Compartments in the Ring.

Sister chromatid cohesion has been thought to be mediated by DNA entrapment within the large cohesin ring. Vazquez Nunez et al. and Chapard et al. now show that the ring is divided up into two sub-compartments, with implications for how these chromosomal organizers entrap DNA.

Sororin mediates sister chromatid cohesion by antagonizing Wapl.

Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.

Drosophila Dalmatian combines sororin and shugoshin roles in establishment and protection of cohesion.

Sister chromatid cohesion is crucial to ensure chromosome bi-orientation and equal chromosome segregation. Cohesin removal via mitotic kinases and Wapl has to be prevented in pericentromeric regions in order to protect cohesion until metaphase, but the mechanisms of mitotic cohesion protection remain elusive in Drosophila Here, we show that dalmatian (Dmt), an ortholog of the vertebrate cohesin-associated protein sororin, is required for protection of mitotic cohesion in flies. Dmt is essential for cohesion establishment during interphase and is enriched on pericentromeric heterochromatin. Dmt is recruited through direct association with heterochromatin protein-1 (HP1), and this interaction is required for cohesion. During mitosis, Dmt interdependently recruits protein phosphatase 2A (PP2A) to pericentromeric regions, and PP2A binding is required for Dmt to protect cohesion. Intriguingly, Dmt is sufficient to protect cohesion upon heterologous expression in human cells. Our findings of a hybrid system, in which Dmt exerts both sororin-like establishment functions and shugoshin-like heterochromatin-based protection roles, provide clues to the evolutionary modulation of eukaryotic cohesion regulation systems.

Cohesin acetylation and Wapl-Pds5 oppositely regulate translocation of cohesin along DNA.

Cohesin is a ring-shaped protein complex that plays a crucial role in sister chromatid cohesion and gene expression. The dynamic association of cohesin with chromatin is essential for these functions. However, the exact nature of cohesin dynamics, particularly cohesin translocation, remains unclear. We evaluated the dynamics of individual cohesin molecules on DNA and found that the cohesin core complex possesses an intrinsic ability to traverse DNA in an adenosine triphosphatase (ATPase)-dependent manner. Translocation ability is suppressed in the presence of Wapl-Pds5 and Sororin; this suppression is alleviated by the acetylation of cohesin and the action of mitotic kinases. In Xenopus laevis egg extracts, cohesin is translocated on unreplicated DNA in an ATPase- and Smc3 acetylation-dependent manner. Cohesin movement changes from bidirectional to unidirectional when cohesin faces DNA replication; otherwise, it is incorporated into replicating DNA without being translocated or is dissociated from replicating DNA This study provides insight into the nature of individual cohesin dynamics and the mechanisms by which cohesin achieves cohesion in different chromatin contexts.

Wapl is an essential regulator of chromatin structure and chromosome segregation.

Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

Hot debate in hot springs: Report on the second international meeting on SMC proteins.

The second international meeting on "SMC proteins: Chromosomal Organizers from Bacteria to Human" (SMC2017) was held in Nanyo City, Yamagata, Japan, from 13 to 16 June 2017. The meeting was attended by 134 participants (among them, 76 from outside of Japan) who were interested in one of the highly conserved classes of chromosomal proteins regulating large-scale chromosome structure and function. A keynote lecture was followed by 41 oral presentations and 71 poster presentations in the four-day meeting. Diverse topics surrounding eukaryotic SMC protein complexes (cohesins, condensins and SMC5/6) and prokaryotic SMCs, and a wide range of cutting-edge approaches (from polymer physics through medical genetics) were presented. Dominant themes discussed in the meeting included mechanistically how the SMC protein complexes might form chromatin loops and domains. The participants enjoyed both exciting debate about chromosome organization and warm welcome offered by local people in a small city located in the northern part of Japan.

Aurora B and Cdk1 mediate Wapl activation and release of acetylated cohesin from chromosomes by phosphorylating Sororin.

Sister chromatid cohesion depends on Sororin, a protein that stabilizes acetylated cohesin complexes on DNA by antagonizing the cohesin release factor Wings-apart like protein (Wapl). Cohesion is essential for chromosome biorientation but has to be dissolved to enable sister chromatid separation. To achieve this, the majority of cohesin is removed from chromosome arms in prophase and prometaphase in a manner that depends on Wapl and phosphorylation of cohesin's subunit stromal antigen 2 (SA2), whereas centromeric cohesin is cleaved in metaphase by the protease separase. Here we show that the mitotic kinases Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize interactions between Sororin and the cohesin subunit precocious dissociation of sisters protein 5 (Pds5) by phosphorylating Sororin, leading to release of acetylated cohesin from chromosome arms and loss of cohesion. At centromeres, the cohesin protector shugoshin (Sgo1)-protein phosphatase 2A (PP2A) antagonizes Aurora B and Cdk1 partly by dephosphorylating Sororin and thus maintains cohesion until metaphase. We propose that the stepwise loss of cohesion between chromosome arms and centromeres is caused by local regulation of Wapl activity, which is controlled by the phosphorylation state of Sororin.

Sororin is an evolutionary conserved antagonist of WAPL.

Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORIN's functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1Δ mutants display cohesion defects, which are partially alleviated by wpl1Δ. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants.

Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs.

Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.

Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis.

At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.

Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit.

Condensin I is a pentameric protein complex that plays an essential role in mitotic chromosome assembly in eukaryotic cells. Although it has been shown that condensin I loading is mitosis specific, it remains poorly understood how the robust cell cycle regulation of condensin I is achieved. Here, we set up a panel of in vitro assays to demonstrate that cell cycle-specific loading of condensin I is regulated by the N-terminal tail (N-tail) of its kleisin subunit CAP-H. Deletion of the N-tail accelerates condensin I loading and chromosome assembly in Xenopus egg mitotic extracts. Phosphorylation-deficient and phosphorylation-mimetic mutations in the CAP-H N-tail decelerate and accelerate condensin I loading, respectively. Remarkably, deletion of the N-tail enables condensin I to assemble mitotic chromosome-like structures even in interphase extracts. Together with other extract-free functional assays in vitro, our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes.

A loop extrusion-independent mechanism contributes to condensin I-mediated chromosome shaping.

Condensin I is a five-subunit protein complex that is central to mitotic chromosome assembly in eukaryotic cells. Despite recent progress, its molecular mechanisms of action remain to be fully elucidated. By using Xenopus egg extracts as a functional assay, we find that condensin I complexes harboring mutations in its kleisin subunit CAP-H produce chromosomes with confined axes in the presence of topoisomerase IIα (topo IIα) and highly compact structures (termed "beans") with condensin-positive central cores in its absence. The bean phenotype depends on the SMC ATPase cycle and can be reversed by subsequent addition of topo IIα. The HEAT repeat subunit CAP-D2, but not CAP-G, is essential for the bean formation. Notably, loop extrusion activities of the mutant complexes cannot explain the chromosomal defects they exhibit in Xenopus egg extracts, implying that a loop extrusion-independent mechanism contributes to condensin I-mediated chromosome assembly and shaping. We provide evidence that condensin-condensin interactions underlie these processes.

Opening of cohesin's SMC ring is essential for timely DNA replication and DNA loop formation.

The ring-shaped cohesin complex topologically binds to DNA to establish sister chromatid cohesion. This topological binding creates a structural obstacle to genome-wide chromosomal events, such as replication. Here, we examine how conformational changes in cohesin circumvent being an obstacle in human cells. We show that ATP hydrolysis-driven head disengagement, leading to the structural maintenance of chromosome (SMC) ring opening, is essential for the progression of DNA replication. Closure of the SMC ring stalls replication in a checkpoint-independent manner. The SMC ring opening also facilitates sister chromatid resolution and chromosome segregation in mitosis. Single-molecule analyses reveal that forced closure of the SMC ring suppresses the translocation of cohesin on DNA as well as the formation of stable DNA loops. Our results suggest that the ATP hydrolysis-driven SMC ring opening makes topologically bound cohesin dynamic on DNA to achieve replication-dependent cohesion in the S phase and to resolve cohesion in mitosis. Thus, the SMC ring opening could be a fundamental mechanism to modulate both cohesion and higher-order genome structure.

Cohesion and cohesin-dependent chromatin organization.

Cohesin, one of structural maintenance of chromosomes (SMC) complexes, forms a ring-shaped protein complex, and mediates sister chromatid cohesion for accurate chromosome segregation and precise genome inheritance. The cohesin ring entraps one or two DNA molecules to achieve cohesion, which is further regulated by cohesin-binding proteins and modification enzymes in a cell cycle-dependent manner. Recent significant advancements in Hi-C technologies have revealed numerous cohesin-dependent higher-order chromatin structures. Simultaneously, single-molecule imaging has also unveiled the detailed dynamics of cohesin on DNA and/or chromatin. Thus, those studies are providing novel visions for the authentic chromatin structure regulated by cohesin.

Sister chromatid cohesion.

During S phase, not only does DNA have to be replicated, but also newly synthesized DNA molecules have to be connected with each other. This sister chromatid cohesion is essential for the biorientation of chromosomes on the mitotic or meiotic spindle, and is thus an essential prerequisite for chromosome segregation. Cohesion is mediated by cohesin complexes that are thought to embrace sister chromatids as large rings. Cohesin binds to DNA dynamically before DNA replication and is converted into a stably DNA-bound form during replication. This conversion requires acetylation of cohesin, which in vertebrates leads to recruitment of sororin. Sororin antagonizes Wapl, a protein that is able to release cohesin from DNA, presumably by opening the cohesin ring. Inhibition of Wapl by sororin therefore "locks" cohesin rings on DNA and allows them to maintain cohesion for long periods of time in mammalian oocytes, possibly for months or even years.

A betacellulin mutant promotes differentiation of pancreatic acinar AR42J cells into insulin-producing cells with low affinity of binding to ErbB1.

Betacellulin (BTC) is one of the members of the epidermal growth factor (EGF) ligand family of ErbB receptor tyrosine kinases. It is a differentiation factor as well as a potent mitogen. BTC promotes the differentiation of pancreatic acinar-derived AR42J cells into insulin-producing cells. It independently and preferentially binds to two type I tyrosine kinase receptors, the EGF receptor (ErbB1) and ErbB4. However, the physiochemical characteristics of BTC that are responsible for its preferential binding to these two receptors have not been fully defined. In this study, to investigate the essential amino acid residues of BTC for binding to the two receptors, we introduced point mutations into the EGF domain of BTC employing error-prone PCR. The receptor binding abilities of 190 mutants expressed in Escherichia coli were assessed by enzyme immunoassay. Replacement of the glutamic acid residue at position 88 with a lysine residue in BTC was found to produce a significant loss of affinity for binding to ErbB1, while the affinity of binding to ErbB4 was unchanged. In addition, the mutant of BTC-E/88/K showed less growth-promoting activity on BALB/c 3T3 cells compared with that of the wild-type BTC protein. Interestingly, the BTC mutant protein promoted differentiation of pancreatic acinar AR42J cells at a high frequency into insulin-producing cells compared with AR42J cells that were treated with wild-type BTC protein. These results indicate the possibility of designing BTC mutants, which have an activity of inducing differentiation only, without facilitating growth promotion.

Inactivation of Rho/ROCK signaling is crucial for the nuclear accumulation of FKHR and myoblast fusion.

Myoblast fusion is a critical process for the terminal differentiation of skeletal muscle. To elucidate the intracellular mechanisms regulating myoblast fusion, we studied the roles of signaling through the small GTPase Rho and its effector, the Rho-associated kinase ROCK, in myoblast fusion of mouse C2C12 cells. We found that Rho activity, which was high in proliferating myoblasts, decreased during myogenesis. Expression of a constitutively active form of Rho blocked myoblast fusion, but not the earlier steps of differentiation. Consistently, ROCK activity was also decreased in differentiating C2C12 cells, and an active ROCK mutant prevented their fusion. Furthermore, inactivation of ROCK by the specific inhibitor Y-27632 enhanced myoblast fusion, even in cells expressing the active Rho mutant. Thus, the down-regulation of Rho/ROCK signaling is required for myoblast fusion. We also found that Rho/ROCK signaling was required for retaining FKHR, a transcription factor implicated in myoblast fusion, in the cytoplasm and that inactivation of ROCK was essential for the nuclear accumulation of FKHR that took place just before the onset of myoblast fusion. Moreover, ROCK directly phosphorylated FKHR in vitro. We conclude that the inactivation of Rho/ROCK signaling is a prerequisite for FKHR nuclear translocation and myoblast fusion in C2C12 cells, providing evidence for a novel regulatory role of Rho/ROCK signaling in myogenic differentiation.