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Characterization of vertebrate cohesin complexes and their regulation in prophase.
Sumara I
,
Vorlaufer E
,
Gieffers C
,
Peters BH
,
Peters JM
.
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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.
Figure 1. Fractionation of human cohesin complexes by sucrose density gradient centrifugation. (A) Characterization of SA1 and SA2 antibodies. (Left) PhosphorImager scan of in vitroâtranslated 35S-labeled human SA1 and SA2 (IVT-SA1, IVT-SA2) separated by SDS-PAGE. Other panels, control rabbit reticulocyte lysate (RRL), in vitroâtranslated SA1 and SA2, protein extracts (xt) from HeLa cells, and Xenopus interphase egg extracts, and SA2 (446) immunoprecipitates isolated from HeLa extracts (SA2 IP) were analyzed by SDS-PAGE and immunoblotting with specific SA1 (444) or SA2 (446) antibodies. (B) Sucrose gradient fractions containing proteins from logarithmically growing HeLa cells were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. SA1 and SA2 were detected with antibodies 444 and 446, respectively. Prot, proteasome. (C) Sucrose gradient fractions containing proteins from Xenopus interphase extract were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins.
Figure 2. SA1 and SA2 are subunits of two distinct human 14S cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells or Xenopus interphase egg extracts were analyzed by immunoprecipitation (IP) with either preimmune (P) or immune antibodies (I) against SA1 (444) or SA2 (446) and analyzed by SDS-PAGE and Western blotting (WB) with antibodies to the indicated proteins. (B) HeLa extracts were immunodepleted as in A and the resulting supernatant fractions were analyzed by SDS-PAGE and immunoblotting with SA1 (444) and SA2 (446) antibodies.
Figure 3. Purification of human cohesin complexes. (A) Immunoprecipitates (IP) obtained with either SA1/2 (447) antibodies or as a control with CDC27 antibodies (APC IP) from low-speed supernatant of extracts from logarithmically growing HeLa cells were analyzed by SDS-PAGE and silver staining. The positions of cohesin subunits, p85 and APC subunits as determined by immunoblotting are indicated. (B) Proteins were eluted with antigenic peptides from cohesin immunoprecipitates obtained as in A and further separated by sucrose density gradient centrifugation. Fractions 5â8 were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. (C) Sucrose density gradient fractions from the experiment shown in B were analyzed by SDS-PAGE and silver staining.
Figure 4. PDS5 is found in association with SA1- and SA2-containing cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells was analyzed by immunoprecipitation with either preimmune (P) or immune antibodies (I) to PDS5. Immunoprecipitates (IP) and supernatants (SUP) were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (B) HeLa extracts prepared as in A were analyzed by immunoprecipitation with SA1, SA2, or with nonspecific control (MOCK) antibodies and the immunoprecipitates (IP), extracts before immunoprecipitation (Input) and supernatants (Sup) were analyzed by SDS-PAGE and Western blotting with antibodies to PDS5. (C) HeLa extracts prepared as in A were analyzed by immunoprecipitation with either preimmune (P) or immune (I) SA2 antibodies (446). After washing with buffers containing either 150, 250, or 500 mM NaCl, the immunoprecipitates were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (D) Xenopus interphase extracts were analyzed by immunoprecipitation with either nonspecific (IP-contr), Xenopus PDS5 (IP-PDS5), or SA1 (IP-SA1) antibodies. The precipitates were analyzed by immunoblotting with PDS5, SA1, and SCC1 antibodies. After immunoprecipitation with control (xtÎcontr), PDS5 (xtÎPDS5), or SA1 (xtÎSA1) antibodies the resulting supernatants were analyzed side by side. PDS5 antibody 648 was used for the IP and 647 for immunoblotting.
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.
Figure 6. Immunofluorescence microscopy showing the intracellular distribution of SA1, SA2, and PDS5 in human cells at different stages of mitosis. Caco cells were stained with DAPI and with either the SA1/SA2 antibody 447 (A), or with PDS5 antibodies (B), or with the SA1-specific antibody 444. Similar results were obtained with the antibodies 445 and 446 (data not shown). Bars, 5 μm.
Figure 7
Cyclin B proteolysis is not required for the mitotic dissociation of cohesins from chromatin. (A) High-speed supernatant (S100) fractions of Xenopus interphase extracts were incubated for 60 min at room temperature either with 3,200 Xenopus sperm nuclei/ul (left) or without nuclei (right). Nondegradable cyclin B Δ90 was then added to trigger entry into mitosis. At different time points either S100 samples (top) or chromatin reisolated from the reaction mixture 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 behavior of 35S-labeled CDC25 and cyclin B as in Fig. 5. TOPO II, topoisomerase II.
Figure 7 (cont.)
(B) Immunofluorescence microscopy showing the morphology of sperm nuclei incubated for different time points in S100 fractions as in A and subsequently fixed and stained with DAPI. Note that chromosome condensation still occurs in the S100 fraction.
Figure 8. The APC is not required for the mitotic dissociation of cohesins from chromatin. (A) CDC27 immunoblot showing Xenopus interphase extract before and after depletion with either control or CDC27 antibodies. (B) APC-depleted (left) and control-depleted (right) Xenopus interphase extracts were incubated with sperm nuclei for 30 min and then nondegradable cyclin B Î90 was added to trigger entry into mitosis. At different time points either extract samples (top) or chromatin reisolated from the reaction mixture by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (S35CDC25) or immunoblotting with antibodies to the indicated proteins (all other panels). The cell cycle state of the extracts was analyzed by monitoring the behavior of 35S-labeled CDC25 and of endogenous cyclin B. Data from two different experiments are shown. In experiment 2, the degree of APC depletion and the cell cycle behavior of the extracts were the same as in experiment 1 (data not shown).
Figure 9. Cohesins can dissociate from chromatin in the absence of CDK1 activity. (A) Xenopus sperm nuclei (3,200 nuclei/μl) were incubated for 30 min at room temperature in interphase extracts from cycloheximide-treated Xenopus eggs before either 1 μM okadaic acid (left) or DMSO (right) was added. At different time points either extract samples (top) or chromatin reisolated from the reaction mixture by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (S35CDC25) or immunoblotting with antibodies to the indicated proteins (all other panels). The cell cycle state of the extracts was analyzed by monitoring the behavior of 35S-labeled CDC25 and the phosphorylation of histone H3 on serine 10 (H3P). (B) Phase contrast micrographs of human diploid fibroblasts grown to confluency and then treated for 2.5 h with 10 μg/ml cycloheximide and either with DMSO (right) or with 1 μM okadaic acid (left). Whole cell lysates (WCL) or chromatin pellets (CP) were then analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins (lower panels). Data from two different experiments are shown. OA, okadaic acid; H3P, histone H3 phosphorylated on serine 10.
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