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.

© 2023 S. Karger AG, Basel

Introduction

Aneuploidy can arise by three pathways: premature separation of sister chromatids, non-separation, and reverse separation [Webster and Schuh, 2017; Mikwar et al., 2020]. The abnormal chromosome number caused by incorrect chromosome segregation is called aneuploidy, which significantly increases the incidence of congenital birth defects such as infertility, abortion, Down syndrome, and tumors [Bohr et al., 2018; Mikwar et al., 2020]. Therefore, correct chromosome segregation is essential for transmitting genetic information in whole organisms. During meiosis, cohesin complex degradation in the chromosome arm region of anaphase I (AI) and the centromeric region of anaphase II (AII) sister chromatids is necessary to achieve highly conserved chromosome segregation [Mikwar et al., 2020]. However, before the late trigger, the early decline or loss of cohesin level leads to chromosome segregation errors [Tsutsumi et al., 2014], and the occurrence of unpaired sister chromatids is one of the direct reasons for the premature separation of sister chromatid aneuploidy [Mikwar et al., 2020]. In addition, sister chromatids are captured by the same pole before AI of meiosis, and homologous chromosomes cross-connected are captured by microtubules from two poles of the spindle because the tension produced by the centromeric cohesin resisting the pulling force between sister chromatids can stabilize kinetochore-microtubule connections [Kiburz et al., 2008; Clift and Marston, 2011; Marston, 2015; Miyazaki et al., 2017b; Deng and Kuo, 2018]. At the beginning of AI, the meiosis-specific subunit REC8 at the chromosome arm is cleaved by separase, but the cohesin at the centromeres is protected until metaphase II, which explains homologous chromosome segregation and sister chromatid non-segregation in AI [Marston, 2015; Miyazaki et al., 2017]. Thus, the correct chromosome segregation requires centromeric cohesin existence before AII and degradation in AII to inhibit aneuploidy.

Studies have shown that shugoshin plays a vital role in regulating the existence and degradation of chromosome arm and centromeric cohesin and stabilizing kinetochore-microtubule connections [Mehta et al., 2018; Previato de Almeida et al., 2019]. Shugoshin was first found in Drosophila, and MEI-S332 is its immediate homolog. To date, two members of the shugoshin protein family, SGO1 and SGO2, have been reported. Only SGO1 was found in Drosophila melanogaster, budding yeast and Saccharomyces cerevisiae, while SGO1 and SGO2 were found in Schizosaccharomyces pombe, vertebrates, plants, and mammals. They display similar and different effects on the mitosis and meiosis of different species (Table 1) [Kawashima et al., 2007; Wang et al., 2011; Marston, 2015]. In mitosis, shugoshin/MEI-S332 protects cohesin in vertebrates, drosophila, and mammals without any effect in Schizosaccharomyces pombe, budding yeast, nematodes, and plants. However, shugoshin/MEI-S332 is unnecessary to protect mitotic cohesin in Drosophila (Table 1) [Kawashima et al., 2007]. During meiosis, SGO1 is necessary for Drosophila, budding yeast, and plants, but SGO2 in mammals for meiosis I to protect the centromeric cohesin (Table 1) [Kiburz et al., 2008; Wang et al., 2011; Yin et al., 2013; Nogueira et al., 2014; Peplowska et al., 2014; Grishaeva et al., 2016].

Table 1.

Subtypes and functions of SGO2 in different species

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In Saccharomyces cerevisiae, SGO1 is expressed only during meiosis and plays a crucial role in the centromeric cohesin of sister chromatids in meiosis I, while SGO2 is continuously expressed in the M phase and contributes to accurate chromosome segregation. Interestingly, the SGO2 location changes from centromere to telomere after entering the interphase, and reaches the peak in the middle of the G2 phase. SGO2 and subtelomeres form a new chromatin inhibiting domain different from H3K9me-Swi6 mediated heterochromatin, which plays a vital role in nodules and highly concentrated chromatid formation. SGO2 inhibits the initial replication step by limiting the loading of SLD3 at the late start of the subtelomere, which is necessary for proper replication time at the subtelomeric late regions [Tashiro et al., 2016; Kanoh et al., 2018]. However, SGO1 is located in the centromere from MII until the beginning of AII in budding yeast, drosophila and vertebrates, even though it does not play any role in the anaphase of meiosis [Kawashima et al., 2007].

Nevertheless, in mammals such as bovines and mice, SGO1 mainly plays a role in mitosis, preventing centromeric cohesin from entering the “prophase pathway” during mitosis, and protecting it from separase cleavage [Yin et al., 2013]. The coiled helix domain of SGO2 combines with PP2A to antagonize the phosphorylation of the meiosis-specific subunit REC8 of cohesins, thereby protecting the centromeric cohesin from cleavage by separase during meiosis I [Gomez et al., 2007; Kiburz et al., 2008; Clift and Marston, 2011; Cromer et al., 2013; Zamariola et al., 2013; Marston, 2015; Sane et al., 2021]. In addition, SGO2 is necessary to recruit Aurora B to the kinetochore to sense whether kinetochore-microtubule junctions are under tension during mitosis and meiosis. Thus, shugoshin is necessary for effective sister-kinetochore orientation and can help promote the correct connection of chromosomes in mitotic or meiotic II spindles [Resnick et al., 2006; Kiburz et al., 2008; Asai et al., 2019]. SGO2 can also interact with microtubule depolymerization kinesin (MCAK) that corrects inappropriate kinetochore-microtubule junctions [Rattani et al., 2013]. Hence, this review aimed to explore the specific mechanisms by which shugoshin regulates the presence and degradation of chromosome arm and centromeric cohesin and stabilizes kinetochore-microtubule connections, and their relation to cancer.

Shugoshin Protects Cohesin Subunits REC8 and RAD21/SCC1 from Separase Cleavage

Degradation of the cohesin complex in the anaphase I chromosome arms and the anaphase II sister chromatid centromeres has been widely recognized to achieve highly conserved chromosome segregation [Lister et al., 2010]. A study found that in mitosis, the cohesin protein complex is usually composed of SMC1 and SMC3 cyclic dimers, and auxiliary subunit SCC1/RAD21, α-kleisin subunit, and SCC3. During meiosis, the cohesin protein complex subunit SCC1/RAD21 is replaced by REC8 [Nasmyth et al., 2009; Clift and Marston, 2011; Miyazaki et al., 2017]. At the centromeres, the REC8 subunit of cohesin is safeguarded and not dissolved in the late stage of the first meiosis and removed during anaphase II to ensure proper chromosome division [Mengoli et al., 2021].

In mammalian mitosis, cohesin release follows a two-step process: during the early stage, the SA2 subunit on cohesin is phosphorylated by PLK1 resulting in the release of cohesin from the chromosome arms [Gomez et al., 2014]. Similarly, cohesin promoting factor sororin phosphorylation causes it to be replaced by cohesin inhibiting factor WAPL, resulting in the release of chromosome arm cohesin [Rakkaa et al., 2014]. Centromeric cohesin must be maintained when RAD21/SCC1 is cleaved to prevent untimely segregating sister chromatids [Wang et al., 2011; Zamariola et al., 2013]. Likewise, homologous chromosomes formed chiasmata are linked by cohesin in meiosis I. In anaphase I, chiasmata division dissolves by the specific release of chromosome arm cohesin. However, linked sister chromatids need cohesin, including REC8, to be maintained at the centromeres, to ensure accurately resolving homologous chromosomes through kinetochores connected to the same pole of microtubules. Next, anaphase II results in segregating sister chromatids that kinetochores connect to different poles, leading to the formation of four haploid cells [Wang et al., 2011; Zamariola et al., 2013]. Thus, it suggests that in anaphase I chromosomal arms and centromeric cohesin, and in anaphase II centromeric cohesin are protected from separase cleavage to prevent the wrong division of homologous chromosomes and premature division of sister chromatids.

A study found that cohesin was protected from being cleaved by shugoshin [Miyazaki et al., 2017]. In fission yeast, SGO2 safeguards cohesin at centromeres during mitosis [Clift and Marston, 2011; Zamariola et al., 2013; Sane et al., 2021]. In contrast, SGO1 instead of SGO2 showed similar action in mammals [Zamariola et al., 2013]. During mitosis, BUB1 generates H2A-pT120 at the kinetochores, which is recognized directly by SGO1 and recruits SGO1 into the kinetochores. H2A-pT120 also recruits Pol II to kinetochores, which promotes Pol II-mediated mitotic specific transcription and destroys the interaction between SGO1 and nucleosomes containing H2A-pT120, enabling SGO1 to penetrate dense in the mitotic chromatin and reach the inner centromeres to protect cohesin [Liu et al., 2015; Liu, 2016]. In contrast, during meiosis I, the REC8 on chromosome arms is not defended under the existence of SGO2. When sister chromatids achieve biorientation during the second meiotic division, new cohesin dissociates at centromeres [Marston, 2015]. SGO1, an essential protein required for centromeric cohesin protection during MI, can dephosphorylate REC8 by recruiting PP2A-RTS1 and form separase-resistance during MI in Schizosaccharomyces pombe, fission yeast, and Saccharomyces cerevisiae [Mehta et al., 2018; Ma et al., 2021]. At the same time, phosphorylation of REC8 by Meikin-PLO1 activates PP2A, which causes REC8 dephosphorylation, preventing separase from cleaving REC8 [Gutierrez-Caballero et al., 2012; Kim et al., 2015; Galander et al., 2019; Ma et al., 2021]. In addition, consistently, deletion of the splice variants SGO1A and SGO1C of SGO1 or SGO1A alone results in the loss of sister chromatid cohesin [Wong et al., 2015], which supports the notion that SGO1 is indispensable to defending centromeric cohesin in the first meiotic division. SGO2 is required for sensing whether microtubule-kinetochore attachments are under tension through recruting Aurora B to kinetochores [Kiburz et al., 2008]. However, in mammalian oocytes, SGO2 colocalizes to centromeres with REC8 in the middle of the first meiotic division, which defends REC8 at centromeres not subject to being cleaved by separase. SGO2 is dispensable to maintain pericentromeric REC8 during MII [Ogushi et al., 2021]. Conversely, a tension-dependent redistribution of SGO2 in metaphase II facilitates the removal of REC8, allowing sister chromatids to separate during the meiosis II anaphase [Clift and Marston, 2011; Wassmann, 2013]. In addition, shugoshin also prevents cohesin cleavage through PP2ACDC55-dependent inhibition of separase [Clift et al., 2009]. Therefore, shugoshin is required to protect cohesin from being cleaved by separase.

Shugoshin-PP2A and Sororin Synergistically Protect Centromeric Cohesin from Removal

In vertebrates, the establishment and maintenance of cohesin attachments rely on sororin and shugoshin. Cohesin is loaded onto chromatin during the terminal/G1 phase, while in the course of DNA reproduction in the S/G2 period, SMC3 is acetylated by the cohesin-binding protein sororin and acetyltransferase ECO1, which allows its establishment [Nishiyama et al., 2013; Marston, 2015]. Cohesin is dissociated from the chromosome arms during prophase through the prophase pathway. In the mammalian prophase pathway, phosphorylated sororin disrupts the interaction of sorosin with PDS5, allowing WAPL to interact with PDS5, enabling dissociation of acetylated cohesin from chromosome arms, consistent with vertebrates. WAPL drives the cohesin protein loop opening at the SMC3-SCC1 interaction site, leading to its dissociation along chromosome arms in prophase and prometaphase [Nishiyama et al., 2013; Mohr et al., 2015; Hertz and Nilsson, 2017]. SMC3 acetylation recruits sororin protein to antagonize cohesin dissociation [Liu et al., 2013a; Marston, 2015; Yamada et al., 2017], and the cohesin on the centromere is also defended through SGO1/PP2A. SGO1 holds sororin and SA2 on dephosphoric acid status by competing with WAPL to bind to the conserved site on SA2-SCC1, thereby inhibiting Aurora B, CDK1, and PLK1 kinase activity (Fig. 1) [McGuinness et al., 2005; Llano et al., 2008; Liu et al., 2013a; Nishiyama et al., 2013; Hara et al., 2014; Rakkaa et al., 2014; Pallai et al., 2015; Grishaeva et al., 2016], to protect the centromeric cohesin from being removed by WAPL until the metaphase/anaphase transition [Pinto and Orr-Weaver, 2017].

Fig. 1.

Shugoshin inhibits removal of acetylated cohesin from chromosome arms by counteracting Aurora B activity. Shugoshin (cyan) counteracts Aurora B (brown) activity to inhibit interaction between WAPL (purple) and PDS5 (light grey) by inhibiting sororin (yellow) phosphorylation, which protects acetylated cohesin of chromosome arms from removal. Arrows represent promotion, blunt arrows represent inhibition.

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In the first meiotic division, untimely segregation of sister chromatids was found under phosphatase inhibitor treatment [Clift and Marston, 2011], indicating that phosphatase PP2A is needed for chromosome division during meiosis. During the first meiosis, SGO2 is situated at the centromeres as is PP2A [Kitajima et al., 2006]. The coiled-coil domain of SGO2-PP2A can antagonize phosphorylation of the cohesin meiosis-specific subunit REC8 to defend cohesin at centromeres in the first meiotic division (Table 2) [Kiburz et al., 2008; Clift and Marston, 2011]. It was found that sororin cooperates with shugoshin-PP2A in regulating cohesin at centromeres. During mitosis, shugoshin-PP2A localizes to the centromere and binds to the cohesin to dephosphorylate sororin and defend the cohesin at centromeres from influence of mitotic kinase and cohesin inhibitor WAPL (Table 2) [Liu et al., 2013a, b, 2015]. Moreover, SGO1-PP2A also defends acetylated cohesin at the centromeres by counteracting sororin phosphorylation (Fig. 1; Table 2) [Nishiyama et al., 2013]. Sororin was found in the core area of the synaptonemal complex in the prophase of the first meiotic division, which activates PP2A to accumulate at centromeres and remains until the later stage of second meiotic division. In contrast [Gomez et al., 2016], in meiosis II and prometaphase, centromere/kinetochore-generated tension induces redistribution of shugoshin-PP2A, leading to disaggregation of sororin, that phosphorylates REC8 to allow the cohesin to be dissociated by separase before the onset of the second meiotic division to allow sister chromatid separation [Gomez et al., 2007; Liu et al., 2013].

Table 2./WebMaterial/ShowPic/1496946Anaphase II APC/C Removes Centromeric Cohesin by Silencing SAC and Relocating Shugoshin

Both in mitosis and meiosis, shugoshin plays a critical role around the centromere only when the sister centromeres are in a tension-free state [Nerusheva et al., 2014]. During mitosis, anew fixed position of shugoshin that relies on tension was discovered in mouse oocytes. Several reports indicate that a fixed position of Shugoshin is a core of the procedure of perceiving tension [Marston, 2015]. Tension-dependent relocalization of shugoshin phosphorylated at the kinetochore was initiated, essential to redress chromosome division [Nerusheva et al., 2014; Marston, 2015]. Removal of cohesin relies on spindle force to divide shugoshin from cohesin in the middle of the second meiotic division, and is disrupted depending on APC/C activity in anaphase II. In addition, at metaphase-to-anaphase transition, SET removes SGO1 from the inner centromeres to remove centromeric cohesin by disrupting both the SGO1-cohesin and SGO1-nucleosome, thereby promoting sister chromatid separation.

Nevertheless, SGO1 removal from the kinetochore depends on the tension instead of APC/C activity, which is different from meiosis. Tension has two independent functions: removing centromeric cohesin and silencing SAC. SAC can inhibit APC/CCDC20, inhibiting separase activity and delaying entry into anaphase II (Fig. 1). When all chromosomes reach biorientation, kinetochore tension silences the SAC and allows anaphase chromosome segregation [Orth et al., 2011]. SGO2 was found to have the same distribution as REC8 at centromeres of the first meiotic division, while in the existing tension, SGO2 promotes sister chromatid biorientation and shifts to centromeres and distances REC8 away to permit REC8 phosphorylation. Thus, REC8 is prone to cleavage upon separase activation, which accelerates the onset of the later stage of second meiosis [Marston, 2015]. Besides, studies on mouse oocytes revealed that APC/CCDC20 activity removes PP2A-RTS1 from the kinetochore during meiosis II, thereby triggering sister kinetochore segregation under free-tension, which is different from the bipolar spindle that removes REC8 at centromeres. Spindle force is determined by a topoisomerase II-dependent promotion of centromeric sister DNA, which sterically separates SGO2-PP2A from the centromeric cohesin, phosphorylating REC8 that enables cohesin removal to facilitate sister kinetochore separation [Chambon et al., 2013; Mengoli et al., 2021]. This suggests that anaphase II cohesin removal requires APC/C to silence SAC and relocate shugoshin to allow sister chromatid separation.

Shugoshin Recruits CPC to Centromeres to Correct Inappropriate Kinetochore-Microtubule Attachments

Monotelic, syntelic, and amphitelic attachments may occur in the sister kinetochore. Monotelic attachments are when only one of the two sister kinetochores is attached to the microtubule. The unattached kinetochore triggers the formation of a mitotic checkpoint complex (MCC), which prevents cell cycle progression by capturing the CDC20. In syntelic attachments, both sister kinetochores are captured by microtubules of the same pole. Moreover, wrong monotelic and syntelic attachments before anaphase can lead to aneuploidy [Deng and Kuo, 2018]. Therefore, correcting inappropriate kinetochore-microtubule attachments before entry into anaphase is crucial for appropriate chromosome division.

Shugoshin plays a significant role in correcting the connection between kinetochore and microtubule. Mammalian shugoshin is associated with PP2A and chromosomal passenger complex (CPC) at centromeres [Suzuki et al., 2006; Asai et al., 2019]. CPC is a critical participant that guarantees the appropriate chromosome separation to avoid chromosomal instability (CIN) and aneuploidy [Habib et al., 2018]. Shugoshin promotes CPC recruitment to centromeres by binding to borealin (Fig. 2) [Wang et al., 2021], which ensures kinetochore attachment to microtubules emanating from bipolar spindles, thereby facilitating the correction of improper attachment on the mitotic spindle (Table 2) [Kobayashi and Kawashima, 2019; Sane et al., 2021]. The CPC can dissolve kinetochore-microtubule mis-attachment through phosphorylating several substrates and is one of the highly conserved complexes that sense the lack of kinetochore-microtubule tension (Table 2) [Habib et al., 2018; Cairo and Lacefield, 2020; Wang et al., 2021]. The resulting unconnected kinetochore activates the SAC, stabilizes securin and inhibits separase, thereby delaying entry into anaphase II to ensure proper kinetochore-microtubule attachments (Fig. 2) [Peplowska et al., 2014; Marston, 2015].

Fig. 2.

Shugoshin protects cohesin at kinetochores and corrects inappropriate kinetochore-microtubule attachments. Shugoshin interacts with PP2ACDC55 (light brown), MAD2 (pink), and CPC (light orange) to inhibit separase (blackish green), which protect meiosis-specific subunit REC8 (dark orange) or SCC1/RAD21 (dark green) of cohesin at kinetochores (dark grey) from being cleaved before anaphase. Shugoshin recruits IPl1 (lavender) and CPC to kinetochores by interacting with borealin (green), BIR1/survivin (pink grey), and PP2A (red) to active SAC (grayish green), which inhibts activity of separase, so protecting cohesin at kinetochores from removing before onset of anaphase, and promoting correct attachments of kinetochore-microtubule. Shugoshin recruits PP2A (red) to kinetochores, inhibiting Aurora B (dark brown) activity, which promotes MCAK (reddish brown) accumulation at kinetochores to correct inappropriate attachments of kinetochore-microtubule. Shugoshin interacts with MAD2 to silence SAC to promote onset of anaphase. Arrows represent promotion, blunt arrows represent inhibition. The protein binding to P (yellow) indicates phosphorylation, and P shedding indicates dephosphorylation.

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During mitosis, the CPC is distributed at the centromeres in the middle stage to correct errors in kinetochore-microtubule attachments, regulating cohesin and checkpoint. It then relocates to microtubules to form the intermediate region of the spindle, which is required for anaphase and cytokinesis [Resnick et al., 2006; Wang et al., 2021]. Previous evidence suggests that SGO2 deletion damages the Pic1/INCENP-BIR1/survivin complex and CPC recruited to centromere during mitosis in vertebrates and fission yeast [Vanoosthuyse et al., 2007]. Likewise, the SGO1-PP2A interaction is required to recruit CPC to the centromeres during mammalian mitosis [Liang et al., 2019; Ueki et al., 2021]. SGO1 also indirectly recruits CPC to inner centromeres to offer a connection locus for the histone H3 kinase Haspin by defending cohesin at centromeres to correct wrong attachments and prevent chromosome mis-segregation [Tanno et al., 2010; Meppelink et al., 2015; Asai et al., 2019; Liang et al., 2019]. In mammalian meiosis, SGO2 combines phosphorylated BIR1/survivin CPC to accelerate CPC localization to the centromeres, and interacts with CPC to promote Aurora B kinase distributed at centromeres to ensure proper kinetochore-microtubules connection (Fig. 2) [Zamariola et al., 2013; Cairo and Lacefield, 2020]. This suggests that shugoshin recruits CPC to the kinetochore to correct improper kinetochore-microtubule attachment.

SGO2 Recruits MCAK to Centromeres to Correct Inappropriate Kinetochore-Microtubule Attachments

Correct spindle assembly and chromosome division depend on precise microtubule dynamics, partially regulated by kinesin-13 and mitotic centromere-associated kinesin (MCAK) [Vogt et al., 2010]. MCAK loss causes kinetochore-microtubule attachment errors and chromosomal cohesin defects [Meppelink et al., 2015]. MCAK is a microtubule depolymerase that is phosphorylated by Aurora kinase (AURK) and can disrupt inappropriate kinetochore-microtubule attachments to prevent wrong chromosome division during prophase, metaphase, and early anaphase of chromosomal cohesin [Vogt et al., 2010].

SGO2 promotes MCAK activity in Xenopus laevis [Marston, 2015]. It has been found that during mitosis in mammals such as humans and mice, T537 and T620 on human SGO2 (corresponding to T521 and T600 on mouse SGO2) are phosphorylated by AURKB, facilitating localization of PP2A and MCAK to the centromeres that combine and promote dephosphorylation of the cohesin meiosis-specific subunit REC8 to protect it from separase cleavage (Fig. 2; Table 2) [Tanno et al., 2010; Grishaeva et al., 2016]. SGO2 can also inhibit AURKB/C activity at the kinetochore, which favors MCAK accumulation at the kinetochore (Fig. 2), thereby increasing the microtubule depolymerization activity of MCAK at the kinetochore and correcting inappropriate kinetochore-microtubule attachments (Table 2) [Clift and Marston, 2011; Rattani et al., 2013; Zamariola et al., 2013]. This suggests that shugoshin recruits MCAK to centromeres and rectifies the inappropriate kinetochore-microtubule connection.

Shugoshin Recruits PP2A to Centromeres to Correct Kinetochore-Microtubule Mis-Attachments by Regulating AURKB Activity

Previous studies found that deletion or inhibition of Aurora B kinase disrupts SGO1 accumulation at kinetochores in HeLa cells [Pouwels et al., 2007]. In cells that lack Aurora B kinase, kinetochore-microtubule mis-attachments are prematurely stabilized and SAC is silenced, leading to errors in chromosome separation. Therefore, the correct kinetochore-microtubule connection depends on AURKB activity. The instability of tension-free kinetochore-microtubule connection performed by AURKB generates unattached kinetochores, which activates SAC to sense unattached kinetochores or deletion of tension and inhibits premature entry into anaphase [Kawashima et al., 2007]. AURKB phosphorylates serine residue 10 on histone H3 (H3pS10) releasing phosphorylated HP1 from chromatin, which facilitates shugoshin to localize in the centromeres [van de Werken et al., 2015]. Besides, a tension-sensing motif on histone H3 maintains SGO1 localization to the centromere. The histone H3 tail replenishes the damaged tension-sensing motif to ensure proper chromosome segregation [Luo et al., 2016; Buehl and Kuo, 2018; Buehl et al., 2018]. However, the phosphatase PP2A has a counteracting effect. PP2A-B56 is distributed from centromere to kinetochore under tension, resulting in Aurora B dephosphorylation and PLK1 phosphorylated substrates on the kinetochore. PP2A negatively regulates Aurora B activity by removing phosphorylated Thr232 (Fig. 2) [Asai et al., 2019], thereby allowing initial kinetochore-microtubule attachments to be established in early mitosis.

Shugoshin is required for recruiting Aurora B and PP2A-B56 to centromeres [Kawashima et al., 2007; Bel Borja et al., 2020; Sane et al., 2021; Ueki et al., 2021]. During mitosis, SGO1 promotes CPC accumulation at the centromeres [Verzijlbergen et al., 2014; Williams et al., 2017], and subsequently recruits Aurora B to centromeres through CPC to sense the loss of tension due to sister kinetochores unattached to microtubules from different poles [Kawashima et al., 2007; Kiburz et al., 2008]. In meiosis, the SGO2 and Aurora kinase complexes colocalize at the centromere in the prometaphase and metaphase [Kawashima et al., 2007]. SET on SGO2 interacts with SGO2 in earlier stages and early metaphase to maintain AURKB activity by counteracting PP2A [Qi et al., 2013]. Furthermore, SGO2 deletion and mutation of the Aurora kinase complex significantly enhanced pericentromeric heterochromatin Swi6 deficiency, while increasing the incidence of lagging and missegregation of chromosomes. These defects can be restored by localizing BIR1 to the centromere [Kawashima et al., 2007]. SGO2 interacts with BIR1/survivin and accelerates AURK distribution in pericentromeric regions to redress kinetochore-microtubule mis-attachments for achieving tension-generating attachments (Table 2). However, an excessive increase in Aurora B kinase activity leads to the persistence of unconnected kinetochores and unstable spindles, increasing the frequency of chromosome gains or losses [Rattani et al., 2013; Asai et al., 2019]. Studies have shown that SGO2 can also inhibit the AURKB/C activity by reducing the autophosphorylation of Aurora B/C kinase to promote chromosome biorientation (Table 2) [Rattani et al., 2013]. Thus, shugoshin can promote AURKB activity by recruiting PP2A to centromeres and inhibiting autophosphorylation of AURKB/C kinases, ensuring proper kinetochore-microtubule attachments.

Shugoshin Maintains Ipl1 Kinase Distributed at Centromeres to Correct Wrong Kinetochore-Microtubule Connection

Under tension-free circumstances, the distance between kinetochores is contiguous. In contrast, tension increases the distance between kinetochores, reducing substrate phosphorylation by Aurora B to stabilize the kinetochore attached to the microtubule [Nerusheva et al., 2014]. Tension-free kinetochore-microtubule connections are resolved by activated AURKB/Ipl1, which advances exact chromosome connection. Aurora B/Ipl1 was found to take part in and advance spindle disassembly [Sane et al., 2021].

It was found that shugoshin is necessary to maintain the localization of AURKB/Ipl1 at the kinetochore in metaphase [Peplowska et al., 2014; Sane et al., 2021]. Firstly, pericentromeric chromatin conformation was regulated by shugoshin, which promoted the ability to recruit cohesin in budding yeast. Secondly, shugoshin helped to maintain AURKB/Ipl1 at the kinetochore to correct improper kinetochore-microtubule connection [Peplowska et al., 2014]. In Saccharomyces cerevisiae, SGO1 is responsible for recruiting Aurora B/Ipl1 to the kinetochore, thereby promoting the recruitment of the monopoly protein complex to kinetochore by falling off Ndc80 to prevent merotely (Table 2) [Verzijlbergen et al., 2014; Mehta et al., 2018]. Likewise, shugoshin interacts with CPC in mammals to promote Aurora B/Ipl1 localization around the centromeres. Ipl1 generates unattached kinetochores by severing microtubule-centromere interactions and activating SAC. SAC recognizes unattached kinetochores and postpones the late phase by counteracting APC/C activity until all sister chromatid pairs become biorientated (Fig. 2) [Kiburz et al., 2008]. In addition, SGO1 and Ipl1 phosphorylated Dam1, necessary for maintaining the phosphorylation circumstance of SAC protein without tension, which prevented SAC silence to maintain metaphase arrest until all kinetochore-microtubule attachments are correct (Table 2) [Jin and Wang, 2013]. Thus, shugoshin can correct improper attachments by maintaining centromeric Ipl1. In addition, shugoshin recruits condensin to the pericentromeric regions, which promotes sister kinetochore capture by microtubules from different poles, independent of Aurora B/Ipl1 error attachment [Verzijlbergen et al., 2014].

SUMOylation of Shugoshin Corrects Improper Kinetochore-Microtubule Connection

In addition to regulating kinetochore-microtubule connection by phosphorylation, shugoshin can also subject SUMOylation to correct improper kinetochore-microtubule attachments. SUMOylation is a significant protein post-translational modification, which produces a marked effect on the regulation of the cell cycle, cell proliferation, differentiation, and apoptosis by target protein binding to the SUMO protein [Saitoh and Hinchey, 2000; Ihara et al., 2008; Wang et al., 2010; Rodriguez and Pangas, 2016]. Four subtypes of SUMO protein are involved in regulating many cellular processes [Mattoscio et al., 2013], and their distribution is similar [Geiss-Friedlander and Melchior, 2007]. Inhibition of SUMOylation disrupts proper kinetochore-microtubule attachments [Feitosa et al., 2018]. In the absence of SUMO ligase siz1/siz2, SGO1 was stable, CPC removal was insufficient, and biorientation was unstable. It was found that SUMO modification of SGO1 is necessary to maintain kinetochore-microtubule biorientation (Table 2). The deficiency of siz1/siz2 leading to medium-term delay was caused partly by CPC error correction. After the biorientation, SGO1 loss leads to the increase in CPC, which activates SAC, inhibits APC/C activity, and then inhibits the activity of separase, hindering the beginning of the later stage. The results showed that siz1/siz2 and SGO1 SUMOylation promoted the mid-late transition in a SAC- and CPC-dependent manner to stabilize the kinetochore-microtubule biorientation. In mammalian meiosis, SGO2 colocalizes with SUMO2/3 at the centromere [Ding et al., 2018]. However, it is still unclear whether SGO2 can interact with SUMO2/3 to stabilize sister kinetochore biorientation during meiosis, and the specific mechanism needs further studies.

SGO2 Silences SAC by Recruiting MAD2 to Centromeres to Correct Inappropriate Kinetochore-Microtubule Connection

SAC is a monitor mechanism that promotes accurate chromosome separation. A requirement for satisfying the SAC is appropriate tension between sister chromatids before the onset of anaphase [Kawashima et al., 2007; Rakkaa et al., 2014]. The SAC senses the kinetochore-microtubule connection state and prevents activating APC/C when the kinetochore is unconnected or in a tension-free state [Meadows et al., 2017]. The activation of APC/C destroys securin and cyclin B, thereby activating separase, which cleaves the kleisin subunit of the cohesin complex to hold the sister chromatids together. Thus, SAC silence is required for the first meiosis by activating APC/C to trigger anaphase onset [Meadows et al., 2017]. The study found that silencing SAC requires direct binding of SGO2 to PP2A and the MCC protein MAD2 [Rattani et al., 2013; Marston, 2015; Hellmuth et al., 2020]. However, only dephosphorylated MAD2 can interact with SGO2. PP2A can selectively stabilize the SGO2-MAD2 complex by keeping MAD2 in a dephosphorylated state, thereby silencing SAC to alter the activity of MCC, the APC/C inhibitor (Fig. 2) [Rattani et al., 2013], activating APC/C to trigger anaphase onset (Table 2). APC/C activation initiated ubiquitination of securin and cyclin B, which resulted in the deletion of sister chromatid cohesin and inactivation of cyclin B/Cdk1, respectively, to promote AURKB and CPC redistribution from centromeres to the central spindle area [Meadows et al., 2017], which allows the correct kinetochore-microtubule connection to guarantee an accurate chromosome separation.

Shugoshin Inhibits Chromosomal Instability

Lagging chromosomes are a sign of CIN, usually caused by a continuous error in kinetochore-microtubule connections [Liang et al., 2019]. Incorrect chromosome segregation leads to CIN, resulting in aneuploidy in cancer, developmental disorders, and congenital birth defects [Mishra et al., 2018]. Moreover, CIN is mainly caused by the loss of mitotic fidelity, which leads to aneuploidy. Thus, CIN and aneuploidy are markers of several cancers. The dysfunction of the inner centromere-shugoshin (ICS) network may be the critical mechanism of CIN in human tumorigenesis. The stability of the ICS network requires the combination of centromere HP1a with H3K9me3 and SGO1 with H3K9me3 to inhibit CIN occurrence (Table 2) [Tanno et al., 2015]. Consistently, SGO1 deletion can affect the function of the centrosome, leading to defects in multipolar cell division, thus leading to CIN [Iwaizumi et al., 2009; Rao et al., 2016a, b]. In addition, Bub1 can prevent chromosomal instability by phosphorylating H2A to locate shugoshin [Kawashima et al., 2010]. Moreover, SGO1-PP2A inhibition can cause CIN. CIP2A, an inhibitor of PP2A, can destroy cohesin through interaction with SGO1, leading to premature chromosome separation and aneuploidy [Pallai et al., 2015]. Overexpressed SGO1 recruited excessive PP2A to the centromere without Aurora B, weakening Aurora B function and the consequent premature superstability of kinetochore-microtubule connections, which is related to chromosomal instability (Table 2) [Meppelink et al., 2015]. This shows that shugoshin can inhibit CIN occurrence by stabilizing the ICS network and inhibiting Aurora B activity.

Shugoshin Can Be Used as a Molecular Target for Various Cancers

CIN caused by shugoshin deletion is involved in the occurrence and development of lung cancer, colon tumorigenesis, neuroblastoma, hepatocellular carcinoma (HCC), prostate cancer, glioma, triple-negative breast cancer (TNBC), and acute myeloid leukemia (AML). CIN can be induced by G2/M arrest caused by SGO1 knockout, which inhibits cell proliferation and promotes cell apoptosis [Iwaizumi et al., 2009; Yuan et al., 2022]. SGO1 deletion can activate spindle checkpoint, leading to premature activation of APC/C and separase, resulting in premature separation of cohesin from the centromere [Liu et al., 2012; Rao et al., 2016a]. SGO1 can also interact with CIP2A to reduce the expression levels of PDS5 and SGO1, and promote the cleavage of cohesin, leading to premature separation of sister chromatids and the permanent arrest of mitosis thereby inducing CIN [Pallai et al., 2015]. SGO1 deletion also activates the Wnt signal in the lung, reducing glutathione’s protective effect and increasing DNA damage, which promotes the occurrence and development of lung cancer [Yamada et al., 2016]. Simultaneously, SGO1-B, one of the SGO1 splicing variants, induces abnormal mitosis in non-small cell lung cancer (NSCLC) and resistance to taxane. Therefore, SGO1 is an excellent molecular target for lung cancer treatment [Liu et al., 2012]. In addition, the overexpression of SGOL1-P1, a splicing variant of SGO1, in colon cancer HCT116 cells leads to abnormal chromosome arrangement, inhibits the colocalization of endogenous SGO1 and the subunit RAD21/SCC1 of cohesin, leading to premature separation of sister chromatids, delayed mitotic process, and CIN. These phenomena can also be observed in SGO1 knockout and SGO1 haploid-deficient cells.

The splicing variant of SGOL1 is considered a negative factor of natural SGOL1, indicating that SGO1 can inhibit CIN. Therefore, SGO1 can be used as a molecular target for treating colon cancer [Kahyo et al., 2011]. Similarly, SGO1 downregulation impaired proliferation and induced DNA damage in MYCN-overexpressing neuroblastoma cells. Therefore, SGO1 is also a potential molecular target for treating MYCN-amplified neuroblastoma [Murakami-Tonami et al., 2016]. CIN induced by SGO1 deletion also promotes HCC occurrence and development, which can be used as a potential therapeutic target for HCC [Wang et al., 2015; Yamada et al., 2015]. SGO1 can also induce proliferation and metastasis of prostate cancer through the AKT-mediated signal pathway, and its expression is positively related to the poor prognosis of prostate cancer patients. SGO1 knockdown significantly increased the expression of cleaved caspase-3, and cleaved caspase-9, and cleaved PARP and decreased the phosphorylation of AKT pRWPE1 in PC3 and DU145 cells, indicating that the high expression of SGO1 in prostate cancer cells promotes cell cycle, inhibits cell apoptosis, and promotes the invasion and metastasis of prostate cancer cells. Therefore, SGO1 is combined with AKT inhibitors for targeted prostate cancer therapy [Chen et al., 2019].

SGO2 mRNA and protein expression in glioma were higher than in the normal brain tissue. Inhibiting SGO2 could inhibit cell proliferation and migration. SGO2-expression is also positively correlated with the poor prognosis of patients with high-grade glioma, indicating that SGO2 can be used as a biomarker to predict the disease progression of glioma [Kao et al., 2021]. SGO1 knockdown also regulates the level and localization of epithelial-to-mesenchymal transition, which plays a role in cancer, and reduces the mRNA levels of MMP2, MMP3, and MMP9, leading to decreased cell invasion, migration, and metastasis. Therefore, SGO1 can be a therapeutic target for TNBC [Jusino et al., 2021]. SGO1 inhibition can also reduce the expression of anti-apoptotic proteins (BCL-2, BCL-XL, Mcl-1) and mitochondrial membrane potential, leading to the release of cytochrome c, activating caspase cascade reaction, and inducing apoptosis of leukemia cells. It is suggested that SGOL1 may be a promising therapeutic target for AML [Yang et al., 2013].

Conclusion

The existence and removal of chromosome arm and centromeric cohesin and the stable kinetochore-microtubule connection are essential for correct chromosome segregation. Shugoshin plays a significant role in the gradual removal of cohesin. Shugoshin-PP2A maintains the dephosphorylation state of chromosome arms and centromeric cohesin subunits before mitosis and anaphase of meiosis, which protects cohesin from cleavage. At a later stage, the tension-dependent redistribution of shugoshin-PP2A and its dissociation from sororin phosphorylates the cohesin subunit, which is cleaved by separase to allow the separation of homologous chromosomes and sister chromatids. In anaphase II, APC/CCDC20 activity could remove PP2A-RTS1 from the driven particle, thus causing sister chromatid separation without tension.

Shugoshin is also essential for stabilizing kinetochore-microtubule connections. It can dissolve the incorrect connections by recruiting CPC to multiple substrates at the kinetochore-microtubule interface of centromeres phosphorylation. In addition, it recruits MCAK to centromeres to destroy inappropriate kinetochore-microtubule connections in the prophase, metaphase, and early anaphase of chromosome cohesin. Shugoshin also recruits PP2A to centromeres to promote Aurora B kinase activity and inhibit autophosphorylation of Aurora B/C kinase to resist Aurora B and prevent an excessive increase in Aurora B activity. It recruits Ipl1 kinase to centromeres, breaking the kinetochore-microtubule connections to produce unattached kinetochores, activating SAC, which recognizes unattached kinetochores, and inhibits APC/C from delaying the late start until all sister chromatid pairs become biorientation. The recruitment of SUMO protein to centromeres promotes the metaphase-anaphase transition in SAC and CPC-dependent manner to stabilize kinetochore-microtubule biorientation. Moreover, recruiting MAD2 to centromeres silences SAC, inhibits APC/C activity, hinders the late start of these mechanisms, corrects kinetochore-microtubule misconnection, and promotes sister kinetochores biorientation.

Shugoshin can inhibit the CIN occurrence. SGO1 and HP1a combine with H3K9me3 to inhibit the ICS network, which is a crucial mechanism of CIN. Both overexpression and deletion of shugoshin were found to lead to CIN in many tumors. Thus, shugoshin is a crucial protein in inhibiting tumorigenesis and can be used as a molecular target for therapy and prognosis in various cancers.

In conclusion, shugoshin is essential to ensure correct chromosome segregation, thereby inhibiting the occurrence of aneuploidy. The proportion of elderly pregnant women in obstetric examination clinics has increased yearly. This review discusses the mechanism of shugoshin inhibiting aneuploidy, which provides a theoretical basis for clinical prevention or reduction of aneuploidy diseases such as infertility, abortion, and fetal birth defects in older women in the future. Furthermore, shugoshin can be used as a molecular target for therapy and prognosis in various cancers, providing a potential treatment for cancer patients.

Conflict of Interest Statement

The authors declare that they have no conflict of interest.

Funding Sources

This article is funded by the National Natural Science Foundation of China (No. 31860329), Project of Guizhou Provincial Department of Science and Technology (qkhj [2019] No. 1344), “15851 talent elite project” of Zunyi City (NSFC 31860329).

Author Contributions

Qiqi Sun wrote the manuscript, while Feng Liu, Xiaolong Mo, Bo Yao, Guanghai Liu, Shanshan Chen, and Yanping Ren edited this paper.

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