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A handcuff model for the cohesin complex.

Zhang N, Kuznetsov SG, Sharan SK, Li K, Rao PH, Pati D - J. Cell Biol. (2008)

Bottom Line: Several models for the cohesin complex have been proposed, but the one-ring embrace model currently predominates the field.The results show that three of the four human cohesin core subunits (Smc1, Smc3, and Rad21) interact with themselves in an Scc3 (SA1/SA2)-dependent manner.These data support a two-ring handcuff model for the cohesin complex, which is flexible enough to establish and maintain sister chromatid cohesion as well as ensure the fidelity of chromosome segregation in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Hematology/Oncology, Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The cohesin complex is responsible for the accurate separation of sister chromatids into two daughter cells. Several models for the cohesin complex have been proposed, but the one-ring embrace model currently predominates the field. However, the static configuration of the embrace model is not flexible enough for cohesins to perform their functions during DNA replication, transcription, and DNA repair. We used coimmunoprecipitation, a protein fragment complement assay, and a yeast two-hybrid assay to analyze the protein-protein interactions among cohesin subunits. The results show that three of the four human cohesin core subunits (Smc1, Smc3, and Rad21) interact with themselves in an Scc3 (SA1/SA2)-dependent manner. These data support a two-ring handcuff model for the cohesin complex, which is flexible enough to establish and maintain sister chromatid cohesion as well as ensure the fidelity of chromosome segregation in higher eukaryotes.

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Knockdown of SA1/SA2 disrupts the Rad21–Rad21 interaction and the formation of the two-ring cohesins. (A and B) SA1 and SA2 are knocked down by respective siRNA 24 h after transfection with tagged Rad21 into 293T cells. (A) Myc-Rad21 cannot coimmunoprecipitate endogenous Rad21 when SA2 is knocked down. (B) Myc-Rad21 and Flag-Rad21 cannot reciprocally coimmunoprecipitate each other when SA2 is down-regulated. (C) Sucrose gradient centrifugation to study cohesin–cohesin interaction after SA2 knockdown. 293T cells were transfected with SA2 siRNA or control siRNA, and, 24 h later, the cells were cotransfected with Myc-Rad21 and Flag-Rad21 for 40 h. Cell lysates were prepared and used in sucrose gradient centrifugation. Rad21, Smc3, and SA2 were analyzed using WB. Sedimentation coefficient is shown on the top of the blot. Input, sample before sucrose gradient centrifugation. (D) Dissociation of cohesin from sister chromatids in SA2 knockdown cells. HeLa cells in the mitosis phase were cytospun onto slides, and immunofluorescent microscopy was performed. Rad21 mAb and human CREST antibodies were used to probe Rad21 (red) and centromere (green), respectively. The nuclear material is visualized by DAPI staining (blue). The centromeres of one chromosome shown in the boxes of merge panels are enlarged on the right. (A and C) Black lines indicate that intervening lanes have been spliced out. Bar, 10 μm.
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fig6: Knockdown of SA1/SA2 disrupts the Rad21–Rad21 interaction and the formation of the two-ring cohesins. (A and B) SA1 and SA2 are knocked down by respective siRNA 24 h after transfection with tagged Rad21 into 293T cells. (A) Myc-Rad21 cannot coimmunoprecipitate endogenous Rad21 when SA2 is knocked down. (B) Myc-Rad21 and Flag-Rad21 cannot reciprocally coimmunoprecipitate each other when SA2 is down-regulated. (C) Sucrose gradient centrifugation to study cohesin–cohesin interaction after SA2 knockdown. 293T cells were transfected with SA2 siRNA or control siRNA, and, 24 h later, the cells were cotransfected with Myc-Rad21 and Flag-Rad21 for 40 h. Cell lysates were prepared and used in sucrose gradient centrifugation. Rad21, Smc3, and SA2 were analyzed using WB. Sedimentation coefficient is shown on the top of the blot. Input, sample before sucrose gradient centrifugation. (D) Dissociation of cohesin from sister chromatids in SA2 knockdown cells. HeLa cells in the mitosis phase were cytospun onto slides, and immunofluorescent microscopy was performed. Rad21 mAb and human CREST antibodies were used to probe Rad21 (red) and centromere (green), respectively. The nuclear material is visualized by DAPI staining (blue). The centromeres of one chromosome shown in the boxes of merge panels are enlarged on the right. (A and C) Black lines indicate that intervening lanes have been spliced out. Bar, 10 μm.

Mentions: Our IP data indicate that the interaction of Rad21 with Rad21 (Figs. S2 and S3) or full-length Rad21 with some truncated Rad21 products (not depicted) is always associated with SA1/SA2. We hypothesized that SA1 or SA2 is one of the linkers of the two Rad21 molecules of the cohesin rings and tested this hypothesis using SA1 and SA2 siRNA. SA1 and SA2 proteins are reduced by 65% and 75%, respectively, with siRNA treatment (Fig. 6, A [lanes 3 and 4] and B [lanes 2 and 3]). In multiple experiments, co-IP of endogenous Rad21 by Myc-Rad21 was significantly reduced by SA2 inhibition, whereas SA1 siRNA treatment had a lesser effect (Fig. 6 A). We performed an additional experiment by cotransfecting Flag-Rad21 and Myc-Rad21 and coimmunoprecipitating the epitope-tagged Rad21 (Fig. 6 B). Similar to the results shown in Fig. 6 A, in this cotransfection experiment, the knockdown of SA2 by siRNA blocked the co-IP of Flag-Rad21 and Myc-Rad21 (Fig. 6 B, lanes 7, 8, 11, and 12). These results suggest that SA2 may play a role in locking the two associated Rad21 molecules, and they are also consistent with the data that SA2 is the dominant form of Scc3 in human somatic cells (Losada et al., 2000). It is interesting to note that although the interaction between the two rings is lost, the three core subunits, Rad21, Smc1, and Smc3, remained associated, as Myc-Rad21 could still coimmunoprecipitate Smc1 and Smc3 (Fig. 6, A [lanes 9 and 10] and B [lanes 7, 8, 11, and 12]), suggesting an intact one-ring cohesin complex. The immunofluorescence microscopy also demonstrates that Rad21 and Smc3 colocalize in SA1 and SA2 knockdown cells (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200801157/DC1), further supporting the association of Rad21, Smc1, and Smc3 and suggesting that both one-ring and two-ring cohesin complexes can exist in the same cells.


A handcuff model for the cohesin complex.

Zhang N, Kuznetsov SG, Sharan SK, Li K, Rao PH, Pati D - J. Cell Biol. (2008)

Knockdown of SA1/SA2 disrupts the Rad21–Rad21 interaction and the formation of the two-ring cohesins. (A and B) SA1 and SA2 are knocked down by respective siRNA 24 h after transfection with tagged Rad21 into 293T cells. (A) Myc-Rad21 cannot coimmunoprecipitate endogenous Rad21 when SA2 is knocked down. (B) Myc-Rad21 and Flag-Rad21 cannot reciprocally coimmunoprecipitate each other when SA2 is down-regulated. (C) Sucrose gradient centrifugation to study cohesin–cohesin interaction after SA2 knockdown. 293T cells were transfected with SA2 siRNA or control siRNA, and, 24 h later, the cells were cotransfected with Myc-Rad21 and Flag-Rad21 for 40 h. Cell lysates were prepared and used in sucrose gradient centrifugation. Rad21, Smc3, and SA2 were analyzed using WB. Sedimentation coefficient is shown on the top of the blot. Input, sample before sucrose gradient centrifugation. (D) Dissociation of cohesin from sister chromatids in SA2 knockdown cells. HeLa cells in the mitosis phase were cytospun onto slides, and immunofluorescent microscopy was performed. Rad21 mAb and human CREST antibodies were used to probe Rad21 (red) and centromere (green), respectively. The nuclear material is visualized by DAPI staining (blue). The centromeres of one chromosome shown in the boxes of merge panels are enlarged on the right. (A and C) Black lines indicate that intervening lanes have been spliced out. Bar, 10 μm.
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fig6: Knockdown of SA1/SA2 disrupts the Rad21–Rad21 interaction and the formation of the two-ring cohesins. (A and B) SA1 and SA2 are knocked down by respective siRNA 24 h after transfection with tagged Rad21 into 293T cells. (A) Myc-Rad21 cannot coimmunoprecipitate endogenous Rad21 when SA2 is knocked down. (B) Myc-Rad21 and Flag-Rad21 cannot reciprocally coimmunoprecipitate each other when SA2 is down-regulated. (C) Sucrose gradient centrifugation to study cohesin–cohesin interaction after SA2 knockdown. 293T cells were transfected with SA2 siRNA or control siRNA, and, 24 h later, the cells were cotransfected with Myc-Rad21 and Flag-Rad21 for 40 h. Cell lysates were prepared and used in sucrose gradient centrifugation. Rad21, Smc3, and SA2 were analyzed using WB. Sedimentation coefficient is shown on the top of the blot. Input, sample before sucrose gradient centrifugation. (D) Dissociation of cohesin from sister chromatids in SA2 knockdown cells. HeLa cells in the mitosis phase were cytospun onto slides, and immunofluorescent microscopy was performed. Rad21 mAb and human CREST antibodies were used to probe Rad21 (red) and centromere (green), respectively. The nuclear material is visualized by DAPI staining (blue). The centromeres of one chromosome shown in the boxes of merge panels are enlarged on the right. (A and C) Black lines indicate that intervening lanes have been spliced out. Bar, 10 μm.
Mentions: Our IP data indicate that the interaction of Rad21 with Rad21 (Figs. S2 and S3) or full-length Rad21 with some truncated Rad21 products (not depicted) is always associated with SA1/SA2. We hypothesized that SA1 or SA2 is one of the linkers of the two Rad21 molecules of the cohesin rings and tested this hypothesis using SA1 and SA2 siRNA. SA1 and SA2 proteins are reduced by 65% and 75%, respectively, with siRNA treatment (Fig. 6, A [lanes 3 and 4] and B [lanes 2 and 3]). In multiple experiments, co-IP of endogenous Rad21 by Myc-Rad21 was significantly reduced by SA2 inhibition, whereas SA1 siRNA treatment had a lesser effect (Fig. 6 A). We performed an additional experiment by cotransfecting Flag-Rad21 and Myc-Rad21 and coimmunoprecipitating the epitope-tagged Rad21 (Fig. 6 B). Similar to the results shown in Fig. 6 A, in this cotransfection experiment, the knockdown of SA2 by siRNA blocked the co-IP of Flag-Rad21 and Myc-Rad21 (Fig. 6 B, lanes 7, 8, 11, and 12). These results suggest that SA2 may play a role in locking the two associated Rad21 molecules, and they are also consistent with the data that SA2 is the dominant form of Scc3 in human somatic cells (Losada et al., 2000). It is interesting to note that although the interaction between the two rings is lost, the three core subunits, Rad21, Smc1, and Smc3, remained associated, as Myc-Rad21 could still coimmunoprecipitate Smc1 and Smc3 (Fig. 6, A [lanes 9 and 10] and B [lanes 7, 8, 11, and 12]), suggesting an intact one-ring cohesin complex. The immunofluorescence microscopy also demonstrates that Rad21 and Smc3 colocalize in SA1 and SA2 knockdown cells (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200801157/DC1), further supporting the association of Rad21, Smc1, and Smc3 and suggesting that both one-ring and two-ring cohesin complexes can exist in the same cells.

Bottom Line: Several models for the cohesin complex have been proposed, but the one-ring embrace model currently predominates the field.The results show that three of the four human cohesin core subunits (Smc1, Smc3, and Rad21) interact with themselves in an Scc3 (SA1/SA2)-dependent manner.These data support a two-ring handcuff model for the cohesin complex, which is flexible enough to establish and maintain sister chromatid cohesion as well as ensure the fidelity of chromosome segregation in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Hematology/Oncology, Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The cohesin complex is responsible for the accurate separation of sister chromatids into two daughter cells. Several models for the cohesin complex have been proposed, but the one-ring embrace model currently predominates the field. However, the static configuration of the embrace model is not flexible enough for cohesins to perform their functions during DNA replication, transcription, and DNA repair. We used coimmunoprecipitation, a protein fragment complement assay, and a yeast two-hybrid assay to analyze the protein-protein interactions among cohesin subunits. The results show that three of the four human cohesin core subunits (Smc1, Smc3, and Rad21) interact with themselves in an Scc3 (SA1/SA2)-dependent manner. These data support a two-ring handcuff model for the cohesin complex, which is flexible enough to establish and maintain sister chromatid cohesion as well as ensure the fidelity of chromosome segregation in higher eukaryotes.

Show MeSH
Related in: MedlinePlus