Centromeres License the Mitotic Condensation of Yeast Chromosome Arms.

During mitosis, chromatin condensation shapes chromosomes as separate, rigid, and compact sister chromatids to facilitate their segregation. Here, we show that, unlike wild-type yeast chromosomes, non-chromosomal DNA circles and chromosomes lacking a centromere fail to condense during mitosis. The centromere promotes chromosome condensation strictly in cis through recruiting the kinases Aurora B and Bub1, which trigger the autonomous condensation of the entire chromosome. Shugoshin and the deacetylase Hst2 facilitated spreading the condensation signal to the chromosome arms. Targeting Aurora B to DNA circles or centromere-ablated chromosomes or releasing Shugoshin from PP2A-dependent inhibition bypassed the centromere requirement for condensation and enhanced the mitotic stability of DNA circles. Our data indicate that yeast cells license the chromosome-autonomous condensation of their chromatin in a centromere-dependent manner, excluding from this process non-centromeric DNA and thereby inhibiting their propagation.

Meikin synergizes with shugoshin to protect cohesin Rec8 during meiosis I.

The conserved meiosis-specific kinetochore regulator, meikin (Moa1 in fission yeast) plays a central role in establishing meiosis-specific kinetochore function. However, the underlying molecular mechanisms remain elusive. Here, we show how Moa1 regulates centromeric cohesion protection, a function that has been previously attributed to shugoshin (Sgo1). Moa1 is known to associate with Plo1 kinase. We explore Plo1-dependent Rec8 phosphorylation and identify a key phosphorylation site required for cohesion protection. The phosphorylation of Rec8 by Moa1-Plo1 potentiates the activity of PP2A associated with Sgo1. This leads to dephosphorylation of Rec8 at another site, which thereby prevents cleavage of Rec8 by separase.

Deprotection of centromeric cohesin at meiosis II requires APC/C activity but not kinetochore tension.

Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin's Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin-PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/C-dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.

Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis.

Precise control of sister chromatid separation during mitosis is pivotal to maintaining genomic integrity. Yet, the regulatory mechanisms involved are not well understood. Remarkably, we discovered that linker histone H1 phosphorylated at S/T18 decorated the inter-chromatid axial DNA on mitotic chromosomes. Sister chromatid resolution during mitosis required the eviction of such H1S/T18ph by the chaperone SET, with this process being independent of and most likely downstream of arm-cohesin dissociation. SET also directed the disassembly of Shugoshins in a polo-like kinase 1-augmented manner, aiding centromere resolution. SET ablation compromised mitotic fidelity as evidenced by unresolved sister chromatids with marked accumulation of H1S/T18ph and centromeric Shugoshin. Thus, chaperone-assisted eviction of linker histones and Shugoshins is a fundamental step in mammalian mitotic progression. Our findings also elucidate the functional implications of the decades-old observation of mitotic linker histone phosphorylation, serving as a paradigm to explore the role of linker histones in bio-signaling processes.

The pericentromeric protein shugoshin 2 cooperates with HSF1 in heat shock response and RNA Pol II recruitment.

The recruitment of RNA polymerase II (Pol II) to core promoters is highly regulated during rapid induction of genes. In response to heat shock, heat shock transcription factor 1 (HSF1) is activated and occupies heat shock gene promoters. Promoter-bound HSF1 recruits general transcription factors and Mediator, which interact with Pol II, but stress-specific mechanisms of Pol II recruitment are unclear. Here, we show in comparative analyses of HSF1 paralogs and their mutants that HSF1 interacts with the pericentromeric adaptor protein shugoshin 2 (SGO2) during heat shock in mouse cells, in a manner dependent on inducible phosphorylation of HSF1 at serine 326, and recruits SGO2 to the HSP70 promoter. SGO2-mediated binding and recruitment of Pol II with a hypophosphorylated C-terminal domain promote expression of HSP70, implicating SGO2 as one of the coactivators that facilitate Pol II recruitment by HSF1. Furthermore, the HSF1-SGO2 complex supports cell survival and maintenance of proteostasis in heat shock conditions. These results exemplify a proteotoxic stress-specific mechanism of Pol II recruitment, which is triggered by phosphorylation of HSF1 during the heat shock response.

A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis.

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A-B56 phosphatase through a coiled-coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A-B56-hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A-B56 substrates containing a canonical B56 binding motif. We find that PP2A-B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.

Distinct Clades of Protein Phosphatase 2A Regulatory B'/B56 Subunits Engage in Different Physiological Processes.

Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C), scaffolding (A), and regulatory (B) subunit. Plants have three groups of evolutionary distinct B subunits: B55, B' (B56), and B''. Here, the Arabidopsis B' group is reviewed and compared with other eukaryotes. Members of the B'α/B'ß clade are especially important for chromatid cohesion, and dephosphorylation of transcription factors that mediate brassinosteroid (BR) signaling in the nucleus. Other B' subunits interact with proteins at the cell membrane to dampen BR signaling or harness immune responses. The transition from vegetative to reproductive phase is influenced differentially by distinct B' subunits; B'α and B'ß being of little importance, whereas others (B'γ, B'ζ, B'η, B'θ, B'κ) promote transition to flowering. Interestingly, the latter B' subunits have three motifs in a conserved manner, i.e., two docking sites for protein phosphatase 1 (PP1), and a POLO consensus phosphorylation site between these motifs. This supports the view that a conserved PP1-PP2A dephosphorelay is important in a variety of signaling contexts throughout eukaryotes. A profound understanding of these regulators may help in designing future crops and understand environmental issues.

The zebrafish cohesin protein Sgo1 is required for cardiac function and eye development.

BACKGROUND: Cohesinopathies is a term that refers to/covers rare genetic diseases caused by mutations in the cohesin complex proteins. The cohesin complex is a multiprotein complex that facilitates different aspects of cell division, gene transcription, DNA damage repair, and chromosome architecture. Shugoshin proteins prevent the cohesin complex from premature dissociation from chromatids during cell division. Patients with a homozygous missense mutation in SGO1, which encodes for Shugoshin1, have problems with normal pacing of the heart and gut. RESULTS: To study the role of shugoshin during embryo development, we mutated the zebrafish sgo1 gene. Homozygous sgo1 mutant embryos display various phenotypes related to different organs, including a reduced heart rate accompanied by reduced cardiac function. In addition, sgo1 mutants are vision-impaired as a consequence of structurally defective and partially non-functional photoreceptor cells. Furthermore, the sgo1 mutants display reduced food intake and early lethality. CONCLUSION: We have generated a zebrafish model of Sgo1 that showed its importance during organ development and function.

Shugoshin ensures maintenance of the spindle assembly checkpoint response and efficient spindle disassembly.

Shugoshin proteins are evolutionarily conserved across eukaryotes, with some species-specific cellular functions, ensuring the fidelity of chromosome segregation. They act as adaptors at various subcellular locales to mediate several protein-protein interactions in a spatio-temporal manner. Here, we characterize shugoshin (Sgo1) in the human fungal pathogen Candida albicans. We observe that Sgo1 retains its centromeric localization and performs its conserved functions of regulating the sister chromatid biorientation, centromeric condensin localization, and maintenance of chromosomal passenger complex (CPC). We identify novel roles of Sgo1 as a spindle assembly checkpoint (SAC) component with functions in maintaining a prolonged SAC response by retaining Mad2 and Bub1 at the kinetochores in response to improper kinetochore-microtubule attachments. Strikingly, we discover the in vivo localization of Sgo1 along the length of the mitotic spindle. Our results indicate that Sgo1 performs a hitherto unknown function of facilitating timely disassembly of the mitotic spindle in C. albicans. To summarize, this study unravels a unique functional adaptation of shugoshin in maintaining genomic stability.

Shugoshin Regulates Cohesin, Kinetochore-Microtubule Attachments, and Chromosomal Instability.

Correct regulation of cohesin at chromosome arms and centromeres and accurate kinetochore-microtubule connections are significant for proper chromosome segregation. At anaphase of meiosis I, cohesin at chromosome arms is cleaved by separase, leading to the separation of homologous chromosomes. However, at anaphase of meiosis II, cohesin at centromeres is cleaved by separase, leading to the separation of sister chromatids. Shugoshin-2 (SGO2) is a member of the shugoshin/MEI-S332 protein family in mammalian cells, a crucial protein that protects centromeric cohesin from cleavage by separase and corrects wrong kinetochore-microtubule connections before anaphase of meiosis I. Shugoshin-1 (SGO1) plays a similar role in mitosis. Moreover, shugoshin can inhibit the occurrence of chromosomal instability (CIN), and its abnormal expression in several tumors, such as triple-negative breast cancer, hepatocellular carcinoma, lung cancer, colon cancer, glioma, and acute myeloid leukemia, can be used as biomarker for disease progression and potential therapeutic targets for cancers. Thus, this review discusses the specific mechanisms of shugoshin which regulates cohesin, kinetochore-microtubule connections, and CIN.

Shugoshin protects centromere pairing and promotes segregation of nonexchange partner chromosomes in meiosis.

Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.

Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation.

During mitosis and meiosis, sister chromatid cohesion resists the pulling forces of microtubules, enabling the generation of tension at kinetochores upon chromosome biorientation. How tension is read to signal the bioriented state remains unclear. Shugoshins form a pericentromeric platform that integrates multiple functions to ensure proper chromosome biorientation. Here we show that budding yeast shugoshin Sgo1 dissociates from the pericentromere reversibly in response to tension. The antagonistic activities of the kinetochore-associated Bub1 kinase and the Sgo1-bound phosphatase protein phosphatase 2A (PP2A)-Rts1 underlie a tension-dependent circuitry that enables Sgo1 removal upon sister kinetochore biorientation. Sgo1 dissociation from the pericentromere triggers dissociation of condensin and Aurora B from the centromere, thereby stabilizing the bioriented state. Conversely, forcing sister kinetochores to be under tension during meiosis I leads to premature Sgo1 removal and precocious loss of pericentromeric cohesion. Overall, we show that the pivotal role of shugoshin is to build a platform at the pericentromere that attracts activities that respond to the absence of tension between sister kinetochores. Disassembly of this platform in response to intersister kinetochore tension signals the bioriented state. Therefore, tension sensing by shugoshin is a central mechanism by which the bioriented state is read.

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.

Critical roles of Shugoshin and histones as tension sensors during mitosis.

Biorientation of paired sister chromosomes is required to maintain mitotic fidelity. A critical signal indicative of bipolar attachment is tension between cohesion-linked sister chromatids. Key components of the tension signaling apparatus include the Shugoshin family of proteins and the tension sensing motif of histone H3. Shugoshin proteins are recruited to chromatin to create discrete domains integral to tension sensing. Many factors involved in the chromatin association of Shugoshin proteins are well established, most strikingly through modifications found directly on centromeric and pericentric chromatin. It has been well established that phosphorylation at the centromere is essential to nucleating Shugoshin recruitment, but recent evidence revealed a role for pericentric histones and acetylation in modulating Shugoshin recruitment and activity. These data demonstrate that chromatins are not simply passive cargo during mitosis, but are instead actively involved in their segregation.

Emerging links among Chromosome Instability (CIN), cancer, and aging.

Aneuploidy was predicted to cause cancer. To test the prediction, various Chromosome Instability (CIN) mice models that carry transgenic mutations in mitotic regulators have been created. The availability of these mice has aided researchers in discovering connections between CIN, cancer, and aging. This review will focus on recent interdisciplinary findings regarding how CIN and aneuploidy affect carcinogenesis, immune dysfunction, and aging. High CIN can be generated in vivo by various intrinsic alterations (e.g., gene mutation, epigenetic modification) and extrinsic/environmental challenges (e.g., biological, chemical, biophysical), while immune surveillance, cell death, and natural turnover can remove cells with CIN. CIN itself is mutagenic and may cause further cellular mutations, which can be carcinogenic. Mitotically damaged cells can activate senescence-related tumor suppressors (e.g., p21WAF1 , p27KIP1 , p16INK4A ), which may lead to tissue-level senescence/aging through inflammatory paracrine mechanisms called Senescence-Associated Secretory Phenotype (SASP) and Senescence Inflammatory Response (SIR). Organs with high CIN show altered gene expressions in both organ-specific and non-specific manners. Organ-specific gene expression signatures include activation of oncogenic pathways. Non-organ-specific gene expression signatures include metabolic changes and downregulations in immune functions. Immune surveillance normally targets senescent cells and tetraploid cells, a form of aneuploidy, for elimination. However, with partial immune dysfunction, immune surveillance is weakened with systemic CIN. In this case, more senescent cells and aneuploid cells survive, which further leads to an inflammatory, pro-tumorigenic, and senescent/aging microenvironment. We also discuss how we may intervene in this sequence of events to prevent CIN- or age-related carcinogenesis and/or some aspects of tissue aging. © 2016 Wiley Periodicals, Inc.

Mutations of SGO2 and CLDN14 collectively cause coincidental Perrault syndrome.

Perrault syndrome (PS) is a genetically heterogeneous disorder characterized by primary ovarian insufficiency (POI) in females and sensorineural hearing loss in males and females. In many PS subjects, causative variants have not been found in the five reported PS genes. The objective of this study was to identify the genetic cause of PS in an extended consanguineous family with six deaf individuals. Whole exome sequencing (WES) was completed on four affected members of a large family, and variants and co-segregation was confirmed by Sanger sequencing. All hearing impaired individuals, including the proband, are homozygous for a pathogenic variant of CLDN14, but this only explains the deafness. The PS proband is also homozygous for a frameshift variant (c.1453_1454delGA, p.(Glu485Lysfs*5)) in exon 7 of SGO2 encoding shugoshin 2, which is the likely cause of her concurrent ovarian insufficiency. In mouse, Sgol2a encoding shugoshin-like 2a is necessary during meiosis in both sexes to maintain the integrity of the cohesin complex that tethers sister chromatids. Human SGO2 has not previously been implicated in any disorder, but in this case of POI and perhaps others, it is a candidate for unexplained infertility.

Antagonizing pathways leading to differential dynamics in colon carcinogenesis in Shugoshin1 (Sgo1)-haploinsufficient chromosome instability model.

Colon cancer is the second most lethal cancer. It is predicted to claim 50,310 lives in 2014. Chromosome Instability (CIN) is observed in 80-90% of colon cancers, and is thought to contribute to colon cancer progression and recurrence. However, there are no animal models of CIN that have been validated for studies of colon cancer development or drug testing. In this study, we sought to validate a mitotic error-induced CIN model mouse, the Shugoshin1 (Sgo1) haploinsufficient mouse, as a colon cancer study model. Wild-type and Sgo1(-/+) mice were treated with the colonic carcinogen, azoxymethane (AOM). We tracked colon tumor development 12, 24, and 36 wk after treatment to assess progression of colon tumorigenesis. Initially, more precancerous lesions, Aberrant Crypt Foci (ACF), developed in Sgo1(-/+) mice. However, the ACF did not develop straightforwardly into larger tumors. At the 36-wk endpoint, the number of gross tumors in Sgo1(-/+) mice was no different from that in wild-type controls. However, Copy Number Variation (CNV) analysis indicated that fully developed colon tumor in Sgo1(-/+) mice carried 13.75 times more CNV. Immunohistological analyses indicated that Sgo1(-/+) mice differentially expressed IL-6, Bcl2, and p16(INK4A) . We propose that formation of ACF in Sgo1(-/+) mice is facilitated by the IL6-STAT3-SOCS3 oncogenic pathway and by the Bcl2-anti-apoptotic pathway, yet further development of the ACF to tumors is inhibited by the p16(INK4A) tumor suppressor pathway. Manipulating these pathways would be beneficial for inhibiting development of colon cancer with CIN.

Deciphering the Role of Shugoshin-Like Protein 1 in Lung Adenocarcinoma: A Comprehensive Analysis and In Vitro Study.

Shugoshin-like protein 1 (SGO1) has been characterized in its function in correct cell division and its role in centrosome cohesion in the nucleus. However, the underlying biological function and potential mechanisms of SGO1 driving the progression of lung adenocarcinoma remain unclear. In this study, we found that SGO1 was increased in LUAD tissues and cell lines. Upregulation of SGO1 expression was correlated with poor overall survival (OS), disease-free survival (DSS), and progression-free survival (PFS) in patients with LUAD. ROC curve analysis suggested that the AUC value of SGO1 was 0.983. Correlation analysis showed that SGO1 expression was related to immune infiltration in LUAD. Meanwhile, a potential ceRNA network was constructed to identify the lncRNA-MIR4435-2HG/miR-125a-5p/SGO1 regulatory axis in LUAD. Finally, we determine that SGO1 regulated the cell proliferation and cell apoptosis of lung adenocarcinoma in vitro. In conclusion, our data suggested that SGO1 could be a novel prognostic biomarker for lung adenocarcinoma.

Subtelomeric Chromatin in the Fission Yeast S. pombe.

Telomeres play important roles in safeguarding the genome. The specialized repressive chromatin that assembles at telomeres and subtelomeric domains is key to this protective role. However, in many organisms, the repetitive nature of telomeric and subtelomeric sequences has hindered research efforts. The fission yeast S. pombe has provided an important model system for dissection of chromatin biology due to the relative ease of genetic manipulation and strong conservation of important regulatory proteins with higher eukaryotes. Telomeres and the telomere-binding shelterin complex are highly conserved with mammals, as is the assembly of constitutive heterochromatin at subtelomeres. In this review, we seek to summarize recent work detailing the assembly of distinct chromatin structures within subtelomeric domains in fission yeast. These include the heterochromatic SH subtelomeric domains, the telomere-associated sequences (TAS), and ST chromatin domains that assemble highly condensed chromatin clusters called knobs. Specifically, we review new insights into the sequence of subtelomeric domains, the distinct types of chromatin that assemble on these sequences and how histone H3 K36 modifications influence these chromatin structures. We address the interplay between the subdomains of chromatin structure and how subtelomeric chromatin is influenced by both the telomere-bound shelterin complexes and by euchromatic chromatin regulators internal to the subtelomeric domain. Finally, we demonstrate that telomere clustering, which is mediated via the condensed ST chromatin knob domains, does not depend on knob assembly within these domains but on Set2, which mediates H3K36 methylation.

Loss of sister kinetochore co-orientation and peri-centromeric cohesin protection after meiosis I depends on cleavage of centromeric REC8.

Protection of peri-centromeric (periCEN) REC8 cohesin from Separase and sister kinetochore (KT) attachment to microtubules emanating from the same spindle pole (co-orientation) ensures that sister chromatids remain associated after meiosis I. Both features are lost during meiosis II, resulting in sister chromatid disjunction and the production of haploid gametes. By transferring spindle-chromosome complexes (SCCs) between meiosis I and II in mouse oocytes, we discovered that both sister KT co-orientation and periCEN cohesin protection depend on the SCC, and not the cytoplasm. Moreover, the catalytic activity of Separase at meiosis I is necessary not only for converting KTs from a co- to a bi-oriented state but also for deprotection of periCEN cohesion, and cleavage of REC8 may be the key event. Crucially, selective cleavage of REC8 in the vicinity of KTs is sufficient to destroy co-orientation in univalent chromosomes, albeit not in bivalents where resolution of chiasmata may also be required.