spMis18_1–120 forms a homodimer

The asymmetric unit of the spMis18_1–120 crystals contained three copies of spMis18_1–120 assembled in a linear arrangement via two different interfaces (interface I and interface II), resulting in two potential dimeric structures (dimer I and dimer II) (Fig ​(Fig2A).2A). While the interface stabilizing dimer I is formed by the stacking of loops L5 and L7 of one monomer over their dimeric counterpart, the dimer II interface is formed by the stacking of β‐sheet I (Fig ​(Fig2A).2A). The oligomeric assembly observed in the crystals prompted us to characterize the oligomeric structure of spMis18_1–120 in solution. The molecular weight of spMis18_1–120, as measured using SEC‐MALS (size exclusion combined with multi‐angle light scattering), was 33,338 Da. Given that the calculated molecular weight of an spMis18_1–120 monomer is 16,160 Da whereas a dimer would be 32,320 Da, this analysis independently confirms that spMis18_1–120 forms a dimer in solution (Figs ​(Figs2B2B and EV2A).

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Figure 2

Yippee‐like globular domains of Mis18 proteins possess an intrinsic preference to form homo/heterodimers

1. Cartoon representation of the assembly of spMis18 Yippee‐like domains (spMis18_1–120) as observed in the crystal asymmetric unit. Close‐up views highlight the amino acid composition and interactions stabilizing the dimeric arrangements. Interface I is composed of conserved residues and an extensive binding surface as compared to interface I (see text for details), suggesting dimer II to be a physiologically relevant dimer. Residues mutated in this study are highlighted with circles.

2. Size‐exclusion chromatography (SEC) and SEC‐MALS (SEC combined with multi‐angle light scattering) analyses of the recombinant wild‐type (wt) and dimer‐disrupting mutant (I31A) of spMis18_1–120. Measured molecular weights from SEC‐MALS confirm that spMis18_1–120 wt is a dimer in solution and spMis18_1–120I31A is a monomer.

3. Evaluation of the ability of Yippee‐like domains of human Mis18 proteins (hsMis18α_77–187 and hsMis18β_56–183) to form a heterodimer. SEC and SEC‐MALS analyses demonstrate that while hsMis18α_77–187 elutes at 15.5 ml as a dimer (31.3 kDa), hsMis18β_56–183 elutes at 17.3 ml as a monomer (15.0 kDa). Purified hsMis18α_77–187 and hsMis18β_56–183 when mixed together elute at 15.4 ml as a heterodimer (27.5 kDa).

4. Cartoon representation of the structure of the homology‐modeled human Mis18α_77–187–Mis18β_56–183 heterodimer using the crystal structure of spMis18_1–120 reported here as a template. Residues mutated in this study are highlighted with circles. Modeling was carried out using Phyre2 web server (www.sbg.bio.ic.ac.uk/phyre2/).

5. SDS–PAGE analysis of the GST pull‐down assay where wt and dimer‐disrupting mutants of hsMis18α_77–187 and hsMis18β_56–183 were co‐expressed as His‐ and GST‐tagged proteins, respectively, in E. coli. While wt GST‐hsMis18β_56–183 showed interaction with wt His‐hsMis18α_77–187, hsMis18 proteins harboring dimer‐disrupting mutations, GST‐hsMis18β_56–183V77E/Y172D, and His‐hsMis18α_77–187V82E/Y176D did not show noticeable interaction. The corresponding Ni‐NTA pull‐downs showing the expression of His‐tagged proteins are shown in Fig EV2D.

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Figure EV2

Evaluation of the dimerization ability of Yippee‐like domains of Mis18 proteins

1. SEC‐MALS profiles of His‐spMis18_1–120 (left panel) and His‐spMis18_1–120I31A (right panel). Refractive index (RI, left y‐axis) and molar mass (MW, right y‐axis) profiles show that His‐spMis18_1–120 (predicted MW of a monomer: 16.2 kDa) is a dimer (measured MW: 33.3 kDa) and His‐spMis18_1–120I31A (predicted MW of monomer: 16.2 kDa) is a monomer (measured MW: 15.6 kDa).

2. Native PAGE analysis of dimer interface II mutants. Interface mutants I31A, V22E, Y114E, and Y114A migrated faster than wt spMis18_1–120, confirming the effect these mutations have on the oligomeric structure of spMis18_1–120.

3. SEC‐MALS profiles of His‐hsMis18α_77–187 (top panel), His‐hsMis18β_56–183 (middle panel), and His‐hsMis18α_77–187–His‐hsMis18β_56–183 (bottom panel). While His‐hsMis18α_77–187 (predicted MW of a monomer: 14.7 kDa) formed a homodimer (measured MW: 31.3 kDa), His‐hsMis18β_56–183 (predicted MW of a monomer: 13.6 kDa) remained as a monomer (measured MW: 15.0 kDa). His‐hsMis18α_77–187–His‐hsMis18β_56–183 (predicted MW of a monomer: 28.3 kDa) formed a heterodimer (measured MW: 27.5 kDa).

4. SDS–PAGE analysis of Ni‐NTA pull‐down experiment, where His‐hsMis18α_77–187 and GST‐hsMis18β_56–183 were co‐expressed in E. coli as wt or dimer‐disrupting mutant proteins. While the GST pull‐down assay shown in Fig ​Fig2E2E confirmed the inability of GST‐hsMis18β_56–183V77E/Y172D to interact with His‐hsMis18α_77–187V82E/Y176D, the corresponding Ni‐NTA pull‐down shown here confirms the abundant expression of His‐hsMis18α_77–187V82E/Y176D in the input. We note that His‐hsMis18α_77–187V82E/Y176D migrates as a doublet. Western blot analysis (data not shown) using anti‐His antibody confirmed the presence of His‐tag on both bands suggesting the unstable nature of His‐hsMis18α_77–187V82E/Y176D mutant (possibly due to its inability to homo‐ or heterodimerize).

To identify the physiologically relevant dimer, we compared the extent of amino acid conservation of the interface residues and the buried surface area within the dimer I and dimer II interfaces. Dimer I is mainly stabilized by poorly conserved amino acid residues (F64, R66, L71, V73, S92, and I95 with the exception of R101) and involves 1,077 Ų buried surface area. In contrast, dimer II is stabilized by highly conserved residues (V22, I31, Y114, and L116) and has an extensive binding interface (as compared to dimer I) with a 1,431 Ų buried surface area. Based on this observation, we reasoned that dimer II is likely the physiologically relevant dimer. To test the basis of dimer formation, we generated several single point mutations at the dimer II interface (I31A, Y114A, Y114E, V22E) and determined whether they perturb the ability of spMis18_1–120 to dimerize. In size‐exclusion chromatography (Fig ​(Fig2B),2B), recombinant spMis18_1–120I31A mutant protein eluted later (Ve = 12.4 ml) than wild‐type spMis18_1–120 (Ve = 11.8 ml), suggesting that this mutant protein forms a smaller entity than wild‐type. Further analysis of spMis18_1–120I31A using SEC‐MALS confirmed that it is monomeric with a measured molecular weight of 15,955 Da (calculated molecular weight of a monomer is 16,160 Da) (Figs ​(Figs2B2B and EV2A). Consistent with this, the other dimer II interface mutants Y114A, Y114E, and V22E also behaved as smaller entities compared to the wild‐type protein when analyzed by native PAGE (Fig EV2B), thus demonstrating that the dimerization of spMis18_1–120 is mediated via the dimer II interface (Fig ​(Fig22A).

Yippee‐like globular domains of Mis18 proteins possess an intrinsic ability to form dimers

To test whether Yippee‐like globular domains of other Mis18 orthologs also display an intrinsic ability to form dimers, we expressed and purified recombinant fragments of human Mis18α (residues 77–187; hsMis18α_77–187) and Mis18β (56–183; hsMis18β_56–183) containing their Yippee‐like globular domains. SEC and SEC‐MALS analyses were conducted to assess their ability to form oligomers. While hsMis18α_77–187 eluted as a dimer at 15.5 ml in SEC with a measured molecular weight of 31,254 Da in SEC‐MALS (calculated MW of a dimer is 29,324 Da), the corresponding values for hsMis18β_56‐183 were 17.3 ml and 14,993 Da (calculated MW of a monomer is 13,591 Da), respectively, indicating that hsMis18β_56–183 exists as a monomer in solution (Fig ​(Fig2C).2C). We next tested whether hsMis18α_77–187 and hsMis18β_56–183 could interact to form a heterodimer, by mixing equimolar quantities of recombinant hsMis18α_77–187 and hsMis18β_56–183. In SEC, hsMis18β_56–183 co‐elutes with hsMis18α_77–187 at 15.4 ml suggesting that they form a heterodimer (Fig ​(Fig2C).2C). The molecular weight of this entity as measured by SEC‐MALS was 27,476 Da, confirming that hsMis18α and hsMis18β can heterodimerize through their respective Yippee‐like domains (Figs ​(Figs2C2C and EV2C).

To test whether the mode of dimerization (dimer II mediated) is conserved from fission yeast to humans, we generated a homology model of the human hsMis18α_77–187–hsMis18β_56–183 heterodimer using the spMis18_1–120 crystal structure described above as a template (Phyre2 server: http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (Fig ​(Fig2D).2D). Our analysis of the modeled dimer interface (containing Mis18α residues Val 82, Arg 89, Pro 91, Asp 94, Tyr 176, and Leu 178, and Mis18β residues Val 77, His 84, Val 86, His 92, Tyr 172, Leu 174, Lys 175, and Thr 176) did not show any steric clashes and involved 1,387 Ų buried surface area, similar to the spMis18_1–120 dimer II interface. Moreover, mutations at the dimer interface (Mis18α V82E/Y176D and Mis18β V77E/Y172D) were sufficient to perturb the ability of hsMis18α_77–187 and hsMis18β_56–183 to form a heterodimer (Figs ​(Figs2E2E and EV2D). This confirms that the Yippee‐like domains within Mis18 proteins employ a conserved mode of dimerization.

Structural and biochemical analyses of Yippee‐like domains from other proteins (Cereblon, RIG‐I and MsrB) have so far yielded no direct evidence that they possess an innate tendency to oligomerize in vitro. We therefore refer to the Yippee‐like domain of Mis18 orthologs as Mis eighteen Dimerization in Yippee (MeDiY) domain hereafter.

The C‐terminal α‐helical domain induces tetramerization of S. pombe Mis18

In addition to the N‐terminal MeDiY domain, Mis18 orthologs possess a C‐terminal α‐helical domain spMis18_{C‐term‐α} (aa residues 121–end). To test whether spMis18_{C‐term‐α} can influence the overall oligomeric state of the protein, we purified recombinant full‐length spMis18 (spMis18_fl). Obtaining intact samples of spMis18_fl proved difficult either with or without His/His‐GFP tag, as it was sensitive to degradation from the C‐terminus (Fig EV3A). The stable partially degraded His‐GFP‐spMis18_fl when analyzed by SEC‐MALS appeared to form a tetramer (Fig ​(Fig3A).3A). Mass spectrometric peptide sequence coverage analysis of this sample revealed the loss of approximately 20 amino acids at the C‐terminus. Close examination of the amino acid sequence revealed the presence of a low‐complexity region at the extreme C‐terminus (amino acid residues 171–end, 15 out of 20 amino acid residues being Lys/Arg) that is unique to spMis18 (Fig EV3B). We therefore expressed and purified recombinant spMis18 with a C‐terminal truncation (spMis18ΔC; amino acids 1–168; Fig EV3C). SEC‐MALS analysis of spMis18ΔC revealed that it predominantly formed a tetramer (Fig ​(Fig3B)3B) and demonstrated a role for the α‐helical region downstream of the MeDiY domain (residues 121–168; Fig ​Fig1B)1B) in spMis18 tetramerization. Since this α‐helical region is structurally conserved (based on secondary structure prediction; Fig ​Fig1B)1B) between Mis18 proteins from evolutionarily distant eukaryotes, we propose that Mis18 oligomerization (either homo or hetero) mediated through the C‐terminus is also likely to be highly conserved.

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Figure EV3

Characterization of the overall oligomeric state of spMis18_fl

• A

  SEC profile and respective SDS–PAGE analysis of fractions (bottom panel) for His‐GFP‐spMis18_fl. Superose 6 10/300 column was used for His‐GFP‐spMis18_fl.

• B

  Multiple sequence alignment of spMis18 with its orthologs highlights the presence of a low‐complexity region (Lys/Arg‐rich region) at the extreme C‐terminus unique to spMis18.

• C, D

  SEC profiles (top panels) and respective SDS–PAGE analyses of their fractions (bottom panels) for His‐spMis18ΔC and His‐GFP‐spMis18_{C‐term‐α}, respectively. Superdex 200 increase 10/300 column was used for His‐spMis18ΔC and His‐GFP‐spMis18_{C‐term‐α}.

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Figure 3

spMis18_fl forms a tetramer in vitro

• A, B

  SEC‐MALS analysis of His‐GFP‐spMis18_fl and His‐spMis18ΔC, respectively. Refractive index (RI, left y‐axis) and molecular mass (kDa, right y‐axis) profiles show that both His‐GFP‐spMis18_fl (predicted MW of a monomer is 51.9 kDa) and His‐spMis18ΔC (predicted MW of a monomer 21.8 kDa) predominantly exist as tetramers (187.6 and 78.6 kDa, respectively) with a small population of dimer (128.5 and 45.8 kDa, respectively).

• C

  SEC profile of a sample containing an equimolar mixture of purified His‐GFP‐spMis18_{C‐term‐α} and His‐spMis18_MeDiY (upper panel) and SDS–PAGE analysis of SEC fractions (bottom panel). His‐GFP‐spMis18_{C‐term‐α} and His‐spMis18_MeDiY eluted separately at 12.3 and 15.3 ml, respectively, demonstrating the inability of these domains to interact with each other.

• D

  SEC‐MALS analysis of His‐GFP‐spMis18_{C‐term‐α} shows that it exists as a homotrimer with a measured molecular weight of 119.8 kDa (theoretically calculated MW of a monomer is 38.3 kDa).

MeDiY and the C‐terminal α‐helical domain are independent structural modules

To obtain further insights into the overall architecture of the spMis18 oligomer, we tested whether spMis18_MeDiY and spMis18_{C‐term‐α} could interact with each other, or whether they existed as structurally independent modules. His‐spMis18_MeDiY and His‐GFP‐spMis18_{C‐term‐α} were purified individually and analyzed for complex formation using size‐exclusion chromatography. His‐GFP‐spMis18_{C‐term‐α} and His‐spMis18_MeDiY eluted separately at distinct elution volumes (12.3 and 15.3 ml, respectively). This demonstrates that the spMis18_MeDiY domain alone is unable to associate with spMis18_{C‐term‐α} (Figs ​(Figs3C3C and EV3D).

We next tested whether spMis18_{C‐term‐α} can self‐associate to form oligomers. SEC‐MALS analysis of His‐GFP‐spMis18_{C‐term‐α} (untagged spMis18_{C‐term‐α} (8,775 Da) is too small for accurate mass detection) revealed that it is a homotrimer with a measured molecular weight of 119,886 Da (theoretical molecular weight of a monomer is 38,337 Da while that of a homotrimer would be 115,011 Da) (Fig ​(Fig3D).3D). We therefore conclude that spMis18 has two structurally independent domains, and they each possess the ability to homo‐oligomerize.

Oligomerization of spMis18 via the MeDiY domain is required for its function

We next tested whether spMis18 dimerization is functionally important in vivo. Co‐immunoprecipitation assays were performed on S. pombe cells expressing endogenous spMis18_fl with a C‐terminal 8xMyc tag, and spMis18_fl or spMis18_flI31A expressed ectopically as a C‐terminally GFP‐tagged fusion protein. Endogenous wild‐type spMis18_fl‐Myc associated efficiently with ectopic wild‐type spMis18_fl‐GFP, confirming the interaction of spMis18_fl with itself in vivo (Fig ​(Fig4A).4A). Such self‐association of wild‐type spMis18 has also been demonstrated previously through yeast two‐hybrid assays 13, 15. In contrast, the spMis18_flI31A dimerization mutant showed significantly reduced association with endogenous spMis18_fl‐Myc. Additionally, spMis18_MeDiY alone was sufficient to associate with spMis18_fl‐Myc (Fig ​(Fig4A).4A). This highlights the role of spMis18_MeDiY in the overall oligomeric state of spMis18 in vivo.

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Figure 4

Dimerization mediated by the MeDiY domain promotes spMis18 function and centromere association

• A

  spMis18_fl‐Myc co‐immunoprecipitates with both GFP‐tagged spMis18_fl and spMis18_MeDiY, but shows reduced association when MeDiY‐mediated dimerization is disrupted in spMis18_flI31A. The asterisk (*) in the top panel denotes the IgG heavy chain.

• B, C

  Dimer II interface mutations I31A and Y114A affect the ability of ectopically expressed spMis18_fl to rescue the temperature sensitivity of mis18‐818 and mis18‐262 cells, while expression of spMis18_MeDiY alone confers a dominant‐negative effect on growth in a MeDiY dimerization‐dependent manner. Fivefold serial dilutions of cells expressing the indicated spMis18 constructs integrated at the leu1 locus in the genome, spotted on complete PMG + phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.

• D

  Mutations that disrupt MeDiY dimerization lead to reduced levels of spMis18_fl association with centromeres. qChIP analyses of spMis18_fl‐GFP association with centromere 2 (cc2) in the indicated strains when grown in complete PMG media supplemented with (repressed) or without (expressed) thiamine. Error bars represent standard deviation between at least three biological replicates.

To test whether spMis18_MeDiY‐mediated oligomerization is essential for spMis18 function, we performed genetic complementation assays in which we evaluated the ability of dimerization mutants I31A and Y114A (in the context of both spMis18_fl and spMis18_MeDiY), to rescue the temperature‐sensitive growth phenotype of mis18‐818 (T49A) and mis18‐262 (G117D) cells in vivo (Fig ​(Fig4B4B and C) 10. Dimer‐disrupting mutants spMis18_flI31A and spMis18_flY114A failed to rescue the viability defect at restrictive temperature in both mis18‐262 and mis18‐818 cells. Expression of spMis18_MeDiY alone, while failing to complement the loss of spMis18 function at 36°C, reproducibly conferred a dominant‐negative effect on growth in both mis18‐262 and mis18‐818 cells at 32°C. This inhibitory effect on growth of mis18‐262 and mis18‐818 cells depended on the ability of spMis18_MeDiY to dimerize, as the spMis18_MeDiYI31A and Y114A mutations caused no such negative influence on growth (Fig ​(Fig4B4B and C). We conclude that dimerization of spMis18 via the MeDiY domain is crucial for spMis18 function in vivo.

Expression and purification of recombinant human proteins

Human Mis18α_fl, Mis18α_77–187, Mis18β_fl, and Mis18β_56–183 codon‐optimized sequences (GeneArt) were cloned into pEC‐K‐3C‐His LIC vector or pGEX‐6P‐1 (GE Healthcare). Human proteins were expressed separately or co‐expressed using pGEX‐6P‐1 and pEC‐K‐3C‐His in BL21 Gold in a similar manner to spMis18ΔC. His‐hsMis18α_77–187 was lysed via sonication in 20 mM Tris pH 8, 500 mM NaCl, 35 mM imidazole, and 4 mM BME with 10 μg/ml DNase, 1 mM PMSF, and complete EDTA‐free. Clarified lysate was loaded onto a 5‐ml HisTrap HP column and washed with lysis buffer and then with 20 mM Tris pH 8, 1 M NaCl, 35 mM imidazole, 50 mM KCl, 10 mM MgCl₂, 2 mM ATP, and 4 mM BME and re‐equilibrated in lysis buffer before eluting with 20 mM Tris pH 8, 500 mM NaCl, 500 mM imidazole, and 4 mM BME. GST‐hsMis18β_56–183 was sonicated in 20 mM Tris pH 8, 500 mM NaCl, and 4 mM DTT with 10 μg/ml DNase, 1 mM PMSF, and complete EDTA‐free. Cleared lysate was loaded on 12 ml glutathione–Sepharose (GE Healthcare) in batch mode and washed in lysis buffer and then with 20 mM Tris pH 8, 1 M NaCl, 50 mM KCl, 10 mM MgCl₂, 2 mM ATP, and 4 mM DTT followed by lysis buffer. 3C protease was added to cleave the tag on the beads overnight and untagged protein collected. To obtain the hsMis18α_77–187/Mis18β_56–183 complex, proteins were co‐expressed and purified as described for GST‐hsMis18β_56–183. All proteins were separated on Superose 6 10/300 pre‐equilibrated with 20 mM Tris pH 8, 200 mM NaCl, and 5 mM DTT.

His/GST pull‐down assays

To test for protein–protein interactions, purified His‐tagged spMis18_MeDiY and His‐GFP‐tagged spMis18_{C‐term‐α} proteins were mixed and applied to a Superdex 200 increase 10/300 column equilibrated with 20 mM Tris pH 8, 150 mM NaCl, and 2 mM DTT and fractions analyzed via SDS–PAGE. To test for interaction of human hsMis18α and β, 10 ml of culture of co‐expressed proteins was grown and sonicated in 20 mM Tris pH 8, 100 mM NaCl, and 2 mM BME. After clarification, supernatants were split and 35 mM imidazole added to half before incubation for 1 h with either glutathione–Sepharose or HisPur Ni‐NTA resin and then washed with buffer. Purified proteins were run on SDS–PAGE.

Native PAGE

12% native PAGE gels (22 mM Tris pH 8.8, 12% acrylamide with stacking 320 mM Tris pH 8.8, 4% acrylamide) were run in 25 mM Tris and 192 mM glycine at 150 V for 30 mins. Proteins in 1× sample buffer (31.25 mM Tris pH 6.8, 12.5% glycerol, 0.5% bromophenol blue) and Native Marker (Life Technologies) were loaded and then run at 150 V for 3 h.

Crystallization and data collection

Crystallization trials were performed using a nanoliter crystallization robot at the Edinburgh crystallization facility. Crystals were grown by vapor diffusion method. Diffraction quality crystals were obtained using well buffer containing 0.2 M ammonium chloride/formate/acetate/phosphate and 20% PEG 3350 (with the measured pH of the solution in the range of 6.2–8). 15–20 mg/ml protein sample was mixed with the well buffer in a 1:1 ratio. Crystals were briefly transferred to cryoprotectant solution (crystallization solutions were supplemented with glycerol or ethylene glycol to a final concentration 25%) before flash cooling in liquid nitrogen. The crystals diffracted to 2.6 Å resolution at the MX beamlines of the Diamond Light Source (Table 1).

Crystal structure solution and refinement

The structure of spMis18_MeDiY was determined by the single anomalous dispersion (SAD) method using the anomalous signal of intrinsically bound Zn²⁺ ion. Data were processed using XIA2 and scaled with SCALA of CCP4 31. SAD phasing and the calculation of the initial map were performed using phenix.autosol from the PHENIX suite of programs 32. The model was built by iterative rounds of manual building with COOT 33, and refinement was done using Refmac5 of CCP4. Data collection, phasing, and refinement statistics are shown in Table 1.

SEC‐MALS

Size‐exclusion chromatography coupled to multi‐angle light scattering (SEC‐MALS) was performed at the Edinburgh Protein Production Facility at room temperature using a ÄKTA FPLC. 100 μl of protein at 1 mg/ml was loaded onto either Superose 6 10/300, Superdex 75 10/300, or Superdex 200 10/300 columns (GE Healthcare) pre‐equilibrated with 50 mM HEPES pH 8, 150 mM NaCl, and 5 mM DTT for His‐spMis18_MeDiY, His‐GFP‐spMis18_{C‐term‐α}, and hsMis18α_77–187/hsMis18β_56–183, or 50 mM HEPES pH 8, 50 mM NaCl, and 5 mM DTT for His‐spMis18ΔC or 50 mM HEPES pH 8, 300 mM NaCl, and 1 mM TCEP for His‐GFP spMis18_fl. A MiniDAWN in‐line detector (Wyatt Technology) was used to measure MALS, while a Viscotek RI Detector (Wyatt Technology) was used to detect refractive index. Data were analyzed using ASTRA^™ software (Wyatt Technology).

Plasmids and S. pombe strains

For in vivo assays in S. pombe, spMis18 cDNA (wt, I31A, Y114A, Y90A or Y74A Y90A T105A S107K in the context of Mis18_fl or Mis18_MeDiY) was PCR‐amplified and cloned into the pDUAL‐GFH41 vector, which allows for expression of C‐terminally GFP‐tagged spMis18 (wt or mutants) under the control of the medium‐strength nmt41 promoter that is induced in the absence of thiamine in culture media 34. spMis18 constructs cloned into the pDUAL‐GFH41 vector were then either transformed as such and selected in the absence of uracil in growth media, or integrated into the genome at the leu1 locus, in wt, mis18‐818, or mis18‐262 cells. Genotypes of S. pombe strains used in this study are listed in Table EV1.

Genetic complementation assays

Fivefold serial dilutions of mis18‐262, mis18‐818, or wt cells expressing GFP‐tagged spMis18_fl or spMis18_MeDiY (wt or mutants) either from an ectopic pDUAL‐GFH41 plasmid (selected for in media lacking uracil; Fig ​Fig1E)1E) or from the leu1 locus (integrated in the genome; Figs ​Figs4B4B and C and EV4B), were spotted onto PMG media containing phloxine B supplemented with or without thiamine and incubated at the indicated temperatures for 3–5 days.

Co‐immunoprecipitation and Western analyses

For co‐immunoprecipitation experiments, cells expressing endogenous spMis18_fl‐Myc and ectopic spMis18‐GFP (wt or mutants; from the leu1 locus) were cultured in complete PMG media lacking thiamine for 21 h and processed as previously described 11. Immunoprecipitation was performed using rabbit anti‐myc antibody A14 (Santa Cruz Biotech) and Western analyses using monoclonal anti‐GFP (Roche) or anti‐myc 9B11 (Cell Signaling).

Quantitative chromatin immunoprecipitation

Anti‐GFP ChIP, anti‐CENP‐A^Cnp1 ChIP, and real‐time PCR analyses were performed as previously described 11, on wild‐type S. pombe cells expressing ectopic GFP‐tagged spMis18_fl (wt or mutants; from the leu1 locus) cultured in complete PMG media lacking thiamine for 21 h.

Cytology

Immunolocalization and microscopy were performed as previously described 11 on wild‐type S. pombe cells expressing ectopic GFP‐tagged spMis18_fl (wt or mutants; from the leu1 locus) cultured in complete PMG media lacking thiamine for 21 h.

We thank D. Foltz for sharing data prior to publication. We thank the staff of Diamond Light Source for assistance during data collection and staff of Edinburgh protein production facility, and particularly M. Wear for his help with SEC‐MALS data generation and analysis. We thank C. Ponting and L. Sanchez‐Pulido for the initial bioinformatics analysis of the Mis18 Yippee‐like domain. We also thank L. Mackay for his help with the MS‐based protein sequence coverage analysis of Mis18. We are grateful to M. Yanagida and the National Bioresource Project (YGRC) Japan for S. pombe strains, and P. Heun, and members of Jeyaprakash and Allshire laboratory for discussions. We also thank W. Earnshaw, A. Cook, K. Hardwick, J. Welburn, A. Pidoux, and M. A. Abad Fernandez for the critical reading of the manuscript. The Wellcome Trust generously supported this work through a Wellcome Trust Career Development Grant to AAJ (095822), a Principal Research Fellowship to RCA (095021 & 065061), a Wellcome Trust Centre Core Grant (092076), an instrument grant (091020), and the Wellcome Trust‐University of Edinburgh Institutional Strategic Support Fund. The European Commission supported this work through a career integration grant to AAJ and through an EpiGeneSys Network of Excellence grant to RCA (HEALTH‐F4‐2010‐257082. LS was supported by an EC FP7 Marie Curie International Incoming Fellowship (PIIF‐GA‐2010‐275280) and an EMBO Long Term Fellowship (ALTF 1491‐2010). RB was supported by a Wellcome Trust 4‐year PhD program.