (either wild-type or the pS145 form) and followed by a Lys-biotin moiety, were synthesized by GL Biochem (Shanghai).

For co-immunoprecipitation, cells were lysed in P150 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 5 mM EDTA) supplemented with the same additives as above. Insoluble material was removed by high-speed centrifugation, and the lysates were pre-cleared with rProtein A/G beads (Smart Life Sciences, Cat. No. SA032100). The precleared lysates were incubated for 4 h at 4°C with Anti-GFP Affinity beads (Smart Life Sciences, SA070005), Anti-Flag M2 Affinity Gel (Sigma, A2220), Anti-Myc Affinity Gel (GNI, GNI4510-MC), or control IgG. After washing with P150 buffer, bound proteins were eluted by boiling in SDS sample buffer for immunoblotting.

Mass spectrometry
For the data presented in Supplementary Fig. 5A and Supplementary Data 1, immunoprecipitated Sororin-GFP were separated by SDS-PAGE (n=1). The gels were minimally stained with Coomassie brilliant blue. Protein bands were excised according to the molecular weight marker range. The excised protein bands were cut into small pieces, then reduced and alkylated in 10 mM DTT and 55 mM IAA, respectively. For digestion, 0.5 μg of sequencing-grade modified trypsin (Promega, USA) was added, followed by incubation overnight at 37°C. Prior to mass spectrometry analysis, the peptide preparations were desalted using StageTip and reconstituted with 0.1% formic acid (FA).

Mass spectrometry analysis was performed using a Thermo Scientific Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer instrument coupled with an Easy-nLC 1200 system. Mobile phase A and B were water and 80% acetonitrile, respectively, each containing 0.1% formic acid. Protein digests were loaded directly onto an analytical column (75 μm × 15 cm, 1.9 μm C18, 1 μm tip) at a flow rate of 450 nl/min. Samples were separated using a linear gradient of 6-8% B for 2 min, 8-27% B for 40 min, 27–40% B for 12 min, 40-90% B for 3 min, 90% B for 3 min. MS/MS data were collected in a data-dependent manner using a top-20 method with a full MS mass range from 400 to 1400 m/z, 60,000 resolution, and an AGC target of 3×106. MS2 scans were triggered when an ion intensity threshold of 4.5×105 was reached. A dynamic exclusion time of 30 s was used. Ions with charge state 1, 6, 8, and > 8 were excluded. The resulting raw data were uploaded into MaxQuant (version 2.0.3.0) and searched against the UniProt KB Sororin (Q96FF9) sequence database. Trypsin/P was specified as the cleavage enzyme, allowing up to two missing cleavages. The mass tolerance for precursor ions was set to 20 parts per million in first search and 5 parts per million in main search, and the mass tolerance for fragment ions was set to 0.02 Da. Carbamidomethylation (C, +57.0215 Da) was used as the fixed modification. Phospho (STY, +79.9663 Da) was included as variable modifications. The false discovery rate (FDR) was adjusted to <1%, with a minimum score for modified peptides set to >20.

Quantitative RT-PCR analysis
Total cellular RNA was extracted with TRIzol (Thermo Fisher Scientific). The homogenate was incubated on ice for 10 min and centrifuged at 13,000×g for 10 min. The supernatant was mixed with chloroform, vigorously shake, and incubated at room temperature. After centrifugation, the upper aqueous phase was mixed with an equal volume of isopropanol, followed by centrifuged. The RNA pellet was washed twice with cold 75% ethanol and dissolved in DEPC-treated water. Next, 1000 ng of RNA was reverse-transcribed using PrimeScript RT Master Mix (Takara, RR047A). Quantitative PCR was carried out using SYBR qPCR SuperMix (Vazyme, Q711-03) according to the manufacturer’s instructions. The Esco2 primer sequences were (5’- ATCCAATCTTCAGTGCATCTTCA-3’ and 5’-TGTTTCTGACCAGCGTCGAT-3’).

Structure prediction
The structure of a complex formed between the Sororin C-terminal fragment (residues 191-252), the SMC3-HD (residues 1-253 and 959-1217 connected by a GGGGS linker), and the RAD21 N-terminal fragment (residues 1-110) was predicted using AlphaFold3. All predictions were performed via the public AlphaFold3 web server (https://www.alphafoldserver.com) using the default settings and without applying any constraints. A representative complex was visualization in PyMOL v3.1.3.

Sequence alignment
Multiple sequence alignments of Sororin, RAD21, and SMC3 were performed using UniProt Align (https://www.uniprot.org/), and the results were visualized using ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/).

Statistics
Statistical analyses were conducted using a two-tailed, unpaired Student’s t-test in GraphPad Prism 6. A P-value of less than 0.05 was considered statistically significant. All representative images, including Western blots and micrographs, were obtained from independent biological replicates that yielded consistent results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a well-designed and carefully executed paper that advances our understanding of Sororin’s role in cohesion. The\nauthors propose that Sororin’s C-terminal region (CTR) directly locks the cohesin ring at the RAD21-SMC3 exit gate, beyond\nits known function as a Wapl inhibitor. The experiments are extensive, but many rely on overexpression of tagged mutants in\nHeLa or 293T cells and pull-down assays. While the data broadly support the model, the evidence for a “molecular lock” at\nthe Smc3-Rad21 gate is still indirect. The conclusion that Sororin’s CTR acts independently of SA2 is also difficult to\nreconcile with earlier reports showing Sororin-SA2 interaction. A more nuanced view may be that Sororin interacts with\nSA2/Pds5 during initial loading or stabilization and then switches to Rad21-Smc3 after cohesin acetylation to\nsecure/strengthen the exit gate. This sequential or context-dependent interaction should be considered and could be tested,\nfor example, with the S145A Sororin mutant.\nThe data redefine Sororin as an “autonomous” cohesin lock, yet Sororin’s function is still initiated by Esco1/2-dependent\nrecruitment and cooperation with Pds5. Emphasizing how Sororin’s gate engagement is spatiotemporally restricted (only\nbecoming critical at centromeres or after cohesin acetylation) would help readers grasp the mechanistic innovation. Finally,\nsome methodological aspects warrant scrutiny. The artificial tethering strategies (centromeric and chromatin-wide) are\npowerful tests of sufficiency, but they represent non-physiological scenarios. For instance, chromatin-wide CTR tethering\nphenocopies Wapl loss with severe condensation and segregation defects, which supports the model but might have off-\ntarget effects (e.g. steric hindrance of other chromatin processes). The centromere-targeting rescue experiment is\nconvincing, yet only partially compensates for Sgo1 loss, suggesting Sororin-CTR alone cannot fully substitute all\ncentromeric cohesion safeguards - this nuance should be discussed. In general, the experimental evidence does support the\ncentral claims that Sororin’s CTR is necessary and sufficient to stabilize cohesion by counteracting Wapl-mediated release;\nhowever, the authors should address these points to ensure the conclusions are fully justified by the data. In its current form,\nthe work is technically sound and quite novel, but bolstering the direct “lock” evidence and clarifying the model would align it\nbetter with Nature Communications’ standards of rigor and conceptual insight.\nFigure-specific comments:\nFig. 2B: The claim of 100% Sororin knockdown is not realistic; provide the full blot.\nFig. 2G,H: Show siRNA depletion efficiency for Esco1/2.\nFig. 4: Can Sororin pull down SA1 in SA2 KO cells? If so, how is this distinguished from the essential Rad21-CTR\ninteraction? Using SA2-deficient cancer lines would strengthen the case.\nThis reviewer remain unconvinced that a Rad21/Smc3 “sub-complex” exists physiologically. This is an intriguing possibility\nbut requires direct verification.\nFig. 4A: A full-length Sororin control is missing. Sororin-CTR pulls down Smc1/3, Rad21, SA2 but not Pds5B; Sororin 1–222\npulls down only Pds5B. Can full-length Sororin immunoprecipitate all cohesin components?\nFig. 4F: GST–Sororin-CTR pulls down overexpressed Smc3-GFP/Rad21-Myc efficiently, but this may artificially generate a\nRad21/Smc3 sub-complex. The authors should repeat with non-overexpressed lysates and probe the stoichiometry of Smc1,\nSmc3, and Rad21 to show whether genuine sub-complexes exist.\nFig. 5: Can the S145A Sororin mutant still bind Rad21?\nRigor and reproducibility: All functional data are limited to HeLa or 293T cells, with no validation in non-transformed or\nadditional lines. The SMC3(KE/KE) experiment shows only a mild phenotype, likely due to viability issues, which weakens\nthe argument for the SMC3 surface. A conditional degron or dTAG system would allow a cleaner test at physiological levels.\nSimilarly, the extent of Wapl suppression across different mutants (Sororin, Rad21, Smc3) should be quantified\nsystematically, and Wapl overexpression tested. It would also be useful to examine Pds5A versus Pds5B specificity.\nFinally, the manuscript is very dense. Simplifying terminology and emphasizing the take-home message would help readers.\nFor instance, the term “gate engagement” is used repeatedly-defining it clearly up front and explaining how Sororin’s gate-\nlocking complements Wapl/Pds5 regulation would prevent confusion.", "reviewer_2": "The manuscript by Chen et al. presents a compelling and mechanistically novel function for Sororin in directly locking the\nRAD21–SMC3 interface of the cohesin ring, thereby acting as a guardian of the DNA exit gate to preserve sister-chromatid\ncohesion. The extreme C-terminal region (CTR) of Sororin is essential for the maintenance of sister chromatid cohesion.\nArtificial tethering of the Sororin CTR to chromosome centromeres can rescue cohesion in Sororin-depleted cells, while\ngenome-wide tethering phenocopies Wapl loss, causing abnormal chromosome condensation, decatenation, and\nsegregation. To further investigate the molecular mechanism of Sororin’s function, the authors combine AlphaFold3-guided\nmodeling with a series of biochemical assays and identify key conserved residues in Sororin, RAD21, and SMC3 that\nmediate this interaction and demonstrate their functional necessity for cohesion. Furthermore, they dissect the regulation of\nSororin, showing that mitotic phosphorylation by Aurora B at S145 selectively disrupts its interaction with Pds5 but not with\nthe RAD21-SMC3 interface. Finally, they demonstrate that the cohesion defects caused by disrupting the Sororin-RAD21-\nSMC3 interaction are entirely Wapl-dependent. The authors therefore propose a dual-function model where Sororin acts\nboth as a competitive displacer of Wapl (via Pds5) and as a direct structural lock for the cohesin exit gate. demonstrated that\nSororin directly binds to the SMC3–RAD21 interface.\nFrom these lines of evidence, the authors conclude that Sororin’s CTR directly binds the SMC3–RAD21 interface to seal\ncohesin’s DNA exit gate. This sealing activity works synergistically with Pds5 competition to balance sister chromatid\ncohesion maintenance and regulated dissociation. Overall, the in vitro and in vivo data are clear and support the\nconclusions. This work provides a comprehensive understanding of how Sororin functions in cohesion and highlights the\nsequential and non​redundant roles of SMC3 acetylation and Sororin gate​locking: acetylation licenses Sororin recruitment,\nwhile Sororin–CTR engagement locks the exit gate. These findings will be of general interest to the fields of chromosome\nbiology and cell cycle regulation. I enthusiastically recommend publication pending the addressing of the points below.\nMajor points:\n1.In Figure 2, acetyltransferases Esco1 and Esco2 acetylate K105 and K106 in the ATPase head of SMC3 (SMC3-HD)\nduring S phase, which promotes Sororin recruitment to stabilize cohesin on chromatin. Overexpression of the Sororin CTR\nfails to rescue cohesion defects caused by ESCO1/2 knockdown. The authors reason that ESCO1/2-mediated acetylation of\nSMC3 licenses Sororin recruitment, and that engagement of Sororin-CTR subsequently locks the cohesin exit gate. Does\nSMC3 acetylation enhance the interaction between Sororin and the SMC3–RAD21 interface? SMC3 interacts with Sororin\nthrough K1034, K1038, and R1099. Do these amino acids spatially localize near the acetylation sites of SMC3? It may be\nworth to testing whether acetylation-deficient (K105A/K106A) and acetylation-mimetic (K105Q/K106Q) mutations in SMC3-\nHD affect the binding of the SMC3-HD/Rad21-N103 subcomplex to Sororin-CTR and/or full-length Sororin.\n2.In Figure 2 (G, H), tethering Sororin-CTR to centromeres had little effect on cohesion in cells co-depleted of Esco1/2. Does\nSororin-CTR affect its binding to chromatin at the cohesion establishment stage? It would be interesting to test whether\nexpression of H2B-fused Sororin-CTR can rescue cohesion defects in Esco1/2-depleted cells.\n3.Sororin is phosphorylated by mitotic kinases PLK1, CDK1, and Aurora B in vivo, which mediates its degradation. The\nauthors found that Aurora B phosphorylation of Sororin at S145 reduces its interaction with Pds5 in vitro. Is this the case in\nvivo? What’s the order of these phosphorylation events which may mediate dissociation from PDS5 and degradation\nsequentially?\n4.Sgo1 plays a well-established role in sustaining Sororin activity at mitotic centromeres. Figure S2 (A-C) shows that\ntethering Sororin-CTR to centromeres, as a fusion protein with a CENP-B fragment, only partially restored cohesion defects\ncaused by Sgo1 depletion. This is somewhat striking, since Sgo1 is generally regarded as a master protector of centromeric\ncohesion. This result suggests that Sgo1 may have an additional role in protecting cohesion along mitotic chromosome\narms. To test this possibility, the authors should assess the effect of chromatin-tethered Sororin-CTR on cohesion in Sgo1-\ndepleted cells.\n5.Figure 3H shows that, compared to H2B-GFP-expressing cells, the frequency of anaphases with chromosome bridges\nwas strongly increased in H2B-Sororin-CTR-expressing cells, whereas the lagging chromosome rates remained\ncomparable. Given the critical role of centromeric Aurora B kinase in correcting erroneous kinetochore-microtubule\nattachments, the authors should compare Aurora B accumulation at mitotic centromeres in cells expressing H2B-GFP\nversus H2B-Sororin-CTR-GFP.\n6.Figures 4G and S3 show that GST-Sororin-CTR interacted with SMC3-HD only when it formed a subcomplex with the N-\nterminal fragment of RAD21. This suggests that Sororin-CTR binds the cohesin core complex in which the DNA-exit gate\n(i.e, the SMC3-HD/RAD21-N interface) is closed, but not when the gate is open. Because cohesin-mediated sister chromatid\ncohesion is established during S phase, maintained in G2, and largely lost in mitosis, the authors should compare the\nrelative binding efficiency of Sororin-CTR to endogenous cohesin core complex in early S phase versus G2, and in G2\nversus mitosis.\n7.Using bacterially expressed and purified proteins, Figure 4H shows that GST-Sororin-CTR pulled down the complex\nformed between RAD21-N103 and SMC3-HD. This indicates a direct interaction between Sororin-CTR and the\nRAD21/SMC3-HD interface. To corroborate this, the authors should test whether the RAD21-N103/SMC3-HD complex can\ndirectly pull down GST-fused full-length Sororin.\n8.It is well known that Sororin phosphorylation during mitosis promotes cohesin release from chromosome arms. Figure 5A\nshows that Sororin efficiently co-immunoprecipitated RAD21-N103, SMC3-HD, and endogenous Pds5B in G2-phase cell.\nIn mitosis, Sororin binding to RAD21-N103 and SMC3-HD persisted, while the Sororin-Pds5B interaction was disrupted.\nFigure 5B demonstrates that co-inhibition of Cdk/Plk1/Aurora kinase activity during mitosis restored the Sororin-Pds5B\ninteraction but did not affect Sororin binding to RAD21-N103/SMC3-HD. These results imply that Sororin-CTR binding to the\nRAD21/SMC3-HD interface is insensitive to mitotic phosphorylation, in contrast to the Sororin-Pds5B interaction. To\nstrengthen this conclusion, the authors should examine the effect of treating mitotic cell lysates with lambda-phosphatase on\nSororin binding to RAD21-N103/SMC3-HD and Pds5B.\n9.Figure 6 (C, D) shows simultaneous mutation of W233 and E252 to alanine and lysine, respectively disrupts Sororin-CTR\nbinding to the SMC3-HD/RAD21-N103 subcomplex and to endogenous cohesin. A Sororin-E252Q mutation is reported in\nthe cancer genome database. It would be interesting to see to what extent this mutation can affect the binding of full-length\nSororin to SMC3 and RAD21 in cells.\nMinor points:\n1.The statistical results lack critical information: such as what the values represent (±SD or ±SEM), the method of\nsignificance analysis, the corresponding p-values, and the number of biological replicates. Fig. 1D and 1G; Fig. 2D and 2E\nlack significance analysis. Fig. 3G lacks error bars, and no significance analysis was performed for Fig. 3H and 3I. Fig. 7B,\n7G, 7I; Fig. 8I; Fig. 9B, 9E lack significance analysis.\n2.The Methods section states that statistical analyses were performed using GraphPad Prism version 6, but do not specify\nthe exact tests used (e.g., unpaired t-test, ANOVA). This should be clarified.\n3.Could the authors provide the 3D structure of the cohesin complex or protein–protein interactions predicted by\nAlphaFold3?\n4.Some grammars and typos, for instance: “phenocopying Wapl loss” in the Abstract better to be changed to “phenocopying\nthe effects of Wapl loss”. “Dysregulation of cohesin underlie developmental disorders and contribute to tumorigenesis” in the\nIntroduction section should be changed to “Dysregulation of cohesin underlies developmental disorders and contributes to\ntumorigenesis”. “Wapl regulates the dynamic association of cohesin with chromatin” in the Results section better to be\nchanged to “Wapl regulates the dynamics of cohesin's association with chromatin”. “To rule out the possibility that the failure\nof Sororin (1-149)​GFP and Sororin (1-222)​GFP to promote cohesion was due to deficient chromatin binding” in the Results\nsection better to be changed to “To rule out the possibility that the failure of Sororin (1-149)​GFP and Sororin (1-222)​GFP to\npromote cohesion was due to defective chromatin binding”.", "reviewer_3": "The manuscript by Chen et al. investigates the mechanisms by which Sororin functions to stabilize cohesive cohesin on\nDNA. While Sororin's antagonistic role against WAPL-mediated cohesin removal has been recognized, the underlying\nmechanisms remained poorly defined. Previous models proposed that Sororin and WAPL compete for PDS5 binding,\nhowever, this study reveals a different mechanism in which, in addition to interacting with PDS5, Sororin directly interacts\nwith the cohesin complex, functioning as a gatekeeper for the SMC3-RAD21 exit gate. The authors demonstrate that the C-\nterminal domain of Sororin is sufficient to maintain sister chromatid cohesion and that tethering of a C-terminal Sororin\nconstruct to centromeres functionally protects sister chromatid cohesion and counters WAPL-mediated removal. Despite this\nprotective effect, knockdown of ESCO1/2 still results in cohesion defects even when Sororin is tethered to centromeres,\nindicating that cohesin acetylation occurs upstream of Sororin binding and that stabilization of Cohesin by acetylation is\nrequired independently to maintain cohesion. Importantly, when Sororin is tethered along the chromosome arms and cannot\nbe removed, it leads to mitotic segregation defects and abnormal chromosome compaction, indicating the importance of\ndynamic regulation and maintenance of cohesive cohesin at centromeres but not at chromosome arms in mitosis. Through\nimmunoprecipitation experiments and elegant structure-function analyses, the authors demonstrate that Sororin directly\ninteracts with cohesin and define the specific interaction interfaces. Using both in vitro and in vivo binding assays, they map\nthe binding interfaces at the SMC3-RAD21 junction and validate these through reciprocal mutagenesis. Critically, co-\ndepletion of both WAPL and Sororin followed by expression of binding mutants restores cohesion, confirming that exit gate\nlocking is required specifically to block WAPL-mediated unlocking rather than to maintain cohesion directly.\nIn addition to its interaction with cohesin, the authors find that Sororin binds PDS5B in an Aurora phosphorylation-dependent\nmanner, identifying the specific phosphorylation residue and testing this through both in vitro and cell-based kinase assays.\nInterestingly, the phosphorylation of Sororin by Aurora regulates Sororin binding to PDS5B but not to cohesin, indicating that\nthe association of Sororin with cohesin and PDS5 is independent, although the Sororin-cohesin interaction was reduced.\nTogether, this work provides a comprehensive answer to the long-standing question of how Sororin counteracts WAPL-\nmediated cohesin removal. The authors employ multiple complementary approaches to test their model and draw balanced\nconclusions from their data. Given its relevance to genome regulation, human health, and its potential to advance the\ncohesin field, I recommend this manuscript for publication in Nature Communications. I have listed a few minor suggestions\nbelow that could strengthen the manuscript.\nSuggestions:\n1. The authors propose a mechanism in which phosphorylation of Sororin by AURKB releases the Sororin-PDS5 interaction\nwhile maintaining the Sororin-cohesin interaction, providing a switch whereby arm cohesin (but not centromeric cohesin)\ncan be released during early mitosis. They suggest that arm cohesin has less Sororin CTR engagement due to lowered\nSororin levels, allowing for exit gate opening. However, I find this model somewhat confusing: if Sororin is still interacting\nwith PDS5 despite lowered Sororin levels, why would the CTR of these same Sororin proteins not also bind to cohesin in\nthese same complexes? An alternative interpretation is that the pull-down of Sororin with cohesin in the ASE mutants\nreflects Sororin bound to soluble cohesin that is being removed from chromatin, rather than chromatin-bound cohesive\ncohesin. Typically, Sororin only interacts with chromatin-bound cohesin except in cases where cohesin cannot be\nacetylated; however, it is possible the authors are capturing this transient interaction when cohesin is being released. To\nhelp support the authors' proposed model, it would be useful to perform the IP experiments coupled with cellular\nfractionation. Additionally, it would be useful to include chromosome spreads in the ASE mutant in the absence of\nendogenous Sororin to show that cohesive cohesin remains intact specifically at centromeres but not at chromosome arms.\nHowever, it is appreciated that these are complicated experiments, and it would be appropriate to alternatively mention other\npossible hypotheses in the discussion.\n2. The authors find that tethering of Sororin to centromeres can only partially rescue Shugoshin knockdown, consistent with\na model in which Shugoshin-mediated PP2A dephosphorylation blocks the release of the Sororin-PDS5 interaction at\ncentromeres, indicating both interaction with PDS5 and cohesin is required to protect cohesin from premature removal.\nTo further test this regulatory pathway, can a non-phosphorylatable Sororin mutant bypass the requirement for Shugoshin\nprotection, functioning to block Aurora phosphorylation? This experiment would provide additional validation of the\nproposed phosphorylation-dependent switch mechanism and the need for both sororin interaction interfaces to mediate\ncohesive cohesin.\n3. It would be useful to add statistics to the figures throughout.\n4. Typo line 129, “whether selectively retention”.", "reviewer_4": "How sister chromatid cohesion is established and maintained is a key question in the field of chromosome segregation.\nExtensive efforts from many labs have identified the major players in these processes and revealed a lot of basics about how\nthese players work together to regulate sister chromatid cohesion. Among these players are Wapl, Pds5 and Sororin. It has\nbeen believed that the positive factor Sororin competes against factor Wapl for Pds5 binding, thus dictating “on and off” of\nsister chromatid cohesion. It has also been shown that the C-terminal domain of Sororin is required for sister chromatid\ncohesion; but the underlying mechanism is unknown. In addition, it remains elusive how these proposed mechanisms work\ntogether to regulate sister chromatid cohesion. Very interestingly, Chen et.al in this study found that the competition between\nWapl and Sororin on Pds5 may not be that important for the regulation of sister chromatid cohesion. Instead, they revealed a\nnovel mechanism, in which Sororin engage its C-terminal domain to directly binds to “the gate” of the cohesin complex that\nforms between Smc3 and Rad21, which competes against the action of Wapl, thus protecting sister chromatid cohesion.\nThe major conclusions in this paper are solid and largely supported by plenty of data. These conclusions will contribute for us to\nfurther understand the molecular mechanisms of sister chromatid cohesion. The explements were well controlled and\ncarried out. And the paper was well organized and written. Especially, a series of point mutants in cohesin and Sororin\nidentified in this study significantly strengthen their conclusion. I congratulate the authors for this great study. I do not have\nany major concerns and support the publication in NC", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Point-to-point response to reviewer comments\nWe sincerely thank all reviewers for their insightful and constructive comments,\nwhich we have now fully addressed. All the important changes in the revised\nmanuscript are underlined. Please find our detailed, point-by-point responses to the\nreviewers’ comments below (in blue).\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis is a well-designed and carefully executed paper that advances our understanding\nof Sororin’s role in cohesion. The authors propose that Sororin’s C-terminal region\n(CTR) directly locks the cohesin ring at the RAD21-SMC3 exit gate, beyond its\nknown function as a Wapl inhibitor. The experiments are extensive, but many rely on\noverexpression of tagged mutants in HeLa or 293T cells and pull-down assays. While\nthe data broadly support the model, the evidence for a “molecular lock” at the Smc3-\nRad21 gate is still indirect.\nResponse: We thank for the thoughtful and constructive feedback, which has been\ninstrumental in strengthening our manuscript. We appreciate the reviewer for\nacknowledging the study’s design and execution, and for the supportive comment\nregarding our model.\nWe agree that strengthening the direct evidence for our model was essential. In\nresponse to this and other reviewers’ comments, we have significantly expanded our\nexperimental evidence beyond overexpression and pull-down assays:\n1) Chromatin-specific interaction: New chromatin fractionation experiments\ndemonstrate that Sororin-GFP co-immunoprecipitates RAD21, SMC3, and Pds5B\nspecifically from the chromatin-bound fraction of mitotic cells, but not from the\nsoluble fraction (Fig. 4M, N; new data). This confirms that our detected interactions\nare physiologically relevant to chromatin-associated cohesin.\n2) Direct binding with recombinant proteins: We now show that the recombinant\nRAD21-N103/SMC3-HD complex pulls down full-length GST-Sororin (Fig. 4J; new\ndata), corroborating the direct nature of the interaction. Moreover, we demonstrate\nthat GST-Sororin-CTR specifically pulls down the recombinant RAD21-N103/SMC3-\nHD complex (Fig. 4I), and that mutations of residues mediating Sororin-CTR binding\nto the RAD21/SMC3 interface disrupt the direct interaction of Sororin-CTR with the\nRAD21/SMC3 complex (Fig. 6C, H).\n3) Cell-cycle regulated interaction: New data reveal that Sororin-CTR binding to\ncohesin is highly cell-cycle-dependent, being strongest in G2 phase and weak in early\nS phase and mitosis (Fig. 4K, L; new data), aligning perfectly with the window of\n1\ncohesion maintenance.\n4) RNAi-rescue assays: By replacing endogenous Sororin (Fig. 7F-J), RAD21\n(Fig. 7C-E), and SMC3 (Fig. 7H-J; new data) with exogenously expressed proteins,\nwe show that the direct binding of Sororin-CTR to the RAD21/SMC3 interface is\nimportant for the protection of sister-chromatid cohesion in cells.\n5) Validation in non-transformed cells: Key findings have been validated in\nhTERT-RPE1 cells, demonstrating that the cohesion-stabilizing effect of Sororin-CTR\nand the defect of its binding mutants are not cell-line-specific (Supplementary Fig.\n7B-D, H-M; new data).\nThe conclusion that Sororin’s CTR acts independently of SA2 is also difficult to\nreconcile with earlier reports showing Sororin-SA2 interaction. A more nuanced view\nmay be that Sororin interacts with SA2/Pds5 during initial loading or stabilization and\nthen switches to Rad21-Smc3 after cohesin acetylation to secure/strengthen the exit\ngate. This sequential or context-dependent interaction should be considered and could\nbe tested, for example, with the S145A Sororin mutant.\nResponse: We appreciate this insightful suggestion for a more nuanced model.\nIt is important to note that the direct Sororin-SA2 interaction was reported in a\nsingle study (Zhang N. & Pati D., Cell Cycle, 2015, PMID: 25608232) and has not\nbeen widely substantiated in the field. Our new data indicate that the reported Sororin-\nSA2 interaction is mediated indirectly through RAD21:\n1) Sororin-CTR retains binding to SMC1, SMC3, and RAD21 in SA2-knockout\ncells (Fig. 4C, D);\n2) The SA2-D793K mutation, which disrupts its binding to RAD21, also\nabolishes its interaction with Sororin-CTR (Supplementary Fig. 4C, D; new data).\nAs suggested, we have integrated a more nuanced model into the revised\nDiscussion. Our new data show that the phospho-deficient Sororin-S145N mutant\nincreases association with Pds5B, SMC3, and RAD21 specifically in mitosis but not\nin G2 phase (Supplementary Fig. 5F, G; new data). This supports a model where\nSororin first engages Pds5 during cohesion establishment and, following cohesin\nacetylation, its CTR locks the SMC3-RAD21 interface for stabilization (revised Fig.\n9J). Given that the Pds5-binding-deficient Sororin-ASE mutant still associates with\ncohesin core subunits, albeit less efficiently (Fig. 5D, E), we clarify that while the\nPds5 interaction contributes to Sororin’s association, the CTR-mediated gate lock is\nessential and can function independently.\nThe data redefine Sororin as an “autonomous” cohesin lock, yet Sororin’s function is\nstill initiated by Esco1/2-dependent recruitment and cooperation with Pds5.\nEmphasizing how Sororin’s gate engagement is spatiotemporally restricted (only\nbecoming critical at centromeres or after cohesin acetylation) would help readers\ngrasp the mechanistic innovation.\nmechanistic innovation. We have added new data and explicit discussion on this point:\n1) Temporal regulation: As mentioned above, new experiments demonstrate cell\ncycle-specific binding of Sororin-CTR to cohesin (Fig. 4K; new data);\n2) Spatial regulation: Sororin-GFP co-immunoprecipitates RAD21, SMC3, and\nPds5B specifically from the chromatin fraction, but not the soluble fraction, of mitotic\ncells (Fig. 4M, N; new data); moreover, centromere-tethered Sororin-CTR bypasses\nthe requirement for endogenous Sororin in cohesion protection (Fig. 2B-F).\nWe now explicitly discuss this regulated, context-dependent engagement in the\nrevised Discussion, clarifying how Sororin’s role as a lock is deployed specifically at\nthe right time and place.\nFinally, some methodological aspects warrant scrutiny. The artificial tethering\nstrategies (centromeric and chromatin-wide) are powerful tests of sufficiency, but they\nrepresent non-physiological scenarios. For instance, chromatin-wide CTR tethering\nphenocopies Wapl loss with severe condensation and segregation defects, which\nsupports the model but might have off-target effects (e.g. steric hindrance of other\nchromatin processes). The centromere-targeting rescue experiment is convincing, yet\nonly partially compensates for Sgo1 loss, suggesting Sororin-CTR alone cannot fully\nsubstitute all centromeric cohesion safeguards - this nuance should be discussed. In\ngeneral, the experimental evidence does support the central claims that Sororin’s CTR\nis necessary and sufficient to stabilize cohesion by counteracting Wapl-mediated\nrelease; however, the authors should address these points to ensure the conclusions are\nfully justified by the data. In its current form, the work is technically sound and quite\nnovel, but bolstering the direct “lock” evidence and clarifying the model would align\nit better with Nature Communications’ standards of rigor and conceptual insight.\nResponse: We thank the reviewer for appreciating that our evidence supports the\ncentral claims and that the work is technically sound and novel.\nRegarding the potential off-target effects of chromatin tethering, we agree that\nthis is an important consideration. To rule out non-specific steric hindrance, we show\nthat tethering mutants of H2B-Sororin or H2B-Sororin-CTR (FA/FA or WA/EK) that\nare defective in binding the SMC3-RAD21 interface do not cause chromosome\ncondensation or segregation defects (Fig. 7A-C; Supplementary Fig. 7A-J with new\npanels of B-D and H-J). The phenotype is therefore specific to the gate-locking\nfunction.\nRegarding the partial rescue of cohesion in Sgo1-depleted cells by centromere-\ntethered Sororin-CTR, we have added the suggested nuance. In the revised\nmanuscript, we state that “This partial rescue suggests that while centromeric Sororin-\nCTR can mitigate Sgo1 depletion, it cannot fully substitute for Sgo1, which also\nprotects cohesin through PP2A-mediated dephosphorylation”. Furthermore, new data\nshow that constitutive chromatin tethering of Sororin-CTR largely bypasses the need\nfor Sgo1 (Supplementary Fig. 3A, B; new data). We therefore state in the revised\n3\nmanuscript that “This indicates that chromatin-tethered Sororin-CTR can largely\nbypass the requirement for Sgo1, supporting the model that Sgo1 protects cohesin\nboth at centromeres and on chromosome arms”.\nFigure-specific comments:\nFig. 2B: The claim of 100% Sororin knockdown is not realistic; provide the full blot.\nResponse: Corrected. The revised Fig. 2B now shows full blots with longer exposure,\nconfirming that knockdown, while efficient, is not 100%.\nFig. 2G,H: Show siRNA depletion efficiency for Esco1/2.\nResponse: Added. Depletion efficiency for Esco1 is shown by immunoblotting (Fig.\n2I; new data). For Esco2, due to a lack of a suitable antibody, we demonstrate\nknockdown via RT-PCR (Fig. 2J; new data).\nFig. 4: Can Sororin pull down SA1 in SA2 KO cells? If so, how is this distinguished\nfrom the essential Rad21-CTR interaction? Using SA2-deficient cancer lines would\nstrengthen the case.\nResponse: Added. GST-Sororin-CTR retains the ability to bind SA1 in SA2-knockout\ncells (Supplementary Fig. 4A, B; new data). Since both SA1 and SA2 bind RAD21\n(Sumara I. et al., J Cell Biol, 2000, PMID: 11076961; Canudas S. & Smith S., J Cell\nBiol, 2009, PMID: 19822671), we propose that Sororin-CTR pulls down SA1\nindirectly via RAD21, consistent with our finding that the Sororin-SA2 interaction is\nRAD21-dependent.\nThis reviewer remain unconvinced that a Rad21/Smc3 “sub-complex” exists\nphysiologically. This is an intriguing possibility but requires direct verification.\nResponse: We apologize for the misleading terminology. We do not propose a stable,\nfree RAD21-SMC3 sub-complex. The term has been replaced throughout the revised\nmanuscript with “RAD21-SMC3 interface” to accurately reflect our model of Sororin\nbinding to their interaction surface.\nFig. 4A: A full-length Sororin control is missing. Sororin-CTR pulls down Smc1/3,\nRad21, SA2 but not Pds5B; Sororin 1–222 pulls down only Pds5B. Can full-length\nSororin immunoprecipitate all cohesin components?\nResponse: Added. New data show that full-length GST-Sororin pulls down\nendogenous Pds5B, SMC3, SMC1, RAD21, and SA2 (Fig. 4A; new data).\nFurthermore, full-length Sororin-GFP co-immunoprecipitates these cohesin\ncomponents (Fig. 5D, 7E).\nFig. 4F: GST–Sororin-CTR pulls down overexpressed Smc3-GFP/Rad21-Myc\nefficiently, but this may artificially generate a Rad21/Smc3 sub-complex. The authors\nshould repeat with non-overexpressed lysates and probe the stoichiometry of Smc1,\nSmc3, and Rad21 to show whether genuine sub-complexes exist.\nResponse: As clarified above, our model does not involve a stable RAD21-SMC3\n4\nsub-complex. The experiment demonstrates Sororin-CTR’s ability to bind the\nRAD21-SMC3 interface. We have revised the text to avoid any implication of a pre-\nformed sub-complex.\nFig. 5: Can the S145A Sororin mutant still bind Rad21?\nResponse: Added. New co-immunoprecipitation data show that the phospho-deficient\nSororin-S145N mutant exhibits increased association with Pds5B, SMC3, and RAD21\nspecifically in mitosis but not in G2 phase (Supplementary Fig. 5F, G; new data).\nRigor and reproducibility: All functional data are limited to HeLa or 293T cells, with\nno validation in non-transformed or additional lines.\nResponse: We now validate key findings in non-transformed RPE-1 cells:\n1) H2B-Sororin-CTR-GFP (H2B-Sororin-GFP), but not the WA/EK or FA/FA\nmutants, prevented mitotic chromosome arm resolution (Supplementary Fig. 7B-D,\nH-J; new data);\n2) Exogenous wild-type Sororin-SFB, but not the WA/EK or FA/FA mutants,\nrescues cohesion loss after endogenous Sororin depletion (Supplementary Fig. 7K-M;\nnew data).\nThe SMC3(KE/KE) experiment shows only a mild phenotype, likely due to viability\nissues, which weakens the argument for the SMC3 surface. A conditional degron or\ndTAG system would allow a cleaner test at physiological levels. Similarly, the extent\nof Wapl suppression across different mutants (Sororin, Rad21, Smc3) should be\nquantified systematically, and Wapl overexpression tested.\nResponse: We have addressed these points:\n1) After optimizing siRNA transfection to minimize cell death, SMC3 knockdown\nin HeLa cells now yields a clearer cohesion defect (Fig. 8H-J; new data). We state in\nthe revised manuscript that “Depletion of endogenous SMC3 in these cells caused a\ncohesion defect in control cells (Fig. 8H-J), albeit milder than that observed following\nRAD21 depletion, likely due to reduced cell viability upon SMC3 loss. Notably, this\ndefect was rescued by expression of wild-type SMC3-GFP but not by the SMC3-\nKE/KE-GFP mutant, indicating that SMC3 residues mediating Sororin-CTR binding\nare required for proper cohesion”.\n2) We provide new quantification showing that Wapl depletion prevents cohesion\nloss in cells where endogenous SMC3 is replaced with the Sororin-binding-deficient\nSMC3-KE/KE-GFP mutant (Fig. 9G-I; new data).\nIt would also be useful to examine Pds5A versus Pds5B specificity.\nResponse: Added. New data show that GST-Sororin, but not the GST-Sororin-ASE\nmutant, pulls down endogenous Pds5B (but not its paralog Pds5A) from HeLa cell\nlysates (Fig. 5E).\nFinally, the manuscript is very dense. Simplifying terminology and emphasizing the\n5\ntake-home message would help readers. For instance, the term “gate engagement” is\nused repeatedly-defining it clearly up front and explaining how Sororin’s gate-locking\ncomplements Wapl/Pds5 regulation would prevent confusion.\nResponse: We thank the reviewer for this suggestion. We have streamlined\nterminology, clarified key concepts like “gate engagement” upon first use, and\nemphasized the core message throughout the revised text to improve readability and\nconceptual clarity.\nWe believe these comprehensive revisions, which include substantial new data,\nhave significantly bolstered the direct evidence for our model, clarified its nuances,\nand addressed all methodological concerns.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Chen et al. presents a compelling and mechanistically novel\nfunction for Sororin in directly locking the RAD21–SMC3 interface of the cohesin\nring, thereby acting as a guardian of the DNA exit gate to preserve sister-chromatid\ncohesion. The extreme C-terminal region (CTR) of Sororin is essential for the\nmaintenance of sister chromatid cohesion. Artificial tethering of the Sororin CTR to\nchromosome centromeres can rescue cohesion in Sororin-depleted cells, while\ngenome-wide tethering phenocopies Wapl loss, causing abnormal chromosome\ncondensation, decatenation, and segregation. To further investigate the molecular\nmechanism of Sororin’s function, the authors combine AlphaFold3-guided modeling\nwith a series of biochemical assays and identify key conserved residues in Sororin,\nRAD21, and SMC3 that mediate this interaction and demonstrate their functional\nnecessity for cohesion. Furthermore, they dissect the regulation of Sororin, showing\nthat mitotic phosphorylation by Aurora B at S145 selectively disrupts its interaction\nwith Pds5 but not with the RAD21-SMC3 interface. Finally, they demonstrate that the\ncohesion defects caused by disrupting the Sororin-RAD21-SMC3 interaction are\nentirely Wapl-dependent. The authors therefore propose a dual-function model where\nSororin acts both as a competitive displacer of Wapl (via Pds5) and as a direct\nstructural lock for the cohesin exit gate. demonstrated that Sororin directly binds to\nthe SMC3–RAD21 interface.\nFrom these lines of evidence, the authors conclude that Sororin’s CTR directly binds\nthe SMC3–RAD21 interface to seal cohesin’s DNA exit gate. This sealing activity\nworks synergistically with Pds5 competition to balance sister chromatid cohesion\nmaintenance and regulated dissociation. Overall, the in vitro and in vivo data are clear\nand support the conclusions. This work provides a comprehensive understanding of\nhow Sororin functions in cohesion and highlights the sequential and non-redundant\nroles of SMC3 acetylation and Sororin gate-locking: acetylation licenses Sororin\nrecruitment, while Sororin–CTR engagement locks the exit gate. These findings will\nbe of general interest to the fields of chromosome biology and cell cycle regulation. I\nenthusiastically recommend publication pending the addressing of the points below.\nsuggestions. Below are our point-by-point responses and a summary of the\ncorresponding revisions made to the manuscript.\nMajor points:\n1. In Figure 2, acetyltransferases Esco1 and Esco2 acetylate K105 and K106 in the\nATPase head of SMC3 (SMC3-HD) during S phase, which promotes Sororin\nrecruitment to stabilize cohesin on chromatin. Overexpression of the Sororin CTR\nfails to rescue cohesion defects caused by ESCO1/2 knockdown. The authors reason\nthat ESCO1/2-mediated acetylation of SMC3 licenses Sororin recruitment, and that\nengagement of Sororin-CTR subsequently locks the cohesin exit gate. Does SMC3\nacetylation enhance the interaction between Sororin and the SMC3–RAD21 interface?\nSMC3 interacts with Sororin through K1034, K1038, and R1099. Do these amino\nacids spatially localize near the acetylation sites of SMC3? It may be worth to testing\nwhether acetylation-deficient (K105A/K106A) and acetylation-mimetic\n(K105Q/K106Q) mutations in SMC3-HD affect the binding of the SMC3-HD/Rad21-\nN103 subcomplex to Sororin-CTR and/or full-length Sororin.\nResponse: We thank the reviewer for this suggestion. As requested, we tested whether\nSMC3 acetylation influences the Sororin interaction. New data show that neither\nacetylation-deficient (K105A/K106A) nor acetylation-mimetic (K105Q/K106Q)\nmutations in SMC3 affected co-immunoprecipitation of Sororin-GFP with the SMC3-\nHD-Flag/RAD21-N103-Myc complex (Supplementary Fig. 6G; new data).\nConsistently, AlphaFold3 modeling indicates that K105 and K106 are spatially distant\nfrom the Sororin-binding residues (K1034, K1038, R1099) on SMC3 (Supplementary\nFig. 6H; new data). These findings indicate thatSMC3 acetylation licenses Sororin\nrecruitment without directly regulating its binding to the RAD21-SMC3 interface.\n2. In Figure 2 (G, H), tethering Sororin-CTR to centromeres had little effect on\ncohesion in cells co-depleted of Esco1/2. Does Sororin-CTR affect its binding to\nchromatin at the cohesion establishment stage? It would be interesting to test whether\nexpression of H2B-fused Sororin-CTR can rescue cohesion defects in Esco1/2-\ndepleted cells.\nResponse: We appreciate this suggestion. New experiments demonstrate that co-\ndepletion of Esco1/2 causes comparable cohesion defects in both H2B-GFP and H2B-\nSororin-CTR-GFP cells (new Supplementary Fig. 3C-F; new data), demonstrating that\nEsco1/2 perform non-redundant functions that cannot be bypassed by Sororin.\n3. Sororin is phosphorylated by mitotic kinases PLK1, CDK1, and Aurora B in vivo,\nwhich mediates its degradation. The authors found that Aurora B phosphorylation of\nSororin at S145 reduces its interaction with Pds5 in vitro. Is this the case in vivo?\nWhat’s the order of these phosphorylation events which may mediate dissociation\nfrom PDS5 and degradation sequentially?\nResponse: We have addressed the reviewer’s questions as follows:\n7\n1) New co-immunoprecipitation data confirm that the phospho-mimetic Sororin-\nS145E mutant fails to interact with Myc-Pds5B in G2-synchronized cells\n(Supplementary Fig. 5D; new data);\n2) Aurora B inhibition in mitotic cells enhanced Pds5B binding to wild-type\nSororin-GFP, but not to Sororin-S145E-GFP (Supplementary Fig. 5E; new data). This\nconfirms that Aurora B phosphorylation at S145 disrupts the Sororin-Pds5 interaction\nin vivo.\nAdditionally, as the reviewer notes, literature indicates Sororin is phosphorylated\nin prophase/prometaphase (causing Pds5 dissociation) and subsequently degraded in\nanaphase.\n4. Sgo1 plays a well-established role in sustaining Sororin activity at mitotic\ncentromeres. Figure S2 (A-C) shows that tethering Sororin-CTR to centromeres, as a\nfusion protein with a CENP-B fragment, only partially restored cohesion defects\ncaused by Sgo1 depletion. This is somewhat striking, since Sgo1 is generally regarded\nas a master protector of centromeric cohesion. This result suggests that Sgo1 may\nhave an additional role in protecting cohesion along mitotic chromosome arms. To test\nthis possibility, the authors should assess the effect of chromatin-tethered Sororin-\nCTR on cohesion in Sgo1-depleted cells.\nResponse: Thanks for this excellent point. New data show that Sgo1 knockdown\ncaused severe cohesion loss in H2B-GFP cells, but this defect was largely suppressed\nin H2B-Sororin-CTR-GFP cells (Supplementary Fig. 3A, B; new data). This indicates\nthat chromatin-tethered Sororin-CTR can largely bypass the requirement for Sgo1,\nsupporting the model that Sgo1 protects cohesin both at centromeres and on\nchromosome arms.\n5. Figure 3H shows that, compared to H2B-GFP-expressing cells, the frequency of\nanaphases with chromosome bridges was strongly increased in H2B-Sororin-CTR-\nexpressing cells, whereas the lagging chromosome rates remained comparable. Given\nthe critical role of centromeric Aurora B kinase in correcting erroneous kinetochore-\nmicrotubule attachments, the authors should compare Aurora B accumulation at\nmitotic centromeres in cells expressing H2B-GFP versus H2B-Sororin-CTR-GFP.\nResponse: We have added the requested analysis. New data show that centromeric\naccumulation of Aurora B, which is important for correcting erroneous kinetochore-\nmicrotubule attachments, was comparable in cells expressing H2B-GFP and H2B-\nSororin-CTR-GFP (Supplementary Fig. 3G, H; new data), indicating that the\nincreased chromosome bridges do not result from Aurora B mislocalization.\n6. Figures 4G and S3 show that GST-Sororin-CTR interacted with SMC3-HD only\nwhen it formed a subcomplex with the N-terminal fragment of RAD21. This suggests\nthat Sororin-CTR binds the cohesin core complex in which the DNA-exit gate (i.e, the\nSMC3-HD/RAD21-N interface) is closed, but not when the gate is open. Because\ncohesin-mediated sister chromatid cohesion is established during S phase, maintained\nin G2, and largely lost in mitosis, the authors should compare the\nrelative binding efficiency of Sororin-CTR to endogenous cohesin core complex in early S phase\nversus G2, and in G2 versus mitosis.\nResponse: We have performed the suggested experiments. New data demonstrate that\nGST-Sororin-CTR bound endogenous SMC3, SMC1, and RAD21 more efficiently in\nG2-phase cell lysates than in early S-phase lysates (Fig. 4K; new data). Furthermore,\nthis interaction persisted in late-G2 phases but was markedly reduced in mitotic cells\n(Fig. 4L; new data). Thus, Sororin-CTR binding to cohesin peaks during the\ncohesion-maintenance phase of the cell cycle.\n7. Using bacterially expressed and purified proteins, Figure 4H shows that GST-\nSororin-CTR pulled down the complex formed between RAD21-N103 and SMC3-\nHD. This indicates a direct interaction between Sororin-CTR and the\nRAD21/SMC3-HD interface. To corroborate this, the authors should test whether the\nRAD21-N103/SMC3-HD complex can directly pull down GST-fused full-length Sororin.\nResponse: We have added this corroborating experiment. Using purified recombinant\nproteins, we show that the RAD21-N103/SMC3-HD complex directly pulls down\nGST-fused full-length Sororin (Fig. 4J; new data). This confirms that full-length\nSororin directly engages the RAD21-SMC3 interface.\n8. It is well known that Sororin phosphorylation during mitosis promotes cohesin\nrelease from chromosome arms. Figure 5A shows that Sororin efficiently co-\nimmunoprecipitated RAD21-N103, SMC3-HD, and endogenous Pds5B in G2-phase\ncell. In mitosis, Sororin binding to RAD21-N103 and SMC3-HD persisted, while the\nSororin-Pds5B interaction was disrupted. Figure 5B demonstrates that co-inhibition of\nCdk/Plk1/Aurora kinase activity during mitosis restored the Sororin-Pds5B\ninteraction but did not affect Sororin binding to RAD21-N103/SMC3-HD. These results imply\nthat Sororin-CTR binding to the RAD21/SMC3-HD interface is insensitive to mitotic\nphosphorylation, in contrast to the Sororin-Pds5B interaction. To strengthen this\nconclusion, the authors should examine the effect of treating mitotic cell lysates with\nlambda-phosphatase on Sororin binding to RAD21-N103/SMC3-HD and Pds5B.\nResponse: As suggested, we treated nocodazole-treated HEK-293T cell lysates with λ-\nphosphatase. New data show this treatment markedly enhances the Sororin-Pds5B co-\nimmunoprecipitation, without affecting interactions with RAD21-N103 or SMC3-HD\n(Fig. 5B; new data). This strengthens our conclusion that the Sororin-Pds5 interaction\nis phosphorylation-sensitive, while Sororin’s binding to the RAD21-SMC3 interface\nis not.\n9. Figure 6 (C, D) shows simultaneous mutation of W233 and E252 to alanine and\nlysine, respectively disrupts Sororin-CTR binding to the SMC3-HD/RAD21-N103\nsubcomplex and to endogenous cohesin. A Sororin-E252Q mutation is reported in the\ncancer genome database. It would be interesting to see to what extent this mutation\ncan affect the binding of full-length Sororin to SMC3 and RAD21 in cells.\nResponse: We tested a cancer-associated Sororin-E252Q mutation. New data show\nthis mutation reduces the co-precipitation of SMC3 and RAD21 with full-length\nSororin-Flag in cells (Supplementary Fig. 6C; new data), suggesting that this mutation\npartially compromises Sororin’s gate-locking function and may contribute to genomic\ninstability.\nMinor points:\n1. The statistical results lack critical information: such as what the values represent\n(±SD or ±SEM), the method of significance analysis, the corresponding p-values, and\nthe number of biological replicates. Fig. 1D and 1G; Fig. 2D and 2E lack significance\nanalysis. Fig. 3G lacks error bars, and no significance analysis was performed for Fig.\n3H and 3I. Fig. 7B, 7G, 7I; Fig. 8I; Fig. 9B, 9E lack significance analysis.\nResponse: We have updated all figures to include error bars (± standard deviation),\nspecified statistical tests (unpaired two-tailed t-test), provided exact p-values, and\nindicated the number of biological replicates (n).\n2. The Methods section states that statistical analyses were performed using GraphPad\nPrism version 6, but do not specify the exact tests used (e.g., unpaired t-test,\nANOVA). This should be clarified.\nResponse: The Methods section now specifies the exact statistical tests used for each\nanalysis.\n3. Could the authors provide the 3D structure of the cohesin complex or protein–\nprotein interactions predicted by AlphaFold3?\nResponse: An AlphaFold3-predicted 3D structure of a Sororin-CTR peptide binding to\nthe interface between RAD21-NHD and SMC3-HD is now provided as\nSupplementary Data 2.\n4. Some grammars and typos, for instance: “phenocopying Wapl loss” in the Abstract\nbetter to be changed to “phenocopying the effects of Wapl loss”. “Dysregulation of\ncohesin underlie developmental disorders and contribute to tumorigenesis” in the\nIntroduction section should be changed to “Dysregulation of cohesin underlies\ndevelopmental disorders and contributes to tumorigenesis”. “Wapl regulates the\ndynamic association of cohesin with chromatin” in the Results section better to be\nchanged to “Wapl regulates the dynamics of cohesin's association with chromatin”.\n“To rule out the possibility that the failure of Sororin (1-149)-GFP and Sororin (1-\n222)-GFP to promote cohesion was due to deficient chromatin binding” in the Results\nsection better to be changed to “To rule out the possibility that the failure of Sororin (1-149)-GFP and Sororin (1-222)-GFP to promote cohesion was due to defective\nchromatin binding”.\nResponse: All grammatical errors and typos have been corrected.\nWe thank the reviewer again for their constructive feedback, which has\nsignificantly strengthened the manuscript.\nThe manuscript by Chen et al. investigates the mechanisms by which Sororin\nfunctions to stabilize cohesive cohesin on DNA. While Sororin's antagonistic role\nagainst WAPL-mediated cohesin removal has been recognized, the underlying\nmechanisms remained poorly defined. Previous models proposed that Sororin and\nWAPL compete for PDS5 binding, however, this study reveals a different mechanism\nin which, in addition to interacting with PDS5, Sororin directly interacts with the\ncohesin complex, functioning as a gatekeeper for the SMC3-RAD21 exit gate. The\nauthors demonstrate that the C-terminal domain of Sororin is sufficient to maintain\nsister chromatid cohesion and that tethering of a C-terminal Sororin construct to\ncentromeres functionally protects sister chromatid cohesion and counters WAPL-\nmediated removal. Despite this protective effect, knockdown of ESCO1/2 still results\nin cohesion defects even when Sororin is tethered to centromeres, indicating that\ncohesin acetylation occurs upstream of Sororin binding and that stabilization of\nCohesin by acetylation is required independently to maintain cohesion. Importantly,\nwhen Sororin is tethered along the chromosome arms and cannot be removed, it leads\nto mitotic segregation defects and abnormal chromosome compaction, indicating the\nimportance of dynamic regulation and maintenance of cohesive cohesin at\ncentromeres but not at chromosome arms in mitosis. Through immunoprecipitation\nexperiments and elegant structure-function analyses, the authors demonstrate that\nSororin directly interacts with cohesin and define the specific interaction interfaces.\nUsing both in vitro and in vivo binding assays, they map the binding interfaces at the\nSMC3-RAD21 junction and validate these through reciprocal mutagenesis. Critically,\nco-depletion of both WAPL and Sororin followed by expression of binding mutants\nrestores cohesion, confirming that exit gate locking is required specifically to block\nWAPL-mediated unlocking rather than to maintain cohesion directly.\nIn addition to its interaction with cohesin, the authors find that Sororin binds PDS5B\nin an Aurora phosphorylation-dependent manner, identifying the specific\nphosphorylation residue and testing this through both in vitro and cell-based kinase\nassays. Interestingly, the phosphorylation of Sororin by Aurora regulates Sororin\nbinding to PDS5B but not to cohesin, indicating that the association of Sororin with\ncohesin and PDS5 is independent, although the Sororin-cohesin interaction was\nreduced.\nTogether, this work provides a comprehensive answer to the long-standing question of\nhow Sororin counteracts WAPL-mediated cohesin removal. The authors employ\nmultiple complementary approaches to test their model and draw balanced\nconclusions from their data. Given its relevance to genome regulation, human health,\nand its potential to advance the cohesin field, I recommend this manuscript for\npublication in Nature Communications. I have listed a few minor suggestions below\nthat could strengthen the manuscript.\nResponse: We thank the reviewer for the supportive assessment and insightful\nsuggestions, which have helped us refine our model and strengthen the manuscript.\nSuggestions:\n1. The authors propose a mechanism in which phosphorylation of Sororin by AURKB\nreleases the Sororin-PDS5 interaction while maintaining the Sororin-cohesin\ninteraction, providing a switch whereby arm cohesin (but not centromeric cohesin)\ncan be released during early mitosis. They suggest that arm cohesin has less Sororin\nCTR engagement due to lowered Sororin levels, allowing for exit gate opening.\nHowever, I find this model somewhat confusing: if Sororin is still interacting with\nPDS5 despite lowered Sororin levels, why would the CTR of these same Sororin\nproteins not also bind to cohesin in these same complexes? An alternative\ninterpretation is that the pull-down of Sororin with cohesin in the ASE mutants\nreflects Sororin bound to soluble cohesin that is being removed from chromatin,\nrather than chromatin-bound cohesive cohesin. Typically, Sororin only interacts with\nchromatin-bound cohesin except in cases where cohesin cannot be acetylated;\nhowever, it is possible the authors are capturing this transient interaction when\ncohesin is being released. To help support the authors’ proposed model, it would be\nuseful to perform the IP experiments coupled with cellular fractionation. Additionally,\nit would be useful to include chromosome spreads in the ASE mutant in the absence of\nendogenous Sororin to show that cohesive cohesin remains intact specifically at\ncentromeres but not at chromosome arms. However, it is appreciated that these are\ncomplicated experiments, and it would be appropriate to alternatively mention other\npossible hypotheses in the discussion.\nResponse: We thank the reviewer for this thoughtful suggestion, which prompted us to\nfurther test our model. To distinguish chromatin-bound from soluble cohesin pools,\nwe performed chromatin fractionation followed by co-immunoprecipitation. Sororin-\nGFP stably expressed in HeLa cells co-precipitates RAD21, SMC3, and Pds5B\nspecifically from the chromatin-bound fraction of mitotic cells, but not from the\nsoluble fraction (Fig. 4M, N; new data). This confirms that the Sororin-cohesin\ninteractions we detect are primarily associated with chromatin-bound cohesin, not\nwith a soluble pool being removed. This finding supports our proposed model\nwherein centromeric Sororin remains stably engaged with chromatin-bound cohesin\nvia both its Pds5-binding motif and CTR, while on chromosome arms, Aurora B\nphosphorylation promotes its dissociation from Pds5 and cohesin release.\n2. The authors find that tethering of Sororin to centromeres can only partially rescue\nShugoshin knockdown, consistent with a model in which Shugoshin-mediated PP2A\ndephosphorylation blocks the release of the Sororin-PDS5 interaction at\ncentromeres, indicating both interaction with PDS5 and cohesin is required to protect\ncohesin from premature removal. To further test this regulatory pathway, can a non-\nphosphorylatable Sororin mutant bypass the requirement for Shugoshin protection,\nfunctioning to block Aurora phosphorylation? This experiment would provide\nadditional validation of the proposed phosphorylation-dependent switch mechanism\nand the need for both sororin interaction interfaces to mediate cohesive cohesin.\nexhibited enhances association with Pds5B, SMC3, and RAD21 compared with wild-\ntype Sororin, whereas no difference is observed in G2-phase cells (Supplementary\nFig. 5F, G; new data). Because Sgo1-associated PP2A protects centromeric cohesion\nby dephosphorylating Sororin, we examined whether the S145N mutant could bypass\nSgo1 depletion. Although the S145N mutant partially rescued the cohesion defect\ncaused by Sgo1 knockdown, it does not fully restore cohesion (Supplementary Fig.\n5H-J; new data). This indicates that preservation of the Sororin-Pds5 interaction via\nS145 dephosphorylation represents a major, but not exclusive, function of Sgo1-PP2A\nin cohesion protection. We have incorporated this nuance into the revised manuscript.\n3. It would be useful to add statistics to the figures throughout.\nResponse: We have added statistics to the figures throughout.\n4. Typo line 129, “whether selectively retention”.\nResponse: All typos have been corrected.\nReviewer #4 (Remarks to the Author):\nHow sister chromatid cohesion is established and maintained is a key question in the\nfield of chromosome segregation. Extensive efforts from many labs have identified\nthe major players in these processes and revealed a lot of basics about how these\nplayers work together to regulate sister chromatid cohesion. Among these players are\nWapl, Pds5 and Sororin. It has been believed that the positive factor Sororin competes\nagainst factor Wapl for Pds5 binding, thus dictating “on and off” of sister chromatid\ncohesion. It has also been shown that the C-terminal domain of Sororin is required for\nsister chromatid cohesion; but the underlying mechanism is unknown. In addition, it\nremains elusive how these proposed mechanisms work together to regulate sister\nchromatid cohesion. Very interestingly, Chen et.al in this study found that the\ncompetition between Wapl and Sororin on Pds5 may not be that important for the\nregulation of sister chromatid cohesion. Instead, they revealed a novel mechanism, in\nwhich Sororin engage its C-terminal domain to directly binds to “the gate” of the\ncohesin complex that forms between Smc3 and Rad21, which competes against the\naction of Wapl, thus protecting sister chromatid cohesion. The major conclusions in\nthis paper are solid and largely supported by plenty of data. These conclusions will\ncontribute for us to further understand the molecular mechanisms of sister chromatid\ncohesion. The explements were well controlled and carried out. And the paper was\nwell organized and written. Especially, a series of point mutants in cohesin and\nSororin identified in this study significantly strengthen their conclusion. I congratulate\nthe authors for this great study. I do not have any major concerns and support the\npublication in NC.\nResponse: We thank the reviewer for the positive and insightful evaluation of our\nmanuscript. We are grateful for the acknowledgment that our work reveals a novel\n13\nmechanistic role for Sororin’s C-terminal domain and solidifies our understanding of\ncohesion maintenance.\nThe reviewer correctly identifies the key contributions of our study: moving\nbeyond the established model of Sororin-Wapl competition for Pds5 binding, and\ninstead providing direct evidence that Sororin acts as a molecular lock at the SMC3-\nRAD21 exit gate to antagonize Wapl and protect cohesion. We are pleased that the\nreviewer found the conclusions solid, the data plentiful and well-controlled, and the\nuse of precise point mutants to be particularly strengthening.\nMinor points:\n1. The authors already demonstrated that the expression of the two C-terminal\ndomains are able to close the chromosome arm. Especially, for the smaller C-terminal\ndomain (223-252), I believe that most of regulatory domains or residues in Sororin\nhave been deleted out, for example, the Cdk1 and Aurora B phosphorylation sites.\nWhether this fragment will always bind Smc3/Scc1 throughout the cell cycle? Does it\nalso generate anaphase bridges just like the H2B-fused fragment? It would be nice if\nauthor have the data. Otherwise, a brief discussion would also be fine.\nResponse: We appreciate this excellent question regarding the behavior of the\nminimal Sororin-CTR fragment. We have addressed this point with new experiments\nexamining its cell cycle-dependent binding and functional consequences.\n1) New pull-down assays reveal that GST-Sororin-CTR bound endogenous\nSMC3, SMC1, and RAD21 more efficiently in G2-phase cell lysates than in early S-\nphase lysates (Fig. 4K; new data). Furthermore, this interaction persists in late-G2\nphases but is markedly reduced in mitotic cells (Fig. 4L; new data). Thus, Sororin-\nCTR binding to cohesin peaks during the\ncohesion-maintenance phase of the cell cycle.\n2) As the reviewer hypothesized, cells overexpressing the Sororin-CTR-GFP\nfragment (lacking N-terminal regulatory domains) also exhibit a significant increase\nin anaphase chromosome bridges (Supplementary Fig. 3I-K; new data), linking these\ndefects directly to the CTR’s cohesion-stabilizing activity.\nThese data collectively support the model that the Sororin CTR functions as a\ncritical, cell cycle-regulated effector whose engagement with the RAD21-SMC3\ninterface is sufficient to stabilize cohesin and whose dysregulated retention causes\nsegregation defects.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nfor the co-assessment of our manuscript.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my concerns with significant new data that substantially\nimprove the rigor of the manuscript. I have a few general comments. The figure\nlegends require further improvement so that readers can fully appreciate and interpret\nthe data without having to refer back to the Results section. For example, in Fig. 4M\n(top two panels), “Sororin-GFP,” “S.” and “L.” should be explicitly defined in the\nlegend rather than left to assumption.\nResponse: We thank the reviewer for the positive assessment and for highlighting this\nimportant point regarding clarity. We have now thoroughly revised all figure legends\nthroughout the manuscript to ensure they are comprehensive and self-sufficient.\nSpecifically, for Figure 4M, N, we have amended the legend to explicitly state: “HeLa\ncells stably expressing Sororin-GFP were synchronized in mitosis by nocodazole\ntreatment for 16 h and subjected to chromatin fractionation. A portion of the whole\ncell lysate (WCL), supernatant, and chromatin pellet was analyzed by immunoblotting\nfor histone H3, GAPDH, GFP, RAD21, SMC3, and Pds5B (M). The remaining\nchromatin pellet and 5% of the supernatant were subjected to immunoprecipitation\nwith anti-GFP beads, followed by immunoblotting for GFP, RAD21, SMC3, and\nPds5B (N). S., short exposure; L., long exposure.”\nThis revision has been applied consistently across all figures to enhance reader\ncomprehension without necessitating referral to the main text.\nReviewer #2 (Remarks to the Author):\nAll comments have been addressed with new data. It's now solid and ready for\npublication.\nResponse: We thank the reviewer for the positive and conclusive evaluation of our\nrevised work.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Chen et al. addresses a long standing question of how Sororin acts\nto counteract WAPl based cohesin removal for the establishment of sister chromatid\ncohesion. In this new revised version of the manuscript the authors have added a\nnumber of experiments, included new analysis and statistics, and importantly added in\nadditional discussion points that together has significantly strengthened the work.\nWith these changes all of our concerns have been addressed and we would\n1\nrecommend this manuscript for publication in Nature Communications in its current\nform.\nResponse: We are grateful to the reviewer for recognizing the significance of our\nstudy and for the encouraging summary. We appreciate their affirmation that the new\nexperiments, analyses, and discussions have satisfactorily addressed their previous\nconcerns.\nReviewer #4 (Remarks to the Author):\nI am satisfied with the authors’ response.\nResponse: We thank the reviewer for the time and for confirming their satisfaction.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse: We acknowledge the contribution of the early-career researcher and thank\nthem for their participation in the review process.\n2"}]

Decision letter