Condensin structures chromosomal DNA through topological links

Sara Cuylen, Jutta Metz & Christian H. Haering

European Molecular Biology Laboratory (EMBL), Cell Biology & Biophysics Unit Meyerhofstrasse 1, 69117 Heidelberg, Germany Correspondence should be addressed to C.H.H. (christian.haering@embl.de) This is the unedited version of the manuscript published in final form in Nature Structural and Molecular Biology Vol. 18(8) 894-901, doi: 10.1038/nsmb.2087, online at http://www.nature.com/nsmb/journal/v18/n8/full/nsmb.2087.html The multi-subunit condensin complex is essential for the structural organization of eukaryotic chromosomes during their segregation by the mitotic spindle, but the mechanistic basis for its function is not understood. To address how condensin binds to and structures chromosomes, we have isolated from yeast cells circular minichromosomes linked to condensin. We find that either linearization of minichromosome DNA or proteolytic opening of the ring-like structure formed through the connection of the two ATPase heads of condensin’s structural maintenance of chromosomes (SMC) heterodimer by its kleisin subunit eliminates their association. This suggests that condensin rings encircle chromosomal DNA. We further show that release of condensin from chromosomes by ring opening in dividing cells compromises the partitioning of chromosome regions distal to centromeres. Condensin hence forms topological links within chromatid arms that provide them with the structural rigidity necessary for their segregation. INTRODUCTION

Key for successful chromosome segregation during mitosis and meiosis is the reorganization of chromatin into compact, individualized sister chromatid pairs by a conserved multi-subunit protein complex named condensin, which was identified as one of the major constituents of chromosomes that had been isolated from metaphase-arrested human cells 1 or assembled in mitotic frog egg extracts 2,3. Condensin depletion from such extracts prevents the transformation of added sperm DNA into mitotic chromosome-like structures, suggesting that condensin drives chromosome condensation. Mutation of condensin genes in yeast similarly causes a reduction in the compaction of chromosomes during mitosis, and such cells eventually fail to segregate their genomes 4-6. While mutation or knock-down of subunits of either of the two condensin complexes present in metazoan cells has apparently only minor effects on the ultimate levels of chromosome compaction in vivo, mitotic chromosomes from these cells have a fuzzy appearance, are hypersensitive to mechanical forces, and frequently form anaphase chromatin bridges 7-11. Though condensin may therefore not be required for the initial chromosome compaction process per se, it is essential for the maintenance of the structural integrity of chromosomes during their segregation. Two of the five subunits of the ~630 kDa condensin complex are members of the Structural Maintenance of Chromosomes (SMC) protein family, which form ~40 nm long antiparallel coiled coils that separate ABC-like ATPase ‘head’ domains at

one end of the coiled coil from a central dimerization ‘hinge’ domain at the other end. While condensin’s Smc2 and Smc4 subunits dimerize via the association of their half-doughnut shaped hinge domains 12, the kleisin subunit Brn1 (NCAPH/H2) binds to the head domain of Smc2 via its N terminus and to the head domain of Smc4 via its C terminus 13. In addition to connecting the Smc2–Smc4 ATPase heads, Brn1 recruits the two HEAT repeat subunits Ycs4 (NCAPD2/D3) and Ycg1 (NCAPG/G2). Condensin shares the same fundamental architecture with another SMC complex named cohesin (reviewed in 14), although electron micrographs of both complexes suggest that the conformations of their SMC coiled coil arms may be quite distinct 15. The key question remains how condensin reinforces mitotic chromosome structure during segregation. The findings that DNA circles are converted into positive supercoils or knots in the presence of topoisomerase and condensin 9,16,17 or Smc2–Smc4 dimers 18 raises the possibility that condensin may reshape chromatin by introducing conformational restrains on the DNA fiber that result in its compaction. DNA supercoiling could be the result of wrapping DNA around parts of the complex 19 and may be driven through ATP hydrolysis by the Smc2–Smc4 head domains and activated by the mitotic phosphorylation of condensin 20,21. An alternative hypothesis is based on the observations that Smc2–Smc4 dimers cluster on DNA 18,22,23. Many condensin complexes, each bound to a different segment of the same chromosome, may therefore

associate into the axes of mitotic chromosomes from which chromatin loops emerge (reviewed in 24). A fundamentally different hypothesis originates from the discovery that the condensin-related cohesin complex holds together pairs of sister chromatids by their entrapment within the large tripartite ring structure formed through the connection of the ATPase heads of an Smc1–Smc3 dimer by the kleisin protein Scc1 25. Given the identical ring-like arrangement of the Smc2, Smc4, and Brn1 subunits in the condensin complex 13, it is tempting to speculate that condensin may use a similar topological mechanism to clamp together different parts of one chromosomal fiber. This idea prompted us to investigate the nature of condensin’s binding to chromosomes. RESULTS

A condensin-minichromosome binding assay To test in a defined biochemical system whether condensin binds chromosomes directly or in a topological fashion, we adopted an in vitro assay developed to measure cohesin binding to small circular minichromosomes 26. We inserted a part of the rDNA region that is highly enriched for condensin binding (Supplementary Fig. 1a) into a centromeric circular minichromosome and transformed the resulting minichromosome (Supplementary Fig. 1b, c) into yeast cells expressing an HA6-tagged version of condensin’s Brn1 subunit. We then tested whether condensin had been recruited to the minichromosome in vivo by Brn1 immunoprecipitation from cell lysates and probing for minichromosome co-purification by Southern blotting. Under the stringent conditions of our assay, we found that a fraction of minichromosomes was specifically pulled down with condensin (Fig. 1a). DNA linearization releases minichromosomes from condensin Isolation of condensin-minichromosome complexes allowed us to investigate the nature of condensin’s chromosome association and compare it to that of cohesin, which we used as a control. If minichromosomes were encircled by condensin rings, we would expect that DNA linearization should cause them to slide out of condensin rings (Fig. 1b). DNA linearization should on the other hand have no effect on minichromosome association with condensin if binding were direct (Fig. 1c). To test this, we cleaved minichromosomes linked to immobilized condensin at a unique BglII site and assayed whether minichromosome DNA was released during cleavage or subsequent washing steps with buffers of increasing ionic strengths, or whether minichromosome DNA remained bound to condensin. While circular minichromosomes remained stably bound to condensin throughout the experiment, a total of ~80% of linearized minichromosomes dissociated from condensin (Fig. 1d).

Notably, efficient release of linearized minichromosomes from condensin required higher salt concentrations than their release from cohesin. These findings suggest that the condensin complex makes direct (electrostatic) interactions with chromatin in addition to topologically encircling the DNA fiber.

Figure 1 Linearization releases minichromosome DNA from condensin. (a) Co-immunoprecipitation of 4.3 kb minichromosomes from lysates of asynchronous yeast cultures (strains C2292, C2293, C2350) with condensin (Brn1-HA6) or cohesin (Scc1-HA6) was tested by Southern blotting of input (IN), flow-through (FT), and immunoprecipitated fractions (B, 5× or 25× concentrated relative to input). Relaxed (top) or supercoiled (bottom) monomers and supercoiled concatemers (*) are indicated (see Supplementary Fig. 1c). 1.6% or 0.7% of total minichromosome DNA was co-immunoprecipitated with condensin or cohesin, respectively (compare to 0.02% when neither was tagged). (b, c) Linearization of minichromosome DNAs should cause them to slide out of condensin rings if they were topologically bound but should not affect binding if they were bound via direct protein-chromatin contacts. (d) Linear minichromosome DNA released from immobilized condensin (top, strain C2292) or cohesin (middle, strain C2350) after BglII cleavage into supernatant (SUP), wash fractions of increasing salt concentrations (0-1 M NaCl), or still bound to beads (SB) was detected by Southern blotting and quantified (bottom, mean ± SD). (e) As in (d) after cleavage at a DraIII site opposite to the BglII site. Cleavage at the DraIII site located roughly opposite to the BglII site within the minichromosome circle released DNA

from condensin with an even higher efficiency than cleavage at the BglII site (Fig. 1e), probably because the bulk of condensin may be bound at the rDNA region in close proximity to the DraIII site. Incubation with BglII of minichromosomes lacking a target site for the enzyme (Supplementary Fig. 2a) or mere relaxation of the supercoiling of the minichromosome DNA using a nicking endonuclease (Supplementary Fig. 2b, c) had in contrast no effect on the binding of minichromosomes to condensin. The latter finding is consistent with the efficient co-purification of relaxed minichromosomes with condensin from cell extracts (Fig. 1a). Thus, linearization of minichromosome DNA in any position, but not simply relaxation of its superhelicity, causes its release from condensin. Generation of a cleavable condensin ring If the link between condensin and chromosomes is of topological nature, then not only DNA linearization but also disruption of the condensin ring integrity should release it from minichromosomes. To test this notion, we created a condensin ring that can be proteolytically opened. We inserted cleavage sites for the tobacco etch virus (TEV) protease into different locations within the central region of condensin’s kleisin subunit Brn1. Five different Brn1(TEV) constructs complemented deletion of the essential BRN1 gene (Fig. 2a), suggesting that insertion of the TEV sites did not interfere with condensin function in these constructs. We first tested whether Brn1 cleavage by TEV protease in vivo eliminates condensin function. Western blotting showed that all five Brn1(TEV) constructs had been efficiently cleaved four hours after induction of TEV expression from the GAL1 promoter (Fig. 2b). Notably, four out of the five strains with cleavable Brn1 versions failed to grow under conditions that maintain TEV expression, but were fully viable in the absence of TEV expression (Fig. 2c). Since Brn1(TEV622) was cleaved with the highest efficiency (Fig. 2b), we used this construct for further experiments. One possible explanation for the loss of condensin function may be that Brn1(TEV622) cleavage causes the complex to fall apart. This is however not the case, since all condensin subunits remain associated with Brn1 after TEV cleavage of immunoprecipitated condensin complexes (Supplementary Fig. 3). The alternative explanation is that Brn1 cleavage creates a gap in the ring and thereby allows the escape of encircled chromosomes. To test whether Brn1(TEV622) cleavage opens condensin rings, we measured the dissociation of the two Brn1 cleavage fragments. Since both fragments remain linked after TEV cleavage (Supplementary Fig. 4a), presumably via their binding to the head domains of the same Smc2–Smc4 heterodimer, we engineered additional TEV target sites into the coiled coil domain of Smc4 (Supplementary Fig. 4b), which should allow us to break the Smc2–Smc4 linker. Simultaneous cleavage of Brn1(TEV622) and Smc4(TEV552/971) indeed released a substantial fraction of the N-terminal Brn1

Figure 2 Ring opening by TEV cleavage of Brn1 eliminates condensin function. (a) TEV sites were introduced between the N-terminal Smc2 and C-terminal Smc4 binding motifs (HTH/WHD) into Brn1 at positions of low secondary structure probability. (b) TEV protease expression was induced from pGAL1 in asynchronous cultures (strains C2437, C2320, C2439, C2322, C2455, and C2335) and Brn1 cleavage was monitored by Western blotting against C-terminal HA6 tags in whole cell extracts at the indicated times after induction. (c) Brn1(TEV) constructs complement brn1Δ in the absence of TEV expression but - with the exception of Brn1(TEV251) - not after induction of TEV expression (strains as in (b), plus C2324 and K8758). (d) TEV251 is positioned in the center of the binding site for Ycs4 in Brn1. It is therefore possible that Ycs4 connects the Brn1(TEV251) cleavage fragments to keep condensin rings intact, while cleavage at other TEV sites opens the ring. (e) In vivo TEV cleavage of Brn1(TEV251) and Ycs4(TEV829) after TEV protease induction was monitored by Western blotting against C-terminal HA6 or PK9 tags, respectively (strains C2813, C2820, C2805). (f) Simultaneous cleavage of Brn1(TEV251) and Ycs4(TEV829), but not cleavage of either protein alone, destroys condensin function at 37°C.

fragment together with the central part of Smc4 from the C-terminal Brn1 fragment (Supplementary Fig. 4c). Cleavage of Brn1 at position 622 therefore opens condensin rings as anticipated. Surprisingly, cleavage of Brn1 at position 251 did not result in any detectable growth defects even at 37°C (Fig. 2c and data not shown). We noticed that this position is situated in the center of the binding region for the Ycs4 subunit (M. Walczak and C.H.H., unpublished results). It may hence be possible that Ycs4 bridges the two Brn1(TEV251) fragments and thereby keeps the ring integrity intact (Fig. 2d). If this were true, we would expect that the two Brn1(TEV251) cleavage fragments remain associated even if we eliminate their linkage through the Smc2–Smc4 heterodimer, which is indeed the case (Supplementary Fig. 4c). If linkage of the Brn1(TEV251) were through Ycs4, destabilization of this subunit by TEV cleavage may eventually destroy condensin ring integrity. Strains that express Brn1(TEV251) and Ycs4(TEV829) are indeed unable to grow at 37°C after TEV protease induction (Fig. 2e, f). These data suggest that cleavage of Brn1 at position 251, in contrast to cleavage at position 622, does not open condensin rings. Thus, cleavage of Brn1 causes condensin to become non-functional only when it results in ring opening. Release of circular minichromosomes by condensin ring opening in vitro If chromosomal DNA were topologically entrapped within condensin rings, then ring opening by cleavage of Brn1(TEV622) should release circular minichromosomes from condensin, while cleavage of Brn1(TEV251), which does not result in ring opening, should have no effect on their association (Fig. 3a). Indeed, we found that ~67% of circular minichromosome DNA dissociated from condensin after ring opening by TEV cleavage of Brn1(TEV622), while more than 85% of minichromosomes remained bound to intact condensin rings in the absence of Brn1 cleavage (Fig. 3b, c). Cleavage of Brn1 at position 251 resulted only in a noticeable increase in minichromosome release under 1 M salt conditions, and 60% of the minichromosome DNA remained bound to condensin after TEV cleavage at this position. Similar to the DNA linearization experiments, we observed that efficient release of minichromosomes from opened condensin rings required higher ionic strength conditions than their release from opened cohesin rings 26. These findings support the notion that besides encircling the chromatin fiber topologically, condensin complexes additionally bind to (mini)chromosomes by secondary direct, salt sensitive contacts. Opening of condensin rings causes their dissociation from chromosomes in vivo To test whether opening of condensin rings not only releases them from minichromosomes in vitro but also from chromosomes in vivo, we compared the amounts of minichromosome DNA that co-immunoprecipitated with

Figure 3 Ring opening by Brn1 cleavage releases condensin from minichromosomes. (a) If minichromosome DNAs were encircled within condensin rings, they should be released when rings are opened after cleavage of Brn1(TEV622) but not when ring integrity remains intact after cleavage of Brn1(TEV251). (b) Immobilized condensin- and cohesin-minichromosome complexes (immunoprecipitated from strains C2461, C2460, C2348, and C2349) were incubated with recombinant TEV protease. Cleavage was monitored by Western blotting against the C-terminal HA6 tag on Brn1 or Scc1. (￮) Scc1 separase cleavage fragment. (c) Release of closed circular minichromosome DNA after TEV cleavage was detected by Southern blotting of supernatant (SUP), salt wash (0-1 M NaCl), or still bound (SB) fractions and quantified (mean ± SD). either cleaved or intact Brn1 after TEV induction in yeast cells (Fig. 4a). Condensin ring opening by Brn1(TEV622) cleavage in vivo reduced the amounts of co-immunoprecipitated minichromosomes ~10-fold, which is similar to the reduction in minichromosome co-purification with cohesin after TEV cleavage of Scc1. As expected from the previous experiment, Brn1(TEV251) cleavage reduced the amounts of minichromosomes that co-purify with condensin to a much lesser extend than cleavage of Brn1(TEV622). In a second experiment, we assayed the effect of ring opening on the association of condensin with endogenous chromosomes by immunofluorescence staining of Brn1 on

mitotic chromosome spreads (Fig. 4b). While cleavage of Brn1(TEV251) had no apparent effect on Brn1 levels on chromatin, condensin ring opening by cleavage of Brn1(TEV622) dramatically reduced chromosomal Brn1 staining. To quantify the decrease in condensin binding, we measured the amounts of condensin at two chromosomal binding sites before and after TEV cleavage by chromatin immunoprecipitation followed by real-time PCR (ChIP-qPCR; Fig. 4c). Condensin levels were largely unaffected by cleavage of Brn1(TEV251) at both loci tested but were almost completely diminished by cleavage of Brn1(TEV622). If chromosomal DNA were encircled by condensin rings, opening of the condensin ring not only by Brn1(TEV622) cleavage but also by severing the Smc4 coiled coil should release the complex from chromosomes. To test this notion, we induced TEV expression in yeast strains that express cleavable versions of Smc4 (Fig. 5a, b). In support of the conclusion that ring integrity is essential for condensin function, we found that cells failed to divide after cleavage of both strands of the Smc4 coiled coil at opposite positions Smc4(TEV552/971)) but are viable when only one of the two coiled coil strands is cleaved (Smc4(TEV522) and Smc4(TEV971); Fig. 5c). ChIP-qPCR showed that Smc4(TEV552/971) cleavage drastically reduced the amounts of chromosomal condensin at the two endogenous binding

Figure 4 Ring opening by Brn1 cleavage in vivo releases condensin from chromosomes. (a) Completion of Brn1 or Scc1 cleavage 4 h after TEV induction in asynchronous cultures (strains C2463, C2567, C2443, and C2444) was confirmed by Western blotting and co-immunoprecipitation of 7.9 kb minichromosomes with HA-tagged condensin or cohesin, respectively, was assayed by Southern blotting of input (IN), supernatant (SUP), and bound fractions (B). (b) Cleavage of Brn1 3 h after TEV induction in cells arrested with nocodazole (strains C2783, C2781, and C2864) was tested by Western blotting as in (a), and condensin release from chromosomes was assayed by immunofluorescence staining of chromosome spreads (red, anti-HA; blue, DAPI); scale bar = 5 µm. Chromosomal Brn1-HA6 immunofluorescence signals were quantified; lines define the median, boxes define the 25th and 75th percentiles, whiskers define the 10th and 90th percentiles. (c) TEV protease expression was induced in asynchronous cultures (strains C2439, C2455, C2783, C2781, and K9872) and Brn1 cleavage monitored 3 h post induction as in (a). Levels of Brn1-HA6 or Smc2-PK6 bound to two chromosomal condensin sites (left: 5’ UTR of RDN37 rDNA, right: CEN4 centromere) before and after Brn1 cleavage were measured by anti-HA and anti-PK chromatin immunoprecipitation (ChIP) followed by quantitative PCR (mean ± SD).

sites tested, while ‘nicking’ of the Smc4 coiled coil had only minor effects on condensin levels at these sites (Fig. 5d). Unfortunately we could not test the effect of Smc4 cleavage on condensin’s association with minichromosomes, since complexes containing Smc4(TEV552/971) bound less stably to the minichromosomes in vitro (data not shown). In conclusion, our experiments suggest that opening of condensin rings independent of the position of cleavage releases them from chromosomal DNA in vivo. Requirement of condensin ring integrity for chromosome arm segregation If enclosure of chromosomes within condensin rings were essential for condensin’s function in structuring mitotic chromosomes, we would expect that disruption of its ring integrity should be deleterious for chromosome segregation. Cells that were synchronously released into the cell cycle after condensin ring opening during a G1 phase arrest indeed accumulate with massive aneuploidies (Supplementary Fig. 5a). To characterize chromosome segregation defects in detail, we directly imaged the partitioning of regions ~20 or ~400 kb away from the centromere of chromosome V (Centromere/Telomere V) and of the ~1 Mb ribosomal DNA region on the right arm of chromosome XII (rDNA) in the course of a single round of mitosis following Brn1 cleavage during a pheromone-induced G1 arrest. Yeast cells in which

Brn1(TEV622) had been cleaved during the G1 arrest replicated their DNA and initiated cell division with normal timing as judged by FACScan analysis (Fig. 6a, b) and bud formation (Supplementary Fig. 5b), and neither sister chromatid cohesion nor centromere segregation were affected (Fig. 6c). By contrast, segregation of telomeres and ribosomal DNA was severely compromised: Approximately half of the cells failed to partition the telomeric locus into the bud even by three hours after release from the G1 arrest, although cohesion between sister loci had been resolved (Fig. 6d). Furthermore, only a tiny fraction of cells with cleaved Brn1(TEV622) correctly segregated their rDNA between mother cell and bud, while in the large majority of cells the bulk of the rDNA repeats remained in the mother cell (Fig. 6e). These findings are consistent with the missegregation phenotypes observed in yeast condensin mutants 27-30. We conclude that condensin ring integrity is essential for the segregation of chromosome regions distal but not proximal to centromeres. Chromosome segregation depends on condensin integrity beyond metaphase To test whether condensin’s association with chromosomes is still required during their movement to the cell poles in anaphase, we allowed cells to enter mitosis in the presence of intact condensin rings and cleaved Brn1(TEV622) only after cells had arrested in metaphase by depletion of the anaphase promoting complex activator Cdc20 (Fig. 6f, g). We then released cells from the arrest and recorded centromere, telomere, and rDNA segregation by live cell imaging. While cells released from metaphase with cleaved Brn1(TEV622) segregated their centromeric regions with undistinguishable timing and efficiency compared to cells with intact Brn1 (Fig. 6h), a large fraction of them failed to partition their telomeric regions, even though cohesion between them had been mostly resolved (Fig. 6i and Supplementary Fig. 5c). Previous studies reported that the rDNA region is re-organized into a linear structure during metaphase in a condensin-dependent

Figure 5 Ring opening by cleavage of Smc4 in vivo releases condensin from chromosomes. (a) Opening of condensin rings by TEV cleavage of both strands of Smc4’s coiled coil at juxtaposed positions. (b) Smc4 cleavage was monitored by Western blotting of whole cell extracts 4 h after TEV protease induction in asynchronous cultures (strains C2838, C2857, C2859, and C2864) against C-terminal PK6 tags in whole cell extracts. Blotting against the FLAG epitope preceding the TEV sites confirmed that Smc4(TEV552/971) had been cleaved at both sites. (c) Smc4 coiled coil cleavage (but not merely ‘nicking’) after induction of TEV expression destroys condensin function (strains as in (b)). (d) TEV protease expression was induced in asynchronous cultures (strains C2838, C2859, C2857, and K9872) grown at 25ºC and Smc4 cleavage monitored 4 h post induction by Western blotting against PK6 and FLAG tags. Levels of Smc4 bound to the 5’ UTR of RDN37 rDNA (top) and CEN4 (bottom) before and after Smc4 cleavage were measured by anti-PK ChIP followed by qPCR (mean ± SD).

manner 31,32. We likewise noted that most cells with cleaved Brn1(TEV622) displayed a more diffuse rDNA arrangement and had segregated only a small fraction of the rDNA mass into the bud even by 90 min after release from the metaphase arrest (Fig. 6j and Supplementary Fig. 5c). We conclude that chromosome entrapment within condensin rings beyond metaphase is essential for their segregation. DISCUSSION Topological enclosure of chromosomal DNA within condensin rings How the condensin complex associates with and functions on mitotic chromosomes to enable their proper segregation has been a subject of intense debate. While its subunit geometry is similar to the ring-shaped cohesin complex 13,33, a number of observations suggested that condensin may not encircle chromosomal DNAs like cohesin does 25,26,34. First, the Smc2–Smc4 coiled coil arms appear to associate over their entire length in electron and atomic force micrographs of condensin complexes 15,22, which would presumably not be compatible with the passage of DNA between them. Second, treatment with PreScission protease of isolated chicken chromosomes bearing condensin whose Smc2 coiled coils contained target sites for the protease had no drastic effect on condensin association with these chromosomes 35. Third, electron-spectroscopic images implied that condensin’s ATPase head domains (and probably the non-SMC subunits) bind directly to DNA 19, as also suggested by electrophoretic mobility shift experiments 16,23. Our findings on the other hand demonstrate that the primary molecular mode of condensin’s interaction with chromosomes must be of topological nature. First, if condensin rings close around chromosome fibers, we would expect that such rings were able to slide off the ends of short linear DNA molecules similar to cohesin rings 26. Linearization - but not mere

relaxation - of circular minichromosomes indeed causes their dissociation from condensin complexes (Fig. 1). Second, breakage of the ring integrity should release condensin from chromosomes if it encircled DNA. Condensin ring opening by proteolytic cleavage of Brn1 in vivo or in vitro indeed abrogates its binding to circular minichromosomes or its association with endogenous binding sites on yeast chromosomes (Fig. 3 and 4). Crucially, Brn1 cleavage at a site that does not result in ring opening (presumably due to connection of the cleavage fragments by Ycs4) has little effect on condensin’s association with chromosomes in vivo, even though it may decrease the strength of the ring under stringent in vitro conditions (Fig. 3c and 4a). Third, if chromosomes were topologically encircled by condensin, opening of the ring in any position should cause their release. We find that cleavage not only of Brn1 but also of Smc4’s coiled coil strongly reduces condensin binding to chromosomes (Fig. 5d). It is possible that cleavage at the offset sites within the Smc2 coiled coil used in previous experiments 35 may not have created an opening in the coiled coil and hence did not release condensin from chromosomes, similar to what we observe after merely ‘nicking’ the Smc4 coiled coils. In addition to DNA linearization or proteolytic ring opening, we find that high salt concentrations are required for efficient dissociation of minichromosomes from condensin but not

Figure 6 Condensin release by ring opening prevents chromosome arm segregation. (a - e) Brn1 cleavage during G1 phase. TEV protease expression from pGAL1 was induced in cells arrested in G1 phase by α-factor. Cells were released from the arrest 3 h after protease induction and chromosome segregation was recorded by live cell imaging. Cell cycle progression was measured by FACScan analysis of DNA content and Brn1 cleavage during the α-factor arrest was monitored by Western blotting. Segregation of GFP-labeled repeats ~20 kb (strains C2381 and C2665) or ~400 kb (strains C2481 and C2619) from centromere V, or of the rDNA marked by Net1-GFP (strains C2497 and C2621) was scored according to the categories shown. (f - j) Brn1 cleavage during metaphase. TEV protease expression was induced in cells arrested in metaphase by repression of Cdc20 expression from pMET3. Cells were released into methionine-free media 3 h after protease induction and chromosome segregation was recorded by live cell imaging as in (a) (strains C2628, C2666, C2513, C2618, C2484, and C2620).

from cohesin. This suggests the presence of secondary electrostatic contacts between condensin ring subunits and chromatin. These contacts could be made through DNA binding sites 17,19,36 or through the ‘clamping’ of chromatids between the associated Smc2–Smc4 coiled coil arms 15 and are probably an essential feature that distinguishes the action of condensin from that of cohesin. Chromosome arm segregation during anaphase requires intact condensin rings Is the entrapment of chromosomes by condensin rings essential for their segregation? We find that the re-arrangement of the rDNA region into a linear array as well as its equal partitioning between mother cell and bud are severely compromised when cells enter anaphase after release of condensin from chromosomes by Brn1 cleavage (Fig. 6). This is similar to the defects observed after inactivation of condensin by temperature sensitive mutations 28-30,32. In addition, condensin ring opening results in substantial segregation defects of centromere-distal chromosome regions, which is also consistent with the stretching of the mass of chromatin over the bud neck in condensin yeast mutants 27-29,37 and lagging chromosomes and chromosome bridges found in animal cells depleted of condensin 8,10,11,38. Enclosure of DNA within condensin rings is hence essential for the maintenance

of a global configuration of chromosome arms that is compatible with their segregation into the daughter cells. Yet we find that centromeres segregate correctly even after condensin ring opening. It is unlikely that Brn1 at centromeres is refractory to TEV cleavage in our experiments, since condensin binding to centromeres is reduced to background levels after TEV expression (Fig. 4c). Similarly, temperature sensitive condensin yeast mutants display no or considerably smaller defects in segregating centromeres than telomeres 5,28,30. This suggests that condensin binding is dispensable for kinetochore bi-orientation during mitosis in budding yeast cells, despite enrichment of the complex on pericentromeric chromatin 39,40. Condensin may however become essential for centromere arrangement and kinetochore co-orientation during meiosis 41 or when the kinetochore or spindle checkpoint are compromised 30. It is also likely that condensin plays an important role for the stability of the (significantly larger) centromeres of animal cells 8,9,42-45. The defects in chromosome segregation following condensin release from chromosomes may be caused either by a failure in sister chromatid resolution (through catenation or cohesion 7,46) or by a loss of chromosome structural integrity. In this case chromosome arms may fail to follow the centromeres when the latter are being pulled to the poles (Fig. 7a). Our finding that sister telomere regions split in more than 75% of cells even after Brn1 cleavage (Fig. 6d, i) suggests that chromatid resolution per se is not prevented by condensin release from chromosomes. This is consistent with efficient cohesin removal from chromosomes and decatenation of minichromosomes in condensin mutants 27,47, even though condensin may be required for disentangling repetitive DNA sequences like those at the rDNA array 48. Instead, we find that both separated sister telomeres remain in the mother cell close to the bud neck and fail to congress into the bud. We argue that condensin is essential for organizing chromosome arms into rigid bodies and thereby allow the transduction of spindle forces from kinetochore to telomere. Such rigid body structures may be the basis for the ‘recoiling’ of chromosome arms during anaphase 49. This hypothesis is also consistent with the increased elasticity of human mitotic chromosomes observed after condensin depletion 11. A model for the organization of mitotic chromosomes by topological condensin linkages How may condensin provide structural integrity to chromatin fibers by encircling them inside its ring? We propose that one condensin complex fastens together two distant segments of one sister chromatid to retain the chromatin fiber in a folded conformation. Fastening may be achieved by passage of one segment through the condensin ring and direct binding to the second segment (Fig. 7b) or by simultaneous passage of both segments through one ring (Fig. 7c) similar to the entrapment

Figure 7 Structuring of chromosomes through topological condensin links. (a) Condensin complexes may structurally reinforce chromosome arms into rigid bodies that can be moved by mitotic spindle microtubules connected to a single kinetochore. Loss of rigidity triggered by release of condensin would cause chromosome arms to get stretched and lag behind centromeres during segregation. (b) Condensin rings may link different chromosome segments by encircling one chromatid segment while binding directly to a second segment of the same chromatid. The topologically bound segment may be free to slide through the ring. (c) Alternatively condensin rings may encircle both segments within their ring structure. of two sister DNAs within cohesin rings 25. While cohesin rings may freely slide along the entrapped DNAs, the direct condensin-chromatin interaction suggested by our experiments and atomic force and electron spectroscopic imaging of condensin bound to DNA 19,22 may maintain the association of condensin with one chromosome segment but may nevertheless allow local chromatin rearrangements through the sliding of the second, topologically bound segment. Another possibility is that multiple condensin complexes, each entrapping a single chromosome segment, associate 24. Entry of chromosomal DNA into condensin rings would presumably require temporary ring opening, possibly through the dissociation of the SMC hinge domains in analogy to what has been suggested for cohesin 50. Future experiments will have to test whether DNA strands pass through the condensin ring once or twice, whether condensin functions as individual or multimeric rings, and how chromosomes gets into and out of the rings. Our discovery that encircling of chromosomal DNA by a large ring structure is fundamental to the action of condensin implies that this unconventional mode of DNA binding may be the basis for the mechanisms of all SMC complexes.

METHODS Methods and any associated references are available in the online version of the paper at http://www.nature.com/nsmb/. Note: Supplementary information is available on the Nature Structural & Molecular Biology website. ACKNOWLEDGEMENTS We thank Jan Ellenberg, Michael Knop , Anne-Claude Gavin, and all members of the Haering group for advice and discussions, Kim Nasmyth (University of Oxford) for strains and plasmids, Dmitri Ivanov for sharing protocols, and the EMBL Advanced Light Microcopy, Flow Cytometry, and Genomics Core Facilities for their assistance. This work was supported by funding from EMBL and the German Research Foundation (DFG) Priority Programme 1384 (C.H.H.). AUTHOR CONTRIBUATIONS S.C., J.M., and C.H.H. yeast strain and plasmid generation, S.C., minichromosome, co-immunoprecipitation, and ChIP-qPCR experiments, S.C. and J.M., live cell imaging experiments, C.H.H., chromosome spreads, S.C. and C.H.H., project design and manuscript preparation, C.H.H. project supervision. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

ONLINE METHODS Yeast strains. All yeast strains are derivatives of W303. Genotypes are listed in Supplementary Table 1. Minichromosomes. 1 kb of the 5´UTR of RDN37 was inserted into a YCplac22 derived centromeric plasmid 26. For minichromosome pull-down assays after Brn1 in vivo cleavage, a kanMX6 cassette was additionally inserted. The pUC19 sequence was removed before transformation of the resulting 4.3 kb (Supplementary Fig. 1b) or 7.9 kb circular minichromosomes into yeast. For DNA nicking, the BglII site was replaced by a Nb.BbvCI site. TEV cleavable condensin subunits. SpeI sites were inserted into the BRN1-HA6 gene in YIplac211 or into the YCS4-PK6 gene in YIplac128 following the indicated amino acid positions by overlap extension PCR. A triple tandem TEV cassette was inserted into the newly generated sites. Constructs were integrated into heterozygous ∆brn1 or ∆ycs4 strains (C1636 or C2791). Tetrad dissection showed that all BRN1(TEV) and YCS4(TEV) constructs shown in Fig. 2 complement deletion of the endogenous BRN1 or YCS4 genes, respectively, at 30ºC and 37ºC. NheI sites were inserted into the SMC2-PK6 or SMC4-PK6 genes in YIplac128 and FLAG-triple tandem TEV cassettes inserted. The resulting plasmids were combined pair wise to yield double cleavable constructs. Plasmids were integrated into heterozygous ∆smc2 or ∆smc4 strains (C1634 or C1635). Tetrad dissection showed that only the constructs indicated in Supplementary Fig. 4b complement deletion of the endogenous genes SMC2 or SMC4 genes, respectively, at 30°C. Minichromosome and condensin subunit co-immuno-precipitations. The minichromosome immunoprecipitation protocol was slightly modified from 34. All co-immunoprecipitation experiments are described in detail in the Supplementary Methods. ChIP-qPCR. Chromatin immunoprecipitation was performed as reported 50 and is described in detail in the Supplementary Methods. Cell synchronization. For arrest in G1 phase, yeast cells were grown at 30ºC in YEP containing 2% (w/v) raffinose (YEPR) to mid-log phase, collected by filtration, washed with water, and diluted to an OD600 of 0.15 in pre-warmed YEPR. α-factor was added to 3 µg ml-1 for 1 h. Additional α-factor was added to 2 µg ml-1 after 1 h and galactose was added to 2% (YEPRG) for TEV protease induction. After another hour, cells were collected by filtration, washed with water, and resuspended in fresh YEPRG pre-warmed to 30°C and containing 3 µg ml-1 α-factor. After 1 h fresh α-factor was added to 2 µg ml-1. To release cells 4 h after the first addition of α-factor, cells were collected by filtration, washed with

YEPRG, and resuspended in YEPRG without α-factor. After 15 min cells with Tet operator arrays integrated 17.8 kb or ~400 kb away from the centromere on the right arm of chromosome V 51,52 or expressing Net1 fused to GFP 53 were transferred to glass-bottom dishes coated with concavalin A (Sigma) for microscopy. For metaphase arrest, cells that express Cdc20 under control of pMET3 were grown at 30ºC in -MET to mid-log phase. Cells were collected by filtration, washed with water, and resuspended in YEP with 2 mM methionine to an OD600 of 0.2 and grown at 30°C. After 105 min galactose was added to 2% (w/v) for TEV protease induction. After 4 h of arrest, a fraction of cells were settled onto a glass-bottom dish for live microscopy, washed 3 times with -MET media, and covered with the same medium. For FACScan analysis, cells were filtered, washed with -MET, and resuspended in the same medium. FACScan analysis. Cells were collected by centrifugation, fixed with 70% (v/v) ethanol overnight, and treated with 0.2 mg ml-1 RNaseA in 50 mM TRIS-HCl pH 7.5 for 2-4 h. Fixed cells were then stained for 30 min at room temperature with 50 µg ml-1 propidium iodide in 200 mM TRIS-HCl pH 7.5, 211 mM NaCl, 78 mM MgCl2. Before analysis in a FACScan flow cytometer (Becton Dickinson), cells were sonicated (3 pulses at 40%, 25 W) and diluted 1:5 in 50 mM TRIS-HCl pH 7.5 and 50 µg ml-1 propidium iodide. 10,000 events were acquired with the CellQuest software and analysed with FlowJo (TreeStar). Chromosome spreading. Asynchronous yeast cultures grown in YEPR were arrested in a metaphase-like state by addition of nocodazole to a final concentration of 10 µg ml-1 for 2 h at 30°C. Cultures were split into two and TEV protease expression was induced in one half by addition of galactose to 2% (w/v) followed by additional 3 h incubation. Chromosome spreads were prepared as described 54 and stained for Brn1-HA6 with 16B12 (Covance, 1:500) and Alexa Fluor 594 labeled anti-mouse IgG (Invitrogen, 1:600) antibodies and for DNA with DAPI. At least 100 nuclei were recorded for every sample on a DeltaVision Spectris Restoration microscope (Applied Precision) with an 100× NA 1.35 oil immersion objective. Alexa Fluor 594 fluorescence intensities within a circle of 5 µm radius centered on DAPI masses were measured after background subtraction in ImageJ 55. Live cell microscopy. Cells were transferred to glass-bottom dishes (MatTek), settled for 10 min, washed, and covered with synthetic medium. Time lapse imaging was performed on a DeltaVision microscope as described in the previous paragraph at 30°C, taking 10-20 sections per image with 700 nm step size and exposure times between 0.2 and 0.3 s. Between 75 to 180 individual cells were scored per strain.

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18. Stray, J.E., Crisona, N.J., Belotserkovskii, B.P., Lindsley, J.E. & Cozzarelli, N.R. The Saccharomyces cerevisiae Smc2/4 condensin compacts DNA into (+) chiral structures without net supercoiling. J Biol Chem 280, 34723-34 (2005).

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22. Yoshimura, S.H. et al. Condensin architecture and interaction with DNA: regulatory non-SMC subunits bind to the head of SMC heterodimer. Curr Biol 12, 508-13 (2002).

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24. Hirano, T. At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol 7, 311-22 (2006).

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Condensin structures chromosomal DNA through topological links

European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany. Correspondence should be addressed to C.H.H. (christian.haering@embl.de).

Sara Cuylen, Jutta Metz & Christian H Haering

SUPPLEMENTARY FIGURES

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Supplementary Figure 1 Construction and characterization of a minichromosome with a condensin-binding region. (a) Condensin binding to di�erent chromosomal regions previously identi�ed by ChIP-on-chip, including the 5´UTR of RDN37 (rDNA), a tRNA gene (SUP2), and ~3 kb centromeric regions (CEN3-5), was quanti�ed in strains C540 and K699 by anti-HA ChIP-qPCR using 2-3 di�erent primer pairs per region. A primer pair speci�c to the TUB2 gene served as a negative control. The 5’UTR of RDN37 showed the strongest condensin enrichment and hence was inserted into a circular minichromosome. (b) Map of the 4.3 kb minichromosome containing 1 kb of the 5´UTR region of RDN37 (rDNA), an 850 bp CEN4 region, TRP1, and ARS1. (c) Minichromosome DNA co-puri�ed with condensin from cell extracts was incubated with topoisomerase II (topo II), nicking enzyme Nb.BbvCI, or bu�er only to assign DNA topoisomers to Southern blot bands as indicated on the left.

Supplementary Figure 2 DNA nicking or incubation with restriction enzyme of minichromosomes lacking the restriction site has no e�ect on minichromosome association with condensin. (a) Minichromosomes lacking a BglII site were co-puri�ed with condensin (from strain C2379) and incubated with BglII restriction enzyme. Southern blotting as in Fig. 1 showed that BglII incubation in the absence of DNA cleavage has no e�ect on minichromosome association with condensin. ( ) supercoiled concatemers. (b) Relaxation of the supercoiled state of the minichromosome DNA should not e�ect its binding to condensin rings if it were bound topologically. (c) Minichro-mosome DNA association with immobilized condensin (top, strain C2379) or cohesin (middle, strain C2378) after relaxation with the nicking enzyme Nb.BbvCI was probed by Southern blotting. Nicking does not or only very slightly a�ect minichromosome association with condensin or cohesin, respectively.

*

Supplementary Figure 3 Brn1 cleavage does not disrupt interactions between condensin subunits. (a) Condensin was immobilized on beads via a PK6 epitope on the C terminus of Smc2 (strains C2781 and C2783) and proteins bound to immunoprecipitation beads before (–TEV) and after (+TEV) 1.5 h incubation with TEV protease were analysed by SDS-PAGE and silver staining. All condensin subunits remain associated after Brn1 TEV cleavage. (b) – (d) Condensin complexes were immunoprecipitated from whole cell extracts via a HA6 epitope on the C terminus on Brn1. Immunoprecipitation beads were incubated with TEV protease (+TEV) or bu�er only (–TEV) and release of Smc2-PK6 (strains C2781 and C2783), Ycs4-PK6 (strains C2785 and C2787), or Ycg1-PK9 (strains C3010 and C3008) was probed by Western blotting of bound (B), supernatant (SUP), or still bound (SB) fractions. Brn1 cleavage has no detectable e�ect on the association of Smc2, Ycs4, or Ycg1 with Brn1. (•) IgG.

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Supplementary Figure 4 Cleavage of Smc4’s coiled coil demonstrates condensin ring opening by Brn1(TEV622) cleavage. (a) Condensin complexes were immunoprecipitated from whole cell extracts via a FLAG3 tag on the N terminus of Brn1 (strains C2596 and C2595), and Brn1 was cleaved with TEV protease while bound to the immunoprecipitation beads. Release of the HA6-tagged C-terminal Brn1 fragment was tested by Western blotting in bound (B), supernatant (SUP), or still bound (SB) fractions after incubation with TEV protease (+TEV) or bu�er only (–TEV). The bulk of the C-terminal Brn1 cleavage fragment remains associated with the N-terminal cleavage fragment, suggesting that the two Brn1 fragments are linked through their binding to the Smc2–Smc4 head domains. (b) Coiled coil predictions for Smc2 (top) and Smc4 (bottom) were aligned in an antiparallel orientation and FLAG-TEV3 sites inserted at opposite positions of low coiled coil probability in the two coiled coil strands following the indicated amino acid residues. The ability of single FLAG-TEV3 site insertions or pair wise combinations of insertions to complement deletion of SMC2 or SMC4 after tetrad dissection are indicated (right). The only combination that produces viable spores is Smc4(TEV552/971). (c) Condensin complexes were immunoprecipitated from whole cell extracts of asynchronous cultures (strains C2961, C2896, C2963, and C2900) via an HA6 tag on the C terminus of Brn1 and incubated with TEV protease while bound to the beads. Release of the N-terminal Brn1 cleavage fragment tagged with PC4 and the central Smc4 fragment tagged with FLAG was probed by Western blotting. While N-terminal Brn1 and central Smc4 fragments remain associated with the C-terminal Brn1 fragment after simultaneous cleavage of Smc4(TEV552/971) and Brn1(TEV251), a signi�cant fraction dissociates after cleavage of Smc4(TEV552/971) and Brn1(TEV622), demonstrating that Brn1 cleavage at position 622 but not at position 251 opens condensin rings. (•) IgG.

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P

SB

+TEV –TEV –TEV+TEV

anti-

HA

anti-

PC

c

HA

TEV251

FLAGFLAG

PC

HA

TEV251

TEV552/971

PC

HA

TEV622

PC

HA

TEV622

FLAGFLAG

TEV552/971

13095

72

55

43

34

B SU

P

SB

SU

P

SB

B SU

P

SB

SU

P

SB

Brn11-754

252-754

623-754

1-754

1-251

1-622130

95

72

55

43

170

anti-

HA

anti-

FLA

G

a

+TEV –TEV –TEV+TEV

FLAG

HA

TEV251TEV622

FLAG

HA

Brn1

1.0 0.8 0.6 0.4 0.2 0

coiled coil probability

1.00.80.60.40.20

200

400

600

800

1000

1200

1400

N C

Smc4

Smc2

200

400

800

1000

600

N C

1.0 0.8 0.6 0.4 0.2 0

coiled coil probability

1.00.80.60.40.20

130

170

95

72

55

B SU

P

SB

SU

P

SB

B SU

P

SB

SU

P

SB

+TEV –TEV –TEV+TEV

FLAGFLAG

PC

HA

TEV251

TEV552/971

PC

HA

TEV622

FLAGFLAG

TEV552/971

aa p

ositi

on

aa p

ositi

on

aa p

ositi

on

aa p

ositi

on

kDa

kDa kDa kDa

Supplementary Figure 5 Condensin cleavage results in chromosome missegregation. (a) TEV protease expression from pGAL1 was induced in yeast strains expressing non-cleavable or TEV-cleavable Brn1 (strains C2335, C2324, and C2455) arrested in G1 phase by α-factor. Strains were released from the arrest and DNA content monitored by FACScan analysis over several cell cycles. Accumulation of cells with lower than 1 N and larger than 2 N DNA contents was already apparent after the �rst mitosis following Brn1(TEV363) or Brn1(TEV622) cleavage and increased over subsequent cell divisions as TEV protease expression continued. (b) Still images from live cell microscopy of centromere, telomere, and rDNA segregation after TEV cleavage in α-factor arrested cells shown in Fig. 6c-e, taken at the indicated times after release. (c) Movies and still images from live cell microscopy of centromere, telomere, and rDNA segregation after TEV cleavage in metaphase arrested cells shown in Fig. 6h-j, taken at the indicated times after release.

55 min 95 min 135 min 175 min

cenV

-GFP

Net

1-G

FPte

lV-G

FP

55 min 95 min 135 min 175 min

Brn1(TEV622)Brn1b

10 min 30 min 50 min 90 min

Brn1(TEV622)Brn1

70 min 10 min 30 min 50 min 90 min70 minc

cenV

-GFP

a

telV

-GFP

Net

1-G

FP

1N2N

Brn1 (TEV363)

release

Brn1 Brn1 (TEV622)

7 h

4 h3 h

2 h1 h

5 h

8.5 h11.5 h

1N2N 1N 2N

6 h

SUPPLEMENTARY TABLES

Supplementary Table 1 Yeast Strains

K699 MATa

K8758 MATalpha, ∆scc1::HIS3, leu2::SCC1(TEV268)-HA3::LEU2, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

K9872 MATa, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C540 MATalpha, BRN1-HA6::HIS3

C1634 MATa/alpha, ∆smc2::HIS3/SMC2

C1635 MATa/alpha, ∆smc4::HIS3/SMC4

C1636 MATa/alpha, ∆brn1::HIS3/BRN1

C2292 MATa, leu2::TetR-TAP::LEU2, BRN1-HA6::HIS3, [4.3 kb Minichromosome]

C2293 MATa, leu2::TetR-TAP::LEU2, [4.3 kb Minichromosome]

C2320 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV1413)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2322 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV3633)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2324 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV3639)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2335 MATa, ∆brn1::HIS3, ura3:::BRN1-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2348 MATa, ∆brn1::HIS3, ura3:::BRN1-HA6::URA3, leu2::TetR-TAP::LEU2, [4.3 kb Minichromosome]

C2349 MATa, ∆scc1::SCC1(TEV3)-HA6::HIS3, TetR-TAP::LEU2, [4.3kb Minichromosome]

C2350 MATa, leu2::TetR-TAP::LEU2, SCC1-HA6::HIS3, [4.3 kb Minichromosome]

C2378 MATa, leu2::TetR-TAP::LEU2, SCC1-HA6::HIS3, [4.3 kb Minichromosome(Nb.BvCI)]

C2379 MATa, leu2::TetR-TAP::LEU2, BRN1-HA6::HIS3, [4.3 kb Minichromosome(Nb.BvCI)]

C2381 MATa, 17.8 kb right from CEN5(-18ChV)::tetO224::URA3, leu2::TetR-GFP::LEU2, ∆brn1::HIS3, ura3:::BRN1-HA6::URA3,

10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2437 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV1153)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2439 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV2513)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2443 MATa, ∆brn1::HIS3, ura3:::BRN1-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1, [7.9 kb Minichromosome-kanMX6]

C2444 MAT, ∆scc1::HIS3, SCC1(TEV268)-HA3::LEU2, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1, [7.9 kb Minichromosome-kanMX6]

C2455 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2460 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, [4.3kb Minichromosome]

C2461 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV2513)-HA6::URA3, [4.3kb Minichromosome]

C2463 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV2513)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1, [7.9kb Minichromosome-kanMX6]

C2481 MATa, ∆brn1::HIS3, ura3:::BRN1-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1,

leu2::TetR-GFP::LEU2, 2× tetO224::URA3 integrated between BMH1 and PDA1

C2484 MATa, NET1-GFP::kanMX, cdc20::pMET3-CDC20::TRP1, ∆brn1::HIS3, ura3:::BRN1-HA6::URA3, 10×trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2497 MATa, NET1-GFP::kanMX, ∆brn1::HIS3, ura3:::BRN1-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2513 MATalpha, pMET-CDC20::LEU2, ∆brn1::HIS3, ura3:::BRN1-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1,

leu2::TetR-GFP::LEU2, 2×tetO224::URA3 integrated between BMH1 and PDA1

C2567 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, 10×trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1, [7.9 kb Minichromosome-kanMX6]

C2595 MATa/alpha, ∆brn1::HIS3/BRN1, ura3:::FLAG3-BRN1(TEV2513)-HA6::URA3/ura3

C2596 MATa/alpha, ∆brn1::HIS3/BRN1, ura3:::FLAG3-BRN1(TEV6223)-HA6::URA3/ura3

C2618 MATa, cdc20::pMET-CDC20::LEU2, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1,

leu2::TetR-GFP::LEU2, 2×tetO224::URA3 integrated between BMH1 and PDA1

C2619 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1,

leu2::TetR-GFP::LEU2, 2× tetO224::URA3 integrated between BMH1 and PDA1

C2620 MATa, NET1-GFP::kanMX, cdc20::pMET3-CDC20::TRP1, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3,

10×trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2621 MATa, NET1-GFP::kanMX, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

SUPPLEMENTARY TABLES

Supplementary Table 1 Yeast Strains (continued)

C2628 MATalpha, cdc20::pMET3-CDC20::TRP1, leu2::TetR-GFP::LEU2, ura3:::BRN1-HA6::URA3,

17.8 kb right from CEN5(-18ChV)::tetO224::URA3, ∆brn1::HIS3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2665 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, leu2::TetR-GFP::LEU2, 10×trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1,

17.8 kb right from CEN5(-18ChV)::tetO224::URA3

C2666 MATalpha, cdc20::pMET3-CDC20::TRP1, leu2::TetR-GFP::LEU2, 17.8 kb right from CEN5(-18ChV)::tetO224::URA3,

∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2781 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, SMC2-PK6::kanMX, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2783 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV2513)-HA6::URA3, SMC2-PK6::kanMX, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2785 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, YCS4-PK9::kanMX, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2787 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV2513)-HA6::URA3, YCS4-PK9:kanMX, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2791 MATa/alpha, ∆ycs4::kanMX6/YCS4; 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1/10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2805 MATa, ∆ycs4::kanMX6, leu2::YCS4(TEV8293)-PK6::LEU2, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2813 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV2513)-HA6::URA3, ∆ycs4::kanMX6, leu2::YCS4-PK6::LEU2, 10×trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2820 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV2513)-HA6::URA3, ∆ycs4::kanMX6, leu2::YCS4(TEV8293)-PK6::LEU2,

10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2838 MATa, ∆smc4::HIS3, leu2:::SMC4(FLAG-TEV5523/FLAG-TEV9713)-PK6::LEU2, 10×trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2857 MATa, ∆smc4::HIS3, leu2:::SMC4(FLAG-TEV5523)-PK6::LEU2, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2859 MATa, ∆smc4::HIS3, leu2:::SMC4(FLAG-TEV9713)-PK6::LEU2, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2864 MATa, ∆smc4::HIS3, leu2::SMC4-PK6::LEU2, 10× trp1::pGAL-NLS-myc9-TEVprotease-NLS2::TRP1

C2896 MATa, ∆brn1::HIS3, ura3:::PC4-BRN1(TEV6223)-HA6::URA3

C2900 MATa, ∆brn1::HIS3, ura3:::PC4-BRN1(TEV2513)-HA6::URA3

C2961 MATa, ∆brn1::HIS3, ura3:::PC4-BRN1(TEV6223)-HA6::URA3, ∆smc4::HIS3, leu2:::SMC4(FLAG-TEV5523/FLAG-TEV9713)-PK6

C2963 MATa, ∆brn1::HIS3, ura3:::PC4-BRN1(TEV2513)-HA6::URA3, ∆smc4::HIS3, leu2:::SMC4(FLAG-TEV5523/FLAG-TEV9713)-PK6

C3008 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV2513)-HA6::URA3, YCG1-PK9:kanMX

C3010 MATa, ∆brn1::HIS3, ura3:::BRN1(TEV6223)-HA6::URA3, YCG1-PK9:kanMX

Supplementary Table 2 Primer sequences

TUB2 SC-83 CGGCCAATTGAACTCTGATT

SC-84 AGCCGACCATGAAGAAATGT

SUP2 SC-35 AGTTGGTTTAAGGCGCAAGA

SC-36 AACGCCCGATCTCAAGATTT

SUP2 SC-37 ACACCTCGCAAGAAATCGAC

SC-38 TCTTGCGCCTTAAACCAACT

rDNA SC-39 CACACTTGTACTCCATGACTAAACC

SC-40 GACAGAGAGGGCAAAAGAAAA

rDNA SC-41 TTTCTGCCTTTTTCGGTGAC

SC-42 TGGCATGGATTTCCCTTTAG

CEN3 SC-47 GTTGAGCATCCCATCCAGTT

SC-48 GGGTAATGGCAAATCTGCTT

CEN3 SC-53 TGAAGGGAAGAGGCTCATTT

SC-54 CAATTGGAGGCATCTCAAGC

Locus Name Sequence 5’ 3’

CEN3 SC-57 CTGTTTTCGCCCATTGTTCT

SC-58 CAACCTCATCGATTCCCTGT

CEN5 SC-63 GTTGCATTTGCCTTTGGACT

SC-64 CCCAATTTTAAACGCTCCAA

CEN5 SC-65 AATGTCCGCCACAGGATAAC

SC-66 TTTTGGTGTCAACGGAACAA

CEN5 SC-67 TGGCGCTTGTCTACTGTTTG

SC-68 TTAGGCAATGGCAAAAATCC

CEN4 SC-77 TGGTGTGGAAGTCCTAATATCG

SC-78 TGCATGATCAAAAGGCTCAA

CEN4 SC-81 TCTCAAATACACTTATTAACCGCTTT

SC-82 CACTGTTTGATATTACTGTCAGCGTA

Locus Name Sequence 5’ 3’

SUPPLEMENTARY METHODS

Minichromosome Co-immunoprecipitation. Cultures were diluted into 350 ml YEPD at 30°C from an overnight culture grown in -TRP media and harvested at an OD600 of 1.5. Lysates were prepared as described34, pre-cleared at 12,000 × g, and incubated at 4°C with 100 µl 12CA5 anti-HA antibody for 1 h followed by addition of 200 µl protein A dynabeads (Invitrogen) overnight. Beads were washed 3 times with lysis bu�er (25 mM HEPES-KOH pH 8.0, 200 mM NaCl, 50 mM KCl, 10 mM MgSO4, 0.25% Triton-X100, 1 mM DTT) containing 0.1 mg ml-1 BSA and split into three aliquots after an additional wash step with lysis bu�er without NaCl. One aliquot was set aside for elution (bound), the other two were incubated with enzyme or bu�er only. For DNA cleavage, dynabeads were incubated in lysis bu�er without NaCl with 100 U ml-1 BglII or 140 U ml-1 DraIII (NEB) for 1.5 h at 4°C. For DNA nicking, beads were incubated for 2 h at 16°C with 450 U ml-1 Nb.BbvCI (NEB). For TEV cleavage, lysis bu�er containing 130 µg ml-1 TEV protease was added for 1.5 h at 16°C. Supernatants were collected and beads were washed twice with lysis bu�er containing 0, 0.2, 0.5, and 1 M NaCl. Minichromosomes (still) bound to the beads were eluted twice with 250 μl elution bu�er (50 mM TRIS-HCl pH 8.0, 500 mM NaCl, 10 mM EDTA, 1% SDS). All samples were adjusted to 1% (w/v) SDS, phenol/chloroform extracted, ethanol precipitated in the presence of 20 µg ml-1 glycogen (Roche), and dissolved in 25 µl EB bu�er. Aliquots were separated on a 1% (w/v) agarose gel containing ethidium bromide. Southern transfer was performed under alkaline conditions onto Immobilon-NY+ membranes (Millipore). Blots from 3-4 independent experiments were hybridized with a 32P-labelled probe, exposed to phosphor screens, scanned on an FLA-7000 image analyzer (Fuji�lm), and quanti�ed with Multi Gauge software (Fuji�lm).

Co-immunoprecipitation of Condensin Subunits. Cell extracts were prepared from 400 ml asynchronous cultures either by spheroblasting as described in the previous section or by breaking cells with glass beads extracts in EBX bu�er (50 mM HEPES-KOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 0.25% Triton X-100, and protease inhibitors) and pre-cleared by centrifugation at 16,000 × g. Condensin was immuno- precipitated on protein A or protein G dynabeads (Invitrogen) from cell extracts with 40 µl 12CA5 anti-HA, 20 µl anti-FLAG M2 (Sigma), or 12 µl anti-PK (AbD Serotec) antibodies. Condensin-bound dynabeads were washed and incubated with TEV protease as described in the previous section. Proteins remaining bound to the beads were eluted with 100 µl SDS loading bu�er.

ChIP-qPCR. Fixation, spheroplasting, and lysis were performed as described50 with the exception that 42 ml culture with OD600 of 0.6 were used and �xation was performed in 3% (w/v) formaldehyde at 16°C. Chromatin was sonicated to an average length of 500 bp using a Bioruptor UCD-200 (Diagenode) for 9 min at ‘high level’ with 30 s on, 1 min o� settings. Lysates were cleared by two rounds of centrifugation and incubated with 50 µl protein A dynabeads for 2 h at 4°C. 150 µl cleared lysate were used to check sonication, 175 µl lysate were kept as an input sample, and 1.4 ml were used for immunoprecipitation with 10 µl 12CA5 anti-HA or 2 µl anti-PK (AbD Serotec) antibody overnight at 4°C, followed

by a 4 h incubation with 100 µl protein A dynabeads. Beads were washed as described50 and eluted in TES (50 mM TRIS-HCl pH 8.0, 10 mM EDTA, 1% (w/v) SDS) at 65°C for 8 h. Samples were digested with 100 µg ml-1 RNaseA for 1.5 h and 660 µg ml-1 proteinase K for 2 h, and DNA was �nally puri�ed via spin columns (Qiagen) and eluted in 50 µl EB bu�er. The qPCR reactions were performed with SYBR green PCR Master mix (Applied Biosystems) and contained 5 µl puri�ed DNA, 5 µM primers (see Supple-mentary Table 2) in a total volume of 20 µl. 1:5 and 1:25 dilutions of immunoprecipitated samples and 1:5, 1:50, 1:500, and 1:5,000 dilutions of input samples were used. The reactions were run on a ABI 7500 real-time PCR system (Applied Biosystems). Binding levels were quanti�ed from 3-4 independent cultures with at least two qPCR repeats each.

NSMB_2011Cuylen_supplementary