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Characterization of vertebrate cohesin complexes and their regulation in prophase.

Sumara I, Vorlaufer E, Gieffers C, Peters BH, Peters JM - J. Cell Biol. (2000)

Bottom Line: SA1 is also a subunit of 14S cohesin in Xenopus.The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase.These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.

ABSTRACT
In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

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Reconstitution of mitosis-specific dissociation of human 14S cohesin complexes and of PDS5 from chromatin in Xenopus egg extracts. (A) HeLa chromatin was incubated in Xenopus interphase extract. The extract was either supplemented with nondegradable cyclin B Δ90 to trigger entry into mitosis (left) or left untreated (right). At different time points either extract samples (top) or chromatin reisolated from the extract by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (top) or immunoblotting with antibodies to the indicated proteins (bottom). The cell cycle state of the extracts was analyzed by monitoring the phosphorylation-dependent electrophoretic mobility shift of 35S-labeled CDC25 and the stability of 35S-labeled cyclin B, which were added to the extracts at time zero. TOPO II, topoisomerase II; H3P, histone H3 phosphorylated on serine 10. (B) HeLa chromatin was incubated in mitotic Xenopus egg extract (xtΔ90), or in mitotic extract treated with 0.8 mM roscovitin (XΔ90+ Roscovitin), or in interphase extract (xti). Chromatin bound proteins were isolated at different time points and analyzed as in A.
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Figure 5: Reconstitution of mitosis-specific dissociation of human 14S cohesin complexes and of PDS5 from chromatin in Xenopus egg extracts. (A) HeLa chromatin was incubated in Xenopus interphase extract. The extract was either supplemented with nondegradable cyclin B Δ90 to trigger entry into mitosis (left) or left untreated (right). At different time points either extract samples (top) or chromatin reisolated from the extract by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (top) or immunoblotting with antibodies to the indicated proteins (bottom). The cell cycle state of the extracts was analyzed by monitoring the phosphorylation-dependent electrophoretic mobility shift of 35S-labeled CDC25 and the stability of 35S-labeled cyclin B, which were added to the extracts at time zero. TOPO II, topoisomerase II; H3P, histone H3 phosphorylated on serine 10. (B) HeLa chromatin was incubated in mitotic Xenopus egg extract (xtΔ90), or in mitotic extract treated with 0.8 mM roscovitin (XΔ90+ Roscovitin), or in interphase extract (xti). Chromatin bound proteins were isolated at different time points and analyzed as in A.

Mentions: Yeast cohesin dissociates from chromatin at the onset of anaphase, whereas Xenopus 14S cohesin dissociates from chromatin already in prophase. Our observation that human cells contain two distinct 14S cohesin complexes containing either SA1 or SA2 therefore raised the possibility that in vertebrates different cohesin complexes may dissociate from chromatin at different times in mitosis. To test this hypothesis we analyzed the chromatin association of SA1 and SA2 in Xenopus egg extracts in vitro (Fig. 5) and in cultured cells by immunofluorescence microscopy in vivo (Fig. 6).


Characterization of vertebrate cohesin complexes and their regulation in prophase.

Sumara I, Vorlaufer E, Gieffers C, Peters BH, Peters JM - J. Cell Biol. (2000)

Reconstitution of mitosis-specific dissociation of human 14S cohesin complexes and of PDS5 from chromatin in Xenopus egg extracts. (A) HeLa chromatin was incubated in Xenopus interphase extract. The extract was either supplemented with nondegradable cyclin B Δ90 to trigger entry into mitosis (left) or left untreated (right). At different time points either extract samples (top) or chromatin reisolated from the extract by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (top) or immunoblotting with antibodies to the indicated proteins (bottom). The cell cycle state of the extracts was analyzed by monitoring the phosphorylation-dependent electrophoretic mobility shift of 35S-labeled CDC25 and the stability of 35S-labeled cyclin B, which were added to the extracts at time zero. TOPO II, topoisomerase II; H3P, histone H3 phosphorylated on serine 10. (B) HeLa chromatin was incubated in mitotic Xenopus egg extract (xtΔ90), or in mitotic extract treated with 0.8 mM roscovitin (XΔ90+ Roscovitin), or in interphase extract (xti). Chromatin bound proteins were isolated at different time points and analyzed as in A.
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Related In: Results  -  Collection

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Figure 5: Reconstitution of mitosis-specific dissociation of human 14S cohesin complexes and of PDS5 from chromatin in Xenopus egg extracts. (A) HeLa chromatin was incubated in Xenopus interphase extract. The extract was either supplemented with nondegradable cyclin B Δ90 to trigger entry into mitosis (left) or left untreated (right). At different time points either extract samples (top) or chromatin reisolated from the extract by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (top) or immunoblotting with antibodies to the indicated proteins (bottom). The cell cycle state of the extracts was analyzed by monitoring the phosphorylation-dependent electrophoretic mobility shift of 35S-labeled CDC25 and the stability of 35S-labeled cyclin B, which were added to the extracts at time zero. TOPO II, topoisomerase II; H3P, histone H3 phosphorylated on serine 10. (B) HeLa chromatin was incubated in mitotic Xenopus egg extract (xtΔ90), or in mitotic extract treated with 0.8 mM roscovitin (XΔ90+ Roscovitin), or in interphase extract (xti). Chromatin bound proteins were isolated at different time points and analyzed as in A.
Mentions: Yeast cohesin dissociates from chromatin at the onset of anaphase, whereas Xenopus 14S cohesin dissociates from chromatin already in prophase. Our observation that human cells contain two distinct 14S cohesin complexes containing either SA1 or SA2 therefore raised the possibility that in vertebrates different cohesin complexes may dissociate from chromatin at different times in mitosis. To test this hypothesis we analyzed the chromatin association of SA1 and SA2 in Xenopus egg extracts in vitro (Fig. 5) and in cultured cells by immunofluorescence microscopy in vivo (Fig. 6).

Bottom Line: SA1 is also a subunit of 14S cohesin in Xenopus.The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase.These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.

ABSTRACT
In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

Show MeSH
Related in: MedlinePlus