Molecular Basis for CPC-Sgo1 Interaction: Implications for Centromere Localisation and Function of the CPC

The Chromosomal Passenger Complex (CPC; consisting of Borealin, Survivin, INCENP and Aurora B kinase) and Shugoshin 1 (Sgo1) are key regulators of chromosome bi-orientation, a process essential for error-free chromosome segregation. Their functions rely on their ability to associate with centromeres. Two histone phosphorylations, histone H3 Thr3 (H3T3ph; directly recognised by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognised via Sgo1), together with CPC’s intrinsic ability to bind nucleosome, facilitate CPC centromere recruitment. The molecular basis for CPC-Sgo1 binding and how their direct interaction influences CPC centromere localisation and function are lacking. Here, using an integrative structure-function approach, we show that the histone H3-like Sgo1 N-terminal tail interacts with Survivin acting as a hot-spot for CPC-Sgo1 assembly, while downstream Sgo1 residues, mainly with Borealin contributes for high affinity interaction. Disruption of the Sgo1 N-terminal tail-Survivin interaction abolished CPC-Sgo1 assembly in vitro and perturbed centromere localisation and function of CPC. Our findings provide evidence that CPC binding to Sgo1 and histone H3 N-terminal tail are mutually exclusive, suggesting that these interactions will likely take place in a spatially/temporally restricted manner and provide a rationale for the Sgo1-mediated ‘kinetochore proximal centromere’ pool of CPC.


Introduction
Equal and identical segregation of chromosomes to the daughter cells during mitosis requires physical attachment of duplicated sister chromatids (via their kinetochores) to microtubules emanating from opposite spindle poles and subsequent alignment of chromosomes at the metaphase plate, a state known as bi-orientation (Musacchio and Desai, 2017). Chromosome bi-orientation is achieved and monitored by several processes including sister chromatid cohesion and quality control mechanisms known as error-correction and spindle assembly checkpoint (SAC), all controlled by the spatiotemporal regulation of kinases and phosphatases (Funabiki and Wynne, 2013;Gelens et al., 2018;Saurin, 2018).
Cohesin, a ring-shaped protein complex is a major player that mediates sister chromatid cohesion in S-phase (Haering et al., 2008;Haering et al., 2002). During prophase, the bulk of cohesin is removed from the chromosome arms (Gandhi et al., 2006;Kueng et al., 2006), while centromeric cohesin is maintained until anaphase onset protected by Shugoshin 1 (Sgo1) (Kitajima et al., 2006;Salic et al., 2004). Cdk1 phosphorylation of Sgo1 during mitosis enables the binding of the Sgo1-protein phosphatase 2 (PP2A) complex to cohesin and ensures that the two sister chromatids remain connected until anaphase onset, when separase cleaves the remaining centromeric cohesion allowing the sister chromatids to separate (Kitajima et al., 2006;Liu et al., 2013b;Shintomi and Hirano, 2009;Waizenegger et al., 2000). Sgo1 localisation to centromeres is crucial for its role as cohesion protector. Sgo1 has been suggested to first localise to kinetochores via the Bub1 dependent histone H2A phosphorylation at T120 (H2AT120ph) in order to then efficiently load onto centromeres to protect cohesion and prevent premature sister chromatid separation (Broad et al., 2020;Hengeveld et al., 2017;Kawashima et al., 2010;Liu et al., 2013a).
Error correction is a mechanism that destabilises incorrect kinetochore-microtubule (KT-MT) attachments, such as syntelic (two sister kinetochores attached to microtubules from the same spindle pole) or merotelic (single kinetochore attached to microtubules emanating from both spindle poles) attachments, and stabilises correct bi-polar attachments. The chromosomal passenger complex (CPC), consisting of Aurora B kinase, INCENP, Borealin and Survivin is one of the key players regulating this process (Carmena et al., 2012). The CPC, via its Aurora B enzymatic core, destabilises aberrant KT-MT attachments by phosphorylating outer kinetochore substrates such as the Knl1/Mis12 complex/Ndc80 complex (KMN) network so that new attachments can be formed (Cheeseman et al., 2006;Cimini et al., 2006;DeLuca et al., 2006;Lampson et al., 2004;Welburn et al., 2010). Sgo1 has also been shown to regulate KT-MT attachments via PP2A-B56 recruitment that balances Aurora B activity at the centromeres (Meppelink et al., 2015). In addition to error correction, the CPC is also involved in the regulation of the SAC, a surveillance mechanism that prevents anaphase onset until all kinetochores are attached to microtubules (Foley and Kapoor, 2013;Musacchio, 2015).
CPC function relies on its ability to localise correctly during mitosis. The CPC predominantly localises in the centromeric region between the sister-kinetochores during early mitosis and three independent studies recently suggested that the evolutionary conserved Haspin and Bub1 kinases can recruit independent pools of the CPC along the inter-kinetochore axis. Both recruitment pathways appear redundant for KT-MT error correction and can support faithful chromosome segregation (Broad et al., 2020;Hadders et al., 2020;Liang et al., 2019). Haspin mediates phosphorylation on histone H3 Thr3 (H3T3ph), which is recognised by the BIR domain of Survivin (Jeyaprakash et al., 2011;Kelly et al., 2010;Wang et al., 2010). Bub1 phosphorylates Thr120 of Histone H2A (H2AT120ph) (Bonner et al., 2020;Tsukahara et al., 2010;Wang et al., 2010;Yamagishi et al., 2010) that is recognised by Sgo1 which in turn, is suggested to interact with Borealin via its coiled-coil domain (Bonner et al., 2020;Yamagishi et al., 2010). However, our earlier work showed that Sgo1 N-terminal tail (AKER peptide) can also interact with Survivin BIR domain using a binding mode that is nearly identical to that of the histone H3 tail phosphorylated at Thr3 (Jeyaprakash et al., 2011), suggesting that a direct interaction between Survivin and Sgo1 could also be possible. H3T3ph and H2AT120ph appear to localise to distinct regions within the mitotic centromeres, with H3T3ph localising to the inner centromere and H2AT120ph to the KT-proximal outer centromere (Broad et al., 2020;Liu et al., 2013a;Yamagishi et al., 2010). While CPC-Sgo1 functional interdependency is well established, structural and molecular basis for how CPC and Sgo1 interact and the contribution of this physical interaction to the centromere localisation and function of the CPC remain unclear. Here, we address these questions by combining biochemical, structural, biophysical and cellular approaches.

CPC-Sgo1 forms a robust complex in vitro
The CPC-Sgo1 interaction has been reported to be critical for sister chromatid bi-orientation and subsequent accurate chromosome segregation from yeast to humans (Hengeveld et al., 2017;Hindriksen et al., 2017;Peplowska et al., 2014;Tsukahara et al., 2010). However, the molecular basis of this interaction has not been characterised. To assess whether the CPC can directly interact with Sgo1 in vitro, we purified recombinant CPC containing INCENP1-58, full length Survivin and a stable version of Borealin lacking the first 9 residues, Borealin10-280 (CPCISB10-280, Fig. 1A) and tested its interaction with recombinant Sgo11-415 (just lacking the HP1 binding domain and the Sgo motif) using size exclusion chromatography (SEC) (Fig. 1A and S1A). Our data showed that Sgo11-415 and CPCISB10-280 can form a stable monodisperse complex in vitro as analysed by SEC (Fig. 1B). Using Isothermal Titration Calorimetry experiments (ITC) we assessed the binding affinity of this interaction. CPCISB10-280 and Sgo11-415 exhibited high affinity with a Kd in the low nM range (Kd= 53 ± 7 nM; Fig. 1C and S2C).

Sgo1 makes multipartite interactions with CPC subunits
Previous studies have suggested that the Sgo1-CPC interaction is mediated via the N-terminal coiled-coil of Sgo1 and Borealin (Bonner et al., 2020;Tsukahara et al., 2010). However, our structural data revealed that the very N-terminus of Sgo1 can interact with the BIR domain of Survivin (Jeyaprakash et al., 2011). Together, these studies suggest that multipartite interactions between Sgo1 and different CPC subunits could facilitate CPC-Sgo1 complex formation. To gain further structural insights, we performed chemical cross-linking of the CPCISB10-280-Sgo11-415 complex using a zero-length cross-linker, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), followed by mass spectrometry analysis (Fig.   S1E). Cross-linking-mass spectrometry (CLMS) data showed that: (1) consistent with our previous observations (Jeyaprakash et al., 2011), the N-terminal region of Sgo1 (amino acids 1-34) makes extensive contacts with Survivin BIR domain (amino acids 18-89); (2) the Nterminal coiled-coil of Sgo1 (amino acids 10-120) interacts with the CPC triple helical bundle; (3) consistent with previous findings (Bonner et al., 2020), the N-terminal coiled-coil also contacts the Borealin dimerisation domain; and (4) the Sgo1 region beyond the N-terminal coilcoil region, which is predicted to be unstructured, contacts both Survivin and Borealin with most contacts confined to the Sgo1 central region spanning amino acids (aa) 180-300 ( Fig. 2A and 2B). Thus, our cross-linking results suggests that Sgo1 interacts with CPC, mainly via two regions, the N-terminal coiled-coil domain and the unstructured central region ( Fig. 2A and   2B).
We further analysed the contribution of different Sgo1 regions for CPC binding using a LacO-LacI tethering assay. For this we made use of U-2 OS cells harbouring a LacO array on the short arm of chromosome 1 to which we could recruit Sgo1 fragments as LacI-GFP fusions (U-2 OS-LacO cells; Janicki et al., 2004). To exclude any contribution from H3T3ph on CPC recruitment we made use of a Haspin CRISPR mutant (CM) cell line that displays no discernible Haspin activity (Hadders et al., 2020). Constructs containing Sgo11-130 and full length Sgo11-527 recruited endogenous Aurora B (Fig. 2C) and Borealin (Fig. 2D) to the LacO foci, at comparable levels. This is in line with previous data that suggested the Sgo1 N-terminal region as a major CPC binding site (Bonner et al., 2020;Jeyaprakash et al., 2011;Tsukahara et al., 2010). Surprisingly, Sgo1130-280 fused to LacI-GFP was also able to recruit endogenous Aurora B (Fig. 2C) and Borealin (Fig. 2D) to the LacO foci, although at lower levels compared to Sgo11-130 and Sgo11-527. On the contrary, the Sgo1 fragments Sgo1274-415 and Sgo1415-527 (Sgo1274-415-LacI-GFP and Sgo1415-527-LacI-GFP) failed to recruit either Aurora B or Borealin ( Fig. 2C and 2D). Taken together, our data confirm that the main CPC-interacting regions of Sgo1 lie within the N-terminal coiled-coil region of Sgo1 (Sgo11-130) and the adjacent unstructured region (Sgo1130-280).

The Survivin interaction with the Sgo1 N-terminal tail is essential for CPC-Sgo1 assembly
Our previous study identified a histone H3-like N-terminal tail in Sgo1 (Ala1-Lys2-Glu3-Arg4), which interacted with the Survivin BIR domain with similar affinity as did the histone H3 tail (Jeyaprakash et al., 2011). X-ray crystallographic structural analysis revealed that the mode of Sgo1 tail binding is near identical to that of the histone H3 tail with phosphorylated Threonine 3 (Ala1-Arg2-Thr3ph-Lys4; Jeyaprakash et al., 2011;Fig. S1F). However, whether the Sgo1 N-terminal tail interaction with Survivin is possible in the context of a longer Sgo1 fragment remained an open question. Here, using SEC, we confirmed that Sgo1 spanning aa residues 1-155 (Sgo11-155; a shorter and stabler fragment identified from limited proteolysis of Sgo11-415), can form a stable complex with Survivin ( Fig. 3A), indicating that in the context of Sgo11-155, the Sgo1 N-terminal tail is accessible for binding to Survivin.
Binding of histone H3 tail by Survivin requires anchoring of the small hydrophobic side chain of H3-Ala1 to the hydrophobic pocket of the Survivin BIR domain (Du et al., 2012;Jeyaprakash et al., 2011;Niedzialkowska et al., 2012). This interaction (anchoring of first Ala in the BIR pocket) is conserved in the Survivin-Sgo1 N-terminal peptide interaction (Jeyaprakash et al., 2011). To investigate the contribution of the Sgo1 N-terminal tail for binding to Survivin, we targeted this interaction by mutating the first alanine after the initiator methionine to a methionine (a residue with a long side chain not compatible with the BIR domain hydrophobic pocket; Sgo1Nmut). Remarkably, the Sgo11-155 Nmut mutant was unable to interact with Survivin, indicating that the Sgo1 N-terminal tail interaction with BIR domain is crucial for Survivin binding ( Fig. 3B and S1F). Similarly, a Survivin BIR mutant (Survivin 3A: K62/E65/H80A) not capable of interacting with the histone H3 tail (Fig. S1F), failed to interact with Sgo11-155 (Fig. 3C). These data together show that both the Sgo1 and histone H3 Nterminal tail use the same binding pocket in the Survivin BIR domain. Consistent with our data (Fig. 1B), the shorter Sgo11-155 fragment can also form a complex with CPCISB10-280 in vitro (Fig. 3D). To evaluate the contribution of the Sgo1-Survivin interaction in the context of the CPC where Borealin can still interact with Sgo1 (Bonner et al., 2020;Tsukahara et al., 2010 and Fig. 2A and 2B), we mixed CPCISB10-280 with Sgo11-155 Nmut (Fig.   3E) and Sgo11-415 Nmut (Fig. 3F) and tested their interaction by SEC. Strikingly, neither Sgo11-155 Nmut, nor the longer Sgo11-415 Nmut that includes the second CPC interacting region (aa 130-280) were able to interact with the CPC. This data agrees with the tethering assays where Sgo11-130 Nmut-LacI-GFP showed a drastic reduction in its ability to recruit Aurora B (Fig. S2A) and Borealin (Fig. S2B) to the LacO array as compared to Sgo11-130-LacI-GFP. Together, our results reveal that the Survivin-Sgo1 interaction is essential for the CPC binding to Sgo1 and that the Sgo1 N-terminal tail acts as a hot-spot, whose perturbation abolishes the ability of CPC to form a complex with Sgo1.

Borealin and INCENP are required for a high affinity CPC-Sgo1 interaction
To assess how different CPC subunits contribute to achieve high affinity Sgo1 interaction, we performed a series of Isothermal Calorimetry (ITC) experiments with either Survivin on its own or with CPCISB containing different Borealin truncations. Sgo11-130 interacted with Survivin with mid-nanomolar affinity (Kd of 255 ± 33 nM; Fig. 4A and S2C). This, together with our previous observation that a Sgo1 N-terminal tail peptide bound Survivin with ~ 1 m affinity (Jeyaprakash et al., 2011) suggests that, although the interaction between the alanine and the Survivin BIR domain is essential for Sgo1/Survivin complex formation, the Sgo1-Survivin interaction extends beyond Sgo1 N-terminal tail. Sgo11-130 bound CPCISB10-280 with a Kd of 57 ± 8 nM, a ~ 5 fold higher affinity as compared to the affinity for Survivin alone (Fig. 4B and S2C). This observation together with the CLMS analysis suggests that further interactions involving Borealin, and possibly INCENP, strengthen the affinity between the CPC and Sgo1. Consistent with our CLMS analysis ( Fig. 2A and 2B) and a previous study (Bonner et al., 2020), CPCISB lacking the Borealin dimerisation domain (CPCISB10-221) bound Sgo11-130 with a three-fold lower affinity as compared to the CPCISB10-280 (Kd =163 nM ± 16 nM vs 57 ± 8 nM), highlighting the contribution of the Borealin dimerisation domain for binding to Sgo1 ( Fig. 4C and S2C). The measured affinity of CPC ISB10-280 binding to the near full length Sgo1 (Sgo11-415, Kd = 53 nM ± 7 nM, Fig. 1C), is almost identical to that for Sgo11-130 ( Fig. 4B; Kd = 57 nM ± 8 nM). This confirms that the first 130 amino acids of Sgo1 represent the main CPC-interacting region in vitro. Furthermore, the observation that the affinity goes from a micromolar range for the AKER peptide with Survivin (Jeyaprakash et al., 2011) to the low nanomolar range for the CPCISB10-280-Sgo11-130 complex, indicates that although the interaction between the CPC and Sgo1 depends on the Sgo1 N-terminal tail binding to Survivin, the high-affinity interaction requires Sgo1 binding to Borealin and possibly INCENP. Overall, the ITC data indicates that the interaction between the Sgo1 Nterminal tail and Survivin is electrostatically driven (Fig. S2D), while the high-affinity interaction between the rest of the Sgo1 regions and the CPC is driven by entropic contribution that could be due to a release of water molecules associated with the surface and/or a conformational rearrangement upon binding (Fig. S2C). All these data together suggest that a weak micromolar affinity long-range electrostatic interaction between Survivin and the Sgo1 N-terminal tail is required to establish a high-affinity CPC-Sgo1 interaction mediated by multiple inter-protein contacts and hydrophobic effects.

The Survivin interaction with the Sgo1 N-terminal tail is essential for the centromeric localisation of the CPC and proper chromosome segregation
We next evaluated how the different Sgo1 regions we identified as important for the CPC-Sgo1 interaction contribute to maintaining the centromeric levels of the CPC in cells. Endogenous Sgo1 was depleted by siRNA in HeLa Kyoto cells expressing either wild type Sgo1 (Sgo1-GFP) or mutant Sgo1 (Sgo1Nmut-GFP, Sgo14A-GFP or Sgo1Nmut/4A double mutant) and centromeric levels of Borealin were analysed by quantitative immunofluorescence microscopy ( Fig. 5A, S3C and S3D). Consistent with previous observations (Broad et al., 2020;Kawashima et al., 2007;Meppelink et al., 2015;Tsukahara et al., 2010;van der Waal et al., 2012;Wang et al., 2010), depletion of Sgo1 led to a two-fold reduction in the centromeric levels of Borealin.
As expected, expression of wild type Sgo1 (Sgo1-GFP) rescued the centromeric abundance of Borealin (Fig. 5A). In line with our in vitro binding studies and cellular tethering data, expression of Sgo1 mutants (Sgo1Nmut-GFP, Sgo14A-GFP or Sgo1Nmut/4A-GFP, Fig. S3D) aimed to perturb either the Sgo1 N-terminal tail-Survivin interaction or the Sgo1 188-191-Borealin interaction did not rescue the centromeric levels of Borealin, demonstrating direct contributions of these regions for efficient CPC recruitment to centromeres (Fig. 5A).
We further analysed the effects of disrupting the CPC-Sgo1 interaction on chromosome biorientation. Sgo1-depleted HeLa cells expressing either Sgo1-GFP or mutant Sgo1 (Sgo1Nmut-GFP, Sgo14A-GFP or Sgo1Nmut/4-GFP) were released from a monastrol-induced mitotic arrest into medium with MG132 and chromosome alignment was assessed. Expression of Sgo1 mutants led to around 70 % of cells with severe chromosome misalignment, comparable to the phenotype observed for Sgo1 depletion (Fig. 5B). Unlike Sgo1 knock-down cells, Sgo1-mutant expressing cells did not seem to experience loss of sister chromatid cohesion because sister centromeres remained closely together (Fig. 5B). This suggests that the alignment errors observed are not due to a loss of centromeric cohesion, but a reflection of perturbed kinetochore-microtubule error correction, presumably as a consequence of the reduced centromeric levels of CPC (Fig. 5A).
Considering the ability of Sgo1 to bind H2AT120ph and to recruit CPC to the kinetochore proximal centromere, we analysed the precise localisation of Borealin using chromosomes spreads of nocodazole-arrested HeLa cells expressing the Sgo1 mutants. Sgo1 depletion on Sgo1-GFP expressing HeLa cells showed Borealin mainly localised at the inner centromere but also displayed a small pool localised at the kinetochore proximal centromere (Fig. 5C), consistent with the previously described pattern of CPC localisation in unperturbed mitotic cells (Bekier et al., 2015;Hadders et al., 2020;Liang et al., 2019). On the contrary, depletion of Sgo1 on Sgo1Nmut-GFP or Sgo14A-GFP expressing cells, Borealin was enriched as a single focus between the two sister ACA dots, similar to the inner centromere localisation previously observed for Borealin dimerisation mutants that bind less well to Sgo1 (Bekier et al., 2015).
Quantification of the full width half max values for the Borealin intensity profiles obtained from the line plots of the chromosome spreads, confirmed that rescue of Sgo1 depletion with Sgo1-GFP expression generated a broader Borealin signal at the centromere (most likely the result of the combination of inner centromere and kinetochore proximal outer centromere pools), while expression of Sgo1 mutants (Sgo1Nmut-GFP and Sgo14A-GFP) generated narrower Borealin profiles consistent with CPC localised at the inner centromere only. This data reveals that the interaction of CPC with H2AT120ph-bound Sgo1 is responsible for the kinetochore proximal centromere pool of the CPC.
A multi-pronged approach combining detailed biochemistry, CLMS analysis and available structural data with cellular phenotypic analysis provided us with an excellent opportunity to dissect the contributions of different Sgo1 regions and CPC subunits for CPC-Sgo1 complex formation. We show that both the Sgo1 N-terminal tail and the downstream hydrophobic region comprising aa 188-191 contribute to CPC-Sgo1 complex formation and are essential for efficient CPC centromere recruitment and function. Interestingly, the observation that the Sgo1 and histone H3 N-terminal tails exploit the same binding site in Survivin, suggests that these interactions are mutually exclusive and may explain why the Bub1-dependent CPC pool exists as a kinetochore-proximal centromere pool that is spatially distinct from the Haspin-dependent inner centromere CPC pool (Broad et al., 2020;Hadders et al., 2020;Liang et al., 2019).    Data are a representative of two independent experiments. Scale bar, 5 m.  Data are a representative of four independent experiments. Scale bar, 5 m.

Protein expression and purification of CPC and Sgo1
CPCISB10-280 and CPCISB10-221 were purified as previously described ( and 1 mM PMSF. The lysate was sonicated for 8 minutes, centrifuged at 58,000 x g for 50 minutes at 4°C and the complex was purified by affinity chromatography using His Trap Column (Cytiva). The protein-bound column was washed with lysis buffer followed by a high salt buffer wash (25 mM HEPES pH 7.5, 1 M NaCl, 25 mM Imidazole, 2 mM mercaptoethanol, 1mM ATP). The complex was eluted using high imidazole buffer (25 mM HEPES pH 7.5, 500 mM NaCl, 400 mM Imidazole, 2 mM -mercaptoethanol) and the affinity tags were cleaved using TEV and 3C proteases while dialysing the sample in a buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl and 4 mM DTT at 4° C overnight. The Sgo1 fragments  were cloned in the pTYB11 vector (IMPACT system, New England Biolabs) which contains an Intein tag with an embedded chitin binding domain. The Intein tag is a DTT-induced self-cleavable tag that allows purification of proteins with a native N-terminus, as it leaves no extra amino acids after cleavage. Cloning of the Sgo1 in the pTYB11 vector with an N-terminal Intein-tag, allowed the purification of a Sgo1 with a native N-terminus leaving the initiator methionine exposed to be excised by Methionine Aminopeptidases (Giglione et al., 2004). Sgo1 fragments were expressed in the BL21 (DE3) Gold E. coli strain. Cells were grown at 37°C to an OD of 1.5 and induced overnight at 18° C with 0.35 mM IPTG. Cells were resuspended in lysis buffer containing 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1mM EDTA and supplemented with complete EDTAfree cocktail tablets (1 tablet/50ml cells; Roche), 0.01 mg/ml DNase (Sigma) and 1 mM PMSF.
The lysate was sonicated for 8 minutes, centrifuged at 58,000 x g for 50 minutes at 4°C and the protein was batch purified using chitin beads (NEB). Protein-bound chitin beads were washed with lysis buffer and high salt buffer (20 mM Tris-HCl pH 7.5, 1 M NaCl, 1 mM EDTA and 1 mM ATP) and eluted with 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 50 mM DTT overnight at room temperature. The eluted protein was then dialysed into 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 50 mM Glutamate, 50 mM Arginine and 2mM DTT overnight at 4°C and loaded onto a HiTrap SP-HP (Cytiva) ion exchange column. The samples containing Sgo1 were pooled, concentrated and run in a Superdex 200 Increase 10/300 column (Cytiva) preequilibrated with 25 mM HEPES pH 8, 250mM NaCl, 5 % Glycerol and 2 M DTT.

Interaction studies using size exclusion chromatography
All SEC experiments for the purified recombinant proteins were performed on an AKTA Pure 25 High Performance Liquid Chromatography (HPLC) unit (Cytiva) with sample collector. For all interaction studies a Superdex 200 10/300GL 24ml column (Cytiva) was used. Before sample injection, the column was pre-equilibrated in 25 mM HEPES pH 7.5, 150 mM NaCl, 4 mM DTT, 5% glycerol (v/v) for interaction experiments involving Sgo11-155 or pre-equilibrated in 25 mM HEPES pH 8, 250 mM NaCl, 2 mM DTT, 5% glycerol (v/v) for interaction experiments involving Sgo11-415. 0.5 ml fractions were collected with a 0.2 CV delayed fractionation setting. UV 280 nm and 260 nm wavelengths were monitored.

Chemical cross-linking and MS analysis
Cross-linking experiments of Sgo11-415 and CPCISB10-280 were performed using 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC, Thermo Fisher Scientific) in the presence of Nhydroxysulfosuccinimide (NHS, Thermo Fisher Scientific). 25 g of gel filtrated protein complex was cross-linked with 120 g EDC and 44 g of NHS in 25 mM HEPES pH 6.8, 150 mM NaCl for 1h 30min at room temperature. The cross-linking was stopped by the addition of 100 mM Tris-HCl and cross-linking products were briefly resolved using a 4-12 % Bis-Tris NuPAGE (Thermo Fisher Scientific). Bands were visualised by short Instant Blue staining (Abcam), excised, reduced with 10 mM DTT for 30 min at room temperature, alkylated with 5 mM iodoacetamide for 20 min at room temperature and digested overnight at 37 °C using 13 ng/l trypsin (Promega). Digested peptides were loaded onto C18-Stage-tips (Rappsilber et al.,

2007). LC-MS/MS analysis was performed using an Orbitrap Fusion™ Lumos™ Tribrid™
Mass Spectrometer (Thermo Scientific) applying a "high-high" acquisition strategy. Peptide mixtures were injected for each mass spectrometric acquisition. Peptides were separated on a 75 µm x 50 cm PepMap EASY-Spray column (Thermo Scientific) fitted into an EASY-Spray source (Thermo Scientific), operated at 50 ºC column temperature. Mobile phase A consisted of water and 0.1% v/v formic acid. Mobile phase B consisted of 80% v/v acetonitrile and 0.1% v/v formic acid. Peptides were loaded at a flow-rate of 0.3 μl/min and eluted at 0.2 μl/min using a linear gradient going from 2% mobile phase B to 40% mobile phase B over 139 (or 109) minutes, followed by a linear increase from 40% to 95% mobile phase B in 11 minutes. The eluted peptides were directly introduced into the mass spectrometer. MS data were acquired in the data-dependent mode with the top-speed option. For each three-second acquisition cycle, the mass spectrum was recorded in the Orbitrap with a resolution of 120,000. The ions with a precursor charge state between 3+ and 8+ were isolated and fragmented using HCD or EThcD.
The fragmentation spectra were recorded in the Orbitrap. Dynamic exclusion was enabled with single repeat count and 60-second exclusion duration.
Search parameters were MS accuracy, 3 ppm; MS/MS accuracy, 10ppm; enzyme, trypsin; cross-linker, EDC; max missed cleavages, 4; missing mono-isotopic peaks, 2; fixed modification, carbamidomethylation on cysteine; variable modifications, oxidation on methionine; fragments b and y type ions (HCD) or b, c, y, and z type ions (EThcD) with loss of H2O, NH3 and CH3SOH. 1% on link level False discovery rate (FDR) was estimated based on the number of decoy identification using XiFDR (Fischer and Rappsilber, 2017)

Mass Photometry
High precision microscope coverslips (No. 1.5, 24 x 50 mm) were cleaned with Milli-Q water, 100 % isopropanol, Milli-Q water and dried. Silicone gaskets (Grace BioLabs 103250) were placed on the coverslips. Samples were cross-linked with 0.01% glutaraldehyde for 5 min at 4 °C and quenched by addition of 50 mM Tris-HCl pH 7.5 for 1h at 4 °C. Immediately prior to mass photometry measurements, samples were diluted to 100 nM in buffer containing 25mM HEPES pH 8, 250 mM NaCl and 2 mM DTT. For each acquisition, 20 nM of diluted protein was measured following manufacturer's instructions. All data was acquired using a One MP mass photometer instrument (Refeyn Ltd, Oxford, UK) and AcquireMP software (Refeyn Ltd.,v2.4.1). Movies were recorded in the regular field of view using default settings. Data was analysed using Discover MP software (Refeyn Ltd.,v2.4.2).

Tethering Assays
The LacO tethering assays were performed essentially as described before (Hadders et al., 2020). U-2 OS LacO Haspin CM cells (Hadders et al., 2020) were seeded on glass coverslips and directly transduced with recombinant baculovirus expressing LacI-GFP fusion proteins.
After ~ 4-6 hours STLC (20 M) was added and left to incubate overnight. The next morning cells were fixed in 4 % PFA for 15 minutes and permeabilized with ice cold methanol. Prior to staining cells were blocked in PBS supplemented with 0.01 % Tween20 (PBST) and 3 % BSA for 30 minutes followed by staining with primary antibodies in PBST + 3 % BSA for 2-4 hours.
Coverslips were then washed three times with PBST followed by staining with secondary antibodies and DAPI (1 g/ml) for 1 hour. After another three washes with PBST coverslips were mounted using Prolong Diamond. Cells were imaged on a DeltaVision system. The  Shown images are deconvolved and maximum-intensity projections.

Western blot
To study the Sgo1 levels after siRNA oligo treatment and to test the expression levels of each of the Sgo1-GFP constructs, HeLa Kyoto cells were transfected in 12-well dishes as described above and lysed in 1× Laemmli buffer, boiled for 5 min, and analysed by SDS-PAGE followed