Sororin is a master regulator of sister chromatid cohesion and separation

The maintenance of sister chromatid cohesion from S phase to the onset of anaphase relies on a small but evolutionarily conserved protein called Sororin. Sororin is a phosphoprotein and its dynamic localization and function are regulated by protein kinases, such as Cdk1/cyclin B and Erk2. The association of Sororin with chromatin requires cohesin to be preloaded to chromatin and modification of Smc3 during DNA replication. Sororin antagonizes the function of Wapl in cohesin releasing from S to G₂ phase and promotes cohesin release from sister chromatid arms in prophase via interaction with Plk1. This review focuses on progress of the identification and regulation of Sororin during cell cycle; role of post-translational modification on Sororin function; role of Sororin in the maintenance and resolution of sister chromatid cohesion; and finally discusses Sororin’s emerging role in cancer and the potential issues that need be addressed in the future.     

Sororin is a master regulator of sister chromatid cohesion and separation

The maintenance of sister chromatid cohesion from S phase to the onset of anaphase relies on a small but evolutionarily conserved protein called Sororin. Sororin is a phosphoprotein and its dynamic localization and function are regulated by protein kinases, such as Cdk1/cyclin B and Erk2. The association of Sororin with chromatin requires cohesin to be preloaded to chromatin and modification of Smc3 during DNA replication. Sororin antagonizes the function of Wapl in cohesin releasing from S to G₂ phase and promotes cohesin release from sister chromatid arms in prophase via interaction with Plk1. This review focuses on progress of the identification and regulation of Sororin during cell cycle; role of post-translational modification on Sororin function; role of Sororin in the maintenance and resolution of sister chromatid cohesion; and finally discusses Sororin’s emerging role in cancer and the potential issues that need be addressed in the future.     

The hsSsu72 phosphatase is a cohesin‐binding protein that regulates the resolution of sister chromatid arm cohesion

Cohesin is a multiprotein complex that establishes sister chromatid cohesion from S phase until mitosis or meiosis. In vertebrates, sister chromatid cohesion is dissolved in a stepwise manner: most cohesins are removed from the chromosome arms via a process that requires polo‐like kinase 1 (Plk1), aurora B and Wapl, whereas a minor amount of cohesin, found preferentially at the centromere, is cleaved by separase following its activation by the anaphase‐promoting complex/cyclosome. Here, we report that our budding yeast two‐hybrid assay identified hsSsu72 phosphatase as a Rad21‐binding protein. Additional experiments revealed that Ssu72 directly interacts with Rad21 and SA2 in vitro and in vivo, and associates with sister chromatids in human cells. Interestingly, depletion or mutational inactivation of Ssu72 phosphatase activity caused the premature resolution of sister chromatid arm cohesion, whereas the overexpression of Ssu72 yielded high resistance to this resolution. Interestingly, it appears that Ssu72 regulates the cohesion of chromosome arms but not centromeres, and acts by counteracting the phosphorylation of SA2. Thus, our study provides important new evidence, suggesting that Ssu72 is a novel cohesin‐binding protein capable of regulating cohesion between sister chromatid arms.     

Calpain-1 cleaves Rad21 to promote sister chromatid separation

Defining the mechanisms of chromosomal cohesion and dissolution of the cohesin complex from chromatids is important for understanding the chromosomal missegregation seen in many tumor cells. Here we report the identification of a novel cohesin-resolving protease and describe its role in chromosomal segregation. Sister chromatids are held together by cohesin, a multiprotein ring-like complex comprised of Rad21, Smc1, Smc3, and SA2 (or SA1). Cohesin is known to be removed from vertebrate chromosomes by two distinct mechanisms, namely, the prophase and anaphase pathways. First, PLK1-mediated phosphorylation of SA2 in prophase leads to release of cohesin from chromosome arms, leaving behind centromeric cohesins that continue to hold the sisters together. Then, at the onset of anaphase, activated separase cleaves the centromeric cohesin Rad21, thereby opening the cohesin ring and allowing the sister chromatids to separate. We report here that the calcium-dependent cysteine endopeptidase calpain-1 is a Rad21 peptidase and normally localizes to the interphase nuclei and chromatin. Calpain-1 cleaves Rad21 at L192, in a calcium-dependent manner. We further show that Rad21 cleavage by calpain-1 promotes separation of chromosome arms, which coincides with a calcium-induced partial loss of cohesin at several chromosomal loci. Engineered cleavage of Rad21 at the calpain-cleavable site without activation of calpain-1 can lead to a loss of sister chromatid cohesion. Collectively, our work reveals a novel function of calpain-1 and describes an additional pathway for sister chromatid separation in humans.     

C-terminus of Sororin interacts with SA2 and regulates sister chromatid cohesion

Sororin is a conserved protein required for accurate separation of sister chromatids in each cell cycle. Sororin is recruited to chromatin during DNA replication, protects sister chromatid cohesion in S and G2 phase, and regulates the resolution of sister chromatid cohesion in mitosis. Sororin binds to cohesin complex, but how Sororin and cohesin subunits interact remains unclear. Here we report that the C-terminus of Sororin, especially the last 12 amino acid (aa) residues, is important for Sororin to bind cohesin core subunit SA2. Deletion of the last 12aa residues not only inhibits the interactions between Sororin and SA2 but also causes precocious chromosome separation. Our data suggest that the C-terminus of Sororin functions as an anchor binding to SA2, which facilitates other conserved motifs on Sororin to interact with other proteins to regulate sister chromatid cohesion and separation.     

Regulation of sister chromatid cohesion between chromosome arms

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.     

Characterization of vertebrate cohesin complexes and their regulation in prophase

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.     

Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres

Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. While both proteins contribute to telomere and arm cohesion, Pds5B is specifically required for centromeric cohesion. Furthermore, reduced accumulation of Aurora B at the inner centromere region in cells lacking Pds5B impairs its error correction function, promoting chromosome mis‐segregation and aneuploidy. Our work supports a model in which the composition and function of cohesin complexes differs between different chromosomal regions.     

Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2

Cohesin is a protein complex that is required to hold sister chromatids together. Cleavage of the Scc1 subunit of cohesin by the protease separase releases the complex from chromosomes and thereby enables the separation of sister chromatids in anaphase. In vertebrate cells, the bulk of cohesin dissociates from chromosome arms already during prophase and prometaphase without cleavage of Scc1. Polo-like kinase 1 (Plk1) and Aurora-B are required for this dissociation process, and Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, consistent with the possibility that cohesin phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms. However, this hypothesis has not been tested yet, and in budding yeast it has been found that phosphorylation of Scc1 by the Polo-like kinase Cdc5 enhances the cleavability of cohesin, but does not lead to separase-independent dissociation of cohesin from chromosomes. To address the functional significance of cohesin phosphorylation in human cells, we have searched for phosphorylation sites on all four subunits of cohesin by mass spectrometry. We have identified numerous mitosis-specific sites on Scc1 and SA2, mutated them, and expressed nonphosphorylatable forms of both proteins stably at physiological levels in human cells. The analysis of these cells lines, in conjunction with biochemical experiments in vitro, indicate that Scc1 phosphorylation is dispensable for cohesin dissociation from chromosomes in early mitosis but enhances the cleavability of Scc1 by separase. In contrast, our data reveal that phosphorylation of SA2 is essential for cohesin dissociation during prophase and prometaphase, but is not required for cohesin cleavage by separase. The similarity of the phenotype obtained after expression of nonphosphorylatable SA2 in human cells to that seen after the depletion of Plk1 suggests that SA2 is the critical target of Plk1 in the cohesin dissociation pathway.     

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.     

STAG2 and Rad21 mammalian mitotic cohesins are implicated in meiosis

STAG/SA proteins are specific cohesin complex subunits that maintain sister chromatid cohesion in mitosis and meiosis. Two members of this family, STAG1/SA1 and STAG2/SA2,double dagger are classified as mitotic cohesins, as they are found in human somatic cells and in Xenopus laevis as components of the cohesin(SA1) and cohesin(SA2) complexes, in which the shared subunits are Rad21/SCC1, SMC1 and SMC3 proteins. A recently reported third family member, STAG3, is germinal cell-specific and is a subunit of the meiotic cohesin complex. To date, the meiosis-specific cohesin complex has been considered to be responsible for sister chromatid cohesion during meiosis. We studied replacement of the mitotic by the meiotic cohesin complex during mouse germinal cell maturation, and we show that mammalian STAG2 and Rad21 are also involved in several meiosis stages. Immunofluorescence results suggest that a cohesin complex containing Rad21 and STAG2 cooperates with a STAG3-specific complex to maintain sister chromatid cohesion during the diplotene stage of meiosis.     

The Multiple Roles of Cohesin in Meiotic Chromosome Morphogenesis and Pairing

Sister chromatid cohesion, mediated by cohesin complexes, is laid down during DNA replication and is essential for the accurate segregation of chromosomes. Previous studies indicated that, in addition to their cohesion function, cohesins are essential for completion of recombination, pairing, meiotic chromosome axis formation, and assembly of the synaptonemal complex (SC). Using mutants in the cohesin subunit Rec8, in which phosphorylated residues were mutated to alanines, we show that cohesin phosphorylation is not only important for cohesin removal, but that cohesin's meiotic prophase functions are distinct from each other. We find pairing and SC formation to be dependent on Rec8, but independent of the presence of a sister chromatid and hence sister chromatid cohesion. We identified mutations in REC8 that differentially affect Rec8's cohesion, pairing, recombination, chromosome axis and SC assembly function. These findings define Rec8 as a key determinant of meiotic chromosome morphogenesis and a central player in multiple meiotic events.     

Sequential loading of cohesin subunits during the first meiotic prophase of grasshoppers

The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.     

Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast

Sister chromatid cohesion in meiosis is established by cohesin complexes, including the Rec8 subunit. During meiosis I, sister chromatid cohesion is destroyed along the chromosome arms to release connections of recombined homologous chromosomes (homologues), whereas centromeric cohesion persists until it is finally destroyed at anaphase II. In fission yeast, as in mammals, distinct cohesin complexes are used depending on the chromosomal region; Rec8 forms a complex with Rec11 (equivalent to SA3) mainly along chromosome arms, while Psc3 (equivalent to SA1 and SA2) forms a complex mainly in the vicinity of the centromeres. Here we show that separase activation and resultant Rec8 cleavage are required for meiotic chromosome segregation in fission yeast. A non-cleavable form of Rec8 blocks disjunction of homologues at meiosis I. However, displacing non-cleavable Rec8 restrictively from the chromosome arm by genetically depleting Rec11 alleviated the blockage of homologue segregation, but not of sister segregation. We propose that the segregation of homologues at meiosis I and of sisters at meiosis II requires the cleavage of Rec8 along chromosome arms and at the centromeres, respectively.     

Shugoshin: guardian spirit at the centromere

A recently emerging protein family, shugoshin, plays a crucial role in the centromeric protection of cohesin, which is responsible for sister chromatid cohesion. This is especially important at the first meiotic division, where cohesin is cleaved by separase only along chromosome arms while the centromeric cohesin must be preserved. In vertebrate cells, arm cohesion is largely lost during prophase and prometaphase in order to facilitate sister chromatid resolution, whereas centromeric cohesion is preserved until the bipolar attachment of sister chromatids is established. Vertebrate shugoshin plays an essential role in protecting centromeric cohesin from prophase dissociation. In yeast, shugoshin also has a crucial role in sensing the loss of tension at kinetochores and in generating the spindle checkpoint signal.     

Shugoshin 1 Plays a Central Role in Kinetochore Assembly and is Required for Kinetochore Targeting of Plk1

Physical connection between the sister chromatids is mediated by the cohesin protein complex. During prophase, cohesin is removed from the chromosome arms while the centromeres remain united. Shugoshin1 (Sgo1) is required for maintenance of centromeric cohesion from prophase to the metaphase-anaphase transition. Furthermore, Sgo1 has been proposed to regulate kinetochore microtubule stability and sense interkinetochore tension, two tasks which are tightly coupled with the function of the Chromosomal Passenger Complex (CPC) and Polo-like kinase 1 (Plk1). Here we show that depletion or chemical inhibition of Aurora B kinase (AurB), the catalytic subunit of the CPC, disrupts accumulation of Sgo1 on the kinetochores in HeLa cells and causes Sgo1 to localize on the chromosome arms. RNAi assays show that depletion of Sgo1 did not affect AurB localization but diminished Plk1 kinetochore binding. Furthermore, we demonstrate that vertebrate Sgo1 is phosphorylated by both AurB and Plk1 in vitro. The data presented here includes an extensive analysis of kinetochore targeting interdependencies of mitotic proteins that propose a novel branch in kinetochore assembly where Sgo1 and Plk1 have central roles. Furthermore our studies implicate Sgo1 in the tension sensing mechanism of the spindle checkpoint by regulating Plk1 kinetochore affinity.     

A conserved motif at the C terminus of sororin is required for sister chromatid cohesion

Sororin is a positive regulator of sister chromatid cohesion that interacts with the cohesin complex. Sororin is required for the increased stability of the cohesin complex on chromatin following DNA replication and sister chromatid cohesion during G(2). The mechanism by which sororin ensures cohesion is currently unknown. Because the primary sequence of sororin does not contain any previously characterized structural or functional motifs, we have undertaken a structure-function analysis of the sororin protein. Using a series of mutant derivatives of sororin, we show that the ability of sororin to bind to chromatin is separable from both its role in sister chromatid cohesion and its interaction with the cohesin complex. We also show that derivatives of sororin with deletions or mutations in the conserved C terminus fail to rescue the loss-of-cohesion phenotype caused by sororin RNAi and that these mutations also abrogate the association of sororin with the cohesin complex. Our data suggest that the interaction of the highly conserved motif at the C terminus of sororin with the cohesin complex is critical to its ability to mediate sister chromatid cohesion.     

A new role for the mitotic RAD21/SCC1 cohesin in meiotic chromosome cohesion and segregation in the mouse

The evolutionarily conserved cohesin complex is required for the establishment and maintenance of sister chromatid cohesion, in turn essential for proper chromosome segregation. RAD21/SCC1 is a regulatory subunit of the mitotic cohesin complex, as it links together all other subunits of the complex. The destruction of RAD21/SCC1 along chromosomal arms and later at centromeres results in the dissociation of the cohesin complex, facilitating chromosome segregation. Here, we report for the first time that mammalian RAD21/SCC1 associates with the axial/lateral elements of the synaptonemal complex along chromosome arms and on centromeres of mouse spermatocytes. Importantly, RAD21/SCC1 is lost from chromosome arms in late prophase I but persists on centromeres. The loss of centromeric RAD21/SCC1 coincides with the separation of sister chromatids at anaphase II. These findings support a role for mammalian RAD21/SCC1 in maintaining sister chromatid cohesion in meiosis.     

Meiotic cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins SMC1beta and SMC3

In meiotic prophase, the sister chromatids of each chromosome develop a common axial element (AE) that is integrated into the synaptonemal complex (SC). We analyzed the incorporation of sister chromatid cohesion proteins (cohesins) and other AE components into AEs. Meiotic cohesin REC8 appeared shortly before premeiotic S phase in the nucleus and formed AE-like structures (REC8-AEs) from premeiotic S phase on. Subsequently, meiotic cohesin SMC1beta, cohesin SMC3, and AE proteins SCP2 and SCP3 formed dots along REC8-AEs, which extended and fused until they lined REC8-AEs along their length. In metaphase I, SMC1beta, SMC3, SCP2, and SCP3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, REC8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We propose that REC8 provides a basis for AE formation and that the first steps in AE assembly do not require SMC1beta, SMC3, SCP2, and SCP3. Furthermore, SMC1beta, SMC3, SCP2, and SCP3 cannot provide arm cohesion during metaphase I. We propose that REC8 then provides cohesion. RAD51 and/or DMC1 coimmunoprecipitates with REC8, suggesting that REC8 may also provide a basis for assembly of recombination complexes.     

Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.