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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">01374</article-id><article-id pub-id-type="doi">10.7554/eLife.01374</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Genes and chromosomes</subject></subj-group></article-categories><title-group><article-title>Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-7174"><name><surname>Verzijlbergen</surname><given-names>Kitty F</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-7175"><name><surname>Nerusheva</surname><given-names>Olga O</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7176"><name><surname>Kelly</surname><given-names>David</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7177"><name><surname>Kerr</surname><given-names>Alastair</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-7178"><name><surname>Clift</surname><given-names>Dean</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7179"><name><surname>de Lima Alves</surname><given-names>Flavia</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7180"><name><surname>Rappsilber</surname><given-names>Juri</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-7131"><name><surname>Marston</surname><given-names>Adele L</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><aff id="aff1"><institution content-type="dept">Wellcome Trust Centre for Cell Biology, Institute of Cell Biology</institution>, <institution>University of Edinburgh</institution>, <addr-line><named-content content-type="city">Edinburgh</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff2"><institution content-type="dept">Department of Biotechnology</institution>, <institution>Institute of Bioanalytics, Technische Universität Berlin</institution>, <addr-line><named-content content-type="city">Berlin</named-content></addr-line>, <country>Germany</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Pines</surname><given-names>Jon</given-names></name><role>Reviewing editor</role><aff><institution>The Gurdon Institute</institution>, <country>United Kingdom</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>adele.marston@ed.ac.uk</email></corresp><fn fn-type="present-address" id="pa1"><label>†</label><p>MRC Laboratory of Molecular Biology, Cambridge, United Kingdom</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>04</day><month>02</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01374</elocation-id><history><date date-type="received"><day>16</day><month>08</month><year>2013</year></date><date date-type="accepted"><day>11</day><month>12</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Verzijlbergen et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Verzijlbergen et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife01374.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01374.001</object-id><p>To protect against aneuploidy, chromosomes must attach to microtubules from opposite poles (‘biorientation’) prior to their segregation during mitosis. Biorientation relies on the correction of erroneous attachments by the aurora B kinase, which destabilizes kinetochore-microtubule attachments that lack tension. Incorrect attachments are also avoided because sister kinetochores are intrinsically biased towards capture by microtubules from opposite poles. Here, we show that shugoshin acts as a pericentromeric adaptor that plays dual roles in biorientation in budding yeast. Shugoshin maintains the aurora B kinase at kinetochores that lack tension, thereby engaging the error correction machinery. Shugoshin also recruits the chromosome-organizing complex, condensin, to the pericentromere. Pericentromeric condensin biases sister kinetochores towards capture by microtubules from opposite poles. Our findings uncover the molecular basis of the bias to sister kinetochore capture and expose shugoshin as a pericentromeric hub controlling chromosome biorientation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.001">http://dx.doi.org/10.7554/eLife.01374.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01374.002</object-id><title>eLife digest</title><p>When a cell divides to create two new daughter cells, it must produce a copy of each of its chromosomes. It is important that each daughter cell gets just one copy of each chromosome. If an error occurs and one cell gets two copies of a single chromosome, it can lead to cancer or birth defects. Fortunately, there are multiple checks to ensure that this does not happen.</p><p>During cell division the chromosomes line up in a way that increases the likelihood that each daughter cell will have one copy of each chromosome. After this process—which is called biorientation—is completed, microtubules pull the chromosomes to opposite ends of the cell, which then divides.</p><p>Proteins called shugoshin proteins are known to be involved in biorientation in many organisms. These proteins are found in a region called the pericentromere, which surrounds the area on the chromosomes that the microtubules attach to, but the details of their involvement in biorientation are not fully understood. Now Verzijlbergen et al. have exploited sophisticated genetic techniques in yeast to explore how shugoshin proteins work.</p><p>These experiments showed that the shugoshin protein helps to recruit condensin—a protein that keeps the DNA organized within the chromosome—to the pericentromere to assist with biorientation. It also keeps aurora B kinase—one of the enzymes that helps to correct errors during cell division—in the pericentromere when a microtubule attaches to the wrong chromosome. These results help us understand how a ‘hub’ in the pericentromere ensures biorientation. The next challenge will be to understand how this hub is disassembled after biorientation to allow error-free cell division to proceed. As shugoshins have been found to be damaged in some cancers, understanding the workings of this hub could also shed new light on how they contribute to disease.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.002">http://dx.doi.org/10.7554/eLife.01374.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>mitosis</kwd><kwd>biorientation</kwd><kwd>shugoshin</kwd><kwd>condensin</kwd><kwd>aurora B</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>S. cerevisiae</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Wellcome Trust</institution></institution-wrap></funding-source><award-id>[090903], [084229], [077707], [092076], [091020]</award-id><principal-award-recipient><name><surname>Verzijlbergen</surname><given-names>Kitty F</given-names></name><name><surname>Nerusheva</surname><given-names>Olga O</given-names></name><name><surname>Kelly</surname><given-names>David</given-names></name><name><surname>Kerr</surname><given-names>Alastair</given-names></name><name><surname>Clift</surname><given-names>Dean</given-names></name><name><surname>de Lima Alves</surname><given-names>Flavia</given-names></name><name><surname>Rappsilber</surname><given-names>Juri</given-names></name><name><surname>Marston</surname><given-names>Adele L</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>EMBO</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Verzijlbergen</surname><given-names>Kitty F</given-names></name><name><surname>Marston</surname><given-names>Adele L</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Scottish Universities Life Sciences Alliance</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Marston</surname><given-names>Adele L</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Darwin Trust of Edinburgh</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Nerusheva</surname><given-names>Olga O</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Shugoshin proteins help to align chromosomes so that copies of the same chromosome attach to microtubules from opposite poles during cell division.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>The accurate segregation of chromosomes during mitosis relies on the capture of newly duplicated sister chromatids by microtubules emanating from opposite poles. This is possible because on each chromosome a multi-subunit kinetochore assembles on a region of DNA, known as a centromere, to mediate attachment to microtubules. The proper attachment of sister kinetochores to opposite poles, called biorientation, generates tension owing to sister chromatid cohesion (<xref ref-type="bibr" rid="bib52">Tanaka, 2010</xref>). Sister kinetochores are inherently biased towards capture by microtubules from opposite poles, yet how this is achieved is not known (<xref ref-type="bibr" rid="bib22">Indjeian and Murray, 2007</xref>). Kinetochore geometry is thought to position microtubule binding sites in a ‘back-to-back’ orientation during mitosis and this has been hypothesized to contribute to biorientation (<xref ref-type="bibr" rid="bib17">Hauf and Watanabe, 2004</xref>). Where erroneous tension-less attachments do occur, they are destabilized by the aurora B kinase, providing a further opportunity for biorientation to be established (<xref ref-type="bibr" rid="bib52">Tanaka, 2010</xref>). Shugoshin proteins are localized to the region surrounding the centromere, known as the pericentromere, and have a conserved, yet poorly understood, function in biorientation (<xref ref-type="bibr" rid="bib23">Indjeian et al., 2005</xref>; <xref ref-type="bibr" rid="bib20">Huang et al., 2007</xref>; <xref ref-type="bibr" rid="bib28">Kiburz et al., 2008</xref>; <xref ref-type="bibr" rid="bib16">Gutiérrez-Caballero et al., 2012</xref>). In fission yeast, frogs, and human cells, shugoshins enable biorientation, at least in part, through recruitment of the chromosome passenger complex (CPC) containing aurora B to the pericentromere (<xref ref-type="bibr" rid="bib25">Kawashima et al., 2007</xref>; <xref ref-type="bibr" rid="bib56">Vanoosthuyse et al., 2007</xref>; <xref ref-type="bibr" rid="bib27">Kelly et al., 2010</xref>; <xref ref-type="bibr" rid="bib55">Tsukahara et al., 2010</xref>; <xref ref-type="bibr" rid="bib57">Wang et al., 2010</xref>; <xref ref-type="bibr" rid="bib61">Yamagishi et al., 2010</xref>; <xref ref-type="bibr" rid="bib47">Rivera et al., 2012</xref>). Shugoshins also have a more defined role in protecting pericentromeric cohesin from premature loss during meiosis and mammalian mitosis; a function attributed to the recruitment of a specific form of the protein phosphatase 2A (PP2A) to the pericentromere (<xref ref-type="bibr" rid="bib24">Katis et al., 2004</xref>; <xref ref-type="bibr" rid="bib30">Kitajima et al., 2004</xref>, <xref ref-type="bibr" rid="bib31">2006</xref>; <xref ref-type="bibr" rid="bib37">Marston et al., 2004</xref>; <xref ref-type="bibr" rid="bib44">Rabitsch et al., 2004</xref>; <xref ref-type="bibr" rid="bib46">Riedel et al., 2006</xref>; <xref ref-type="bibr" rid="bib54">Tang et al., 2006</xref>; <xref ref-type="bibr" rid="bib60">Xu et al., 2009</xref>).</p><p>Though fundamental for accurate chromosome segregation, the role of shugoshin in biorientation has remained unclear. Budding yeast has a single shugoshin, Sgo1, which protects pericentromeric cohesin during meiosis but does not regulate cohesion during mitosis (<xref ref-type="bibr" rid="bib24">Katis et al., 2004</xref>; <xref ref-type="bibr" rid="bib30">Kitajima et al., 2004</xref>; <xref ref-type="bibr" rid="bib37">Marston et al., 2004</xref>; <xref ref-type="bibr" rid="bib23">Indjeian et al., 2005</xref>; <xref ref-type="bibr" rid="bib28">Kiburz et al., 2008</xref>). We have exploited this system to investigate the sister kinetochore biorientation function of Sgo1, independently of effects on cohesion. Our analysis leads us to the unanticipated discovery that shugoshin collaborates with the chromosome-organising complex, condensin, in chromosome biorientation. Moreover, we provide the first molecular insight into how sister kinetochores are biased towards capture by microtubules from opposite, rather than the same, pole.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Shugoshin is important for chromosome biorientation</title><p>To investigate the role of Sgo1 in biorientation we analyzed <italic>sgo1</italic> null cells (<italic>sgo1Δ)</italic> together with three missense mutants: <italic>sgo1-100, sgo1-700</italic> and <italic>sgo1-3A.</italic> The <italic>sgo1-3A</italic> mutant was engineered to disrupt the binding site for PP2A-Rts1 (<xref ref-type="bibr" rid="bib60">Xu et al., 2009</xref>) whereas <italic>sgo1-100</italic> and <italic>sgo1-700</italic> were isolated in a screen due to their inability to sense a lack of tension (<xref ref-type="bibr" rid="bib23">Indjeian et al., 2005</xref>). All three mutants and <italic>sgo1Δ</italic> cells have previously been reported to affect biorientation after microtubule perturbation (<xref ref-type="bibr" rid="bib11">Fernius and Hardwick, 2007</xref>; <xref ref-type="bibr" rid="bib23">Indjeian et al., 2005</xref>; <xref ref-type="bibr" rid="bib22">Indjeian and Murray, 2007</xref>; <xref ref-type="bibr" rid="bib60">Xu et al., 2009</xref>). The Sgo1-3A protein retains its pericentromeric localization (<xref ref-type="bibr" rid="bib60">Xu et al., 2009</xref>; <xref ref-type="fig" rid="fig1">Figure 1A</xref>). Though the kinetics of cell cycle entry in <italic>sgo1-100</italic> and <italic>sgo1-700</italic> mutants is similar to that of wild-type cells (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>), Sgo1-100 and Sgo1-700 show only residual initial centromeric recruitment (<xref ref-type="fig" rid="fig1">Figure 1B</xref>) and are absent from the pericentromeres of cells arrested in mitosis with microtubule-depolymerizing drugs (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). We compared the ability of these Sgo1 mutants to establish bipolar attachments at metaphase after entering the cell cycle in the absence of microtubules. We used strains with spindle pole bodies (SPBs) labeled with tdTomato (<italic>SPC42-</italic>tdTomato), the centromere of chromosome IV labeled with GFP (<italic>CEN4-</italic>GFP) and with <italic>CDC20</italic> under control of the methionione-repressible promoter (<italic>pMET-CDC20</italic>), to enable metaphase arrest by addition of methionine. All strains were released from a G1 arrest into nocodazole- and methionine-containing medium to depolymerize microtubules and induce a metaphase arrest before nocodazole was washed out to allow metaphase spindle formation (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Under these conditions, initial kinetochore-microtubule attachments are frequently erroneous because they occur before spindle pole bodies have migrated apart, leading to a strong reliance on the error correction process driven by Aurora B. We measured the efficiency of biorientation by scoring the splitting of <italic>CEN4-GFP</italic> signals once metaphase spindles reform after nocodazole washout. The <italic>sgo1-100</italic>, <italic>sgo1-700</italic> and <italic>sgo1-3A</italic> mutants showed a similar delay and lower maximum level of biorientation that was not as pronounced as in <italic>sgo1Δ</italic> cells (<xref ref-type="fig" rid="fig1">Figure 1D,E</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.003</object-id><label>Figure 1.</label><caption><title>Sgo1 alleles that affect biorientation.</title><p>(<bold>A</bold>) Sgo1-3A, but not Sgo1-100 or Sgo1-700 are maintained at the centromere in cells arrested in mitosis by treatment with nocodazole. Cells carrying <italic>SGO1-6HA</italic> (AM906), <italic>SGO1-100-6HA</italic> (AM6956), <italic>SGO1-700-6HA</italic> (AM6957), <italic>SGO1-3A-6HA</italic> (AM10011) and a no tag control (AM1176) were arrested in mitosis by treating with nocodozole for 3 hr. Cells were harvested for anti-HA ChIP and the levels of each Sgo1 protein at <italic>CEN4</italic> were analyzed by qPCR. The mean of three independent experiments is shown with error bars representing standard error. (<bold>B</bold>) Sgo1-100 and Sgo1-700 proteins are initially recruited to centromeres but fail to be maintained there. Strains as in (<bold>A</bold>) were arrested in G1 by treatment with alpha factor. Samples were extracted for analysis by anti-HA ChIP at 15 min intervals after release from G1. The levels of Sgo1-6HA at <italic>CEN4</italic> at the indicated times after release from G1 are shown for a representative experiment. (<bold>C</bold>–<bold>E</bold>) <italic>SGO1</italic> mutants are impaired in biorientation. Wild-type (AM4643), <italic>sgo1-100</italic> (AM8924), <italic>sgo1-700</italic> (AM8925), <italic>sgo1-3A</italic> (AM8923) and <italic>sgo1Δ</italic> (AM6117) cells carrying SPB (Spc42-tdTomato) and <italic>CEN4</italic> (<italic>CEN4-GFP</italic>) markers were released from a G1 arrest into medium containing nocodazole (to depolymerize microtubules) and methionine (to deplete <italic>CDC20</italic>). After 3 hr, nocodazole was washed out, and the number of GFP dots was scored in the metaphase-arrested cells as shown in the schematic diagram (<bold>C</bold>). (<bold>D</bold>) Representative images of cells with one and two GFP dots are shown. (<bold>E</bold>) The percentage of visibly separated centromeres was determined at the indicated intervals after nocodazole washout (<italic>t =</italic> 0). Error bars indicate range (n <italic>= 2</italic>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.003">http://dx.doi.org/10.7554/eLife.01374.003</ext-link></p></caption><graphic xlink:href="elife01374f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>The <italic>sgo1-100</italic> and <italic>sgo1-700</italic> mutations do not affect the timing of cell cycle entry.</title><p>Strains as in <xref ref-type="fig" rid="fig1">Figure 1A</xref> were released from G1 as described in <xref ref-type="fig" rid="fig1">Figure 1B</xref> and DNA content was measured at the indicated times by FACS.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.004">http://dx.doi.org/10.7554/eLife.01374.004</ext-link></p></caption><graphic xlink:href="elife01374fs001"/></fig></fig-group></p></sec><sec id="s2-2"><title>Sgo1 does not promote biorientation through PP2A-Rts1 recruitment to centromeres</title><p>The <italic>sgo1-3A</italic> mutation disrupts the interaction between Sgo1 and PP2A-Rts1 (<xref ref-type="fig" rid="fig2">Figure 2A</xref>), which is important for the protection of centromeric cohesion during meiosis (<xref ref-type="bibr" rid="bib60">Xu et al., 2009</xref>). Although the cohesin complex is properly associated with chromosomes in <italic>sgo1Δ</italic> cells during mitosis and cohesion is not affected (<xref ref-type="bibr" rid="bib23">Indjeian et al., 2005</xref>; <xref ref-type="bibr" rid="bib29">Kiburz et al., 2005</xref>; see below), PP2A-Rts1 could perform additional functions in biorientation. Rts1 enrichment at the centromere during metaphase is virtually abolished in <italic>sgo1Δ</italic>, <italic>sgo1-3A</italic> and <italic>sgo1-100</italic>, and modestly reduced in <italic>sgo1-700</italic> cells (<xref ref-type="fig" rid="fig2">Figure 2B</xref>), even though Sgo1-100 and Sgo1-700 proteins retain the ability to associate with Rts1 (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). However, the biorientation defect of the <italic>sgo1</italic> mutants cannot be caused by a failure to recruit Rts1 to centromeres because <italic>rts1Δ</italic> cells achieved biorientation with indistinguishable efficiency to wild-type cells (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). Therefore, PP2A-Rts1 is not required for sister kinetochore biorientation and the <italic>sgo1-3A</italic> mutation must disrupt functions of Sgo1 other than its association with PP2A-Rts1.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.005</object-id><label>Figure 2.</label><caption><title>PP2A-Rts1 recruitment to the centromere by Sgo1 is not required for biorientation.</title><p>(<bold>A</bold>) Sgo1-100 and Sgo1-700, but not Sgo1-3A, associate with Rts1. Cells carrying <italic>RTS1-9MYC</italic> and <italic>SGO1-SZZ(TAP)</italic> (AM9144), <italic>sgo1-100-SZZ(TAP)</italic> (AM9272), <italic>sgo1-700-SZZ(TAP)</italic> (AM9142)<italic>, sgo1-3A-SZZ(TAP)</italic> (AM9145) or no TAP (AM4721) were arrested in nocodazole for 2 hr and treated with the cross-linking reagent dithiobis(succunimidylpropionate) (DSP) before extract preparation as described in ‘Materials and methods’. Extracts were incubated with IgG-coupled beads and immunoprecipitates analyzed with the indicated antibodies. (<bold>B</bold>) Sgo1 mutants affect the centromeric localization of Rts1. Wild-type (AM8895), <italic>sgo1-100</italic> (AM9439), <italic>sgo1-700</italic> (AM9323), <italic>sgo1-3A</italic> (AM9293) and <italic>sgo1Δ</italic> (AM9624) cells carrying <italic>RTS1-3PK</italic>, as well as a no tag control (AM1176), were treated with nocodazole for 3 hr before harvesting for anti-PK ChIP. The mean level of Rts1-3PK enrichment at <italic>CEN4</italic> from three experimental repeats, determined by qPCR, is shown with bars indicating standard error (*p<0.05, paired <italic>t</italic> test). (<bold>C</bold>) Sister kinetochore biorientation after microtubule depolymerization was measured in wild-type (AM4643) and <italic>rts1Δ</italic> (AM5823) cells as in <xref ref-type="fig" rid="fig1">Figure 1</xref> (<bold>C</bold>). The mean of three experimental repeats with error bars representing standard deviation are shown.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.005">http://dx.doi.org/10.7554/eLife.01374.005</ext-link></p></caption><graphic xlink:href="elife01374f002"/></fig></p></sec><sec id="s2-3"><title>Sgo1 ensures the maintenance of aurora B at centromeres</title><p>In other systems, shugoshins are known to affect the association of the chromosomal passenger complex (CPC) containing aurora B kinase with centromeres (<xref ref-type="bibr" rid="bib25">Kawashima et al., 2007</xref>; <xref ref-type="bibr" rid="bib56">Vanoosthuyse et al., 2007</xref>; <xref ref-type="bibr" rid="bib64">Yu and Koshland, 2007</xref>; <xref ref-type="bibr" rid="bib27">Kelly et al., 2010</xref>; <xref ref-type="bibr" rid="bib55">Tsukahara et al., 2010</xref>; <xref ref-type="bibr" rid="bib57">Wang et al., 2010</xref>; <xref ref-type="bibr" rid="bib61">Yamagishi et al., 2010</xref>; <xref ref-type="bibr" rid="bib47">Rivera et al., 2012</xref>). Budding yeast Sgo1 similarly associates with aurora B (called Ipl1) (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). Conditional inactivation of Sgo1 using the auxin-inducible degron (aid) system (<xref ref-type="bibr" rid="bib41">Nishimura et al., 2009</xref>; <xref ref-type="fig" rid="fig3">Figure 3B</xref>) revealed that Sgo1 is not required for the initial recruitment of Ipl1 to centromeres but is important for its maintenance (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>). Indeed, in Sgo1-aid cells arrested in metaphase by treatment with nocodazole, Ipl1 was absent from <italic>CEN4</italic> (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). Early Ipl1 centromere localization also does not require Alk1 and Alk2 (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>), the homologs of Haspin kinase, which is important for centromeric CPC localization in fission yeast and mammals (<xref ref-type="bibr" rid="bib27">Kelly et al., 2010</xref>; <xref ref-type="bibr" rid="bib57">Wang et al., 2010</xref>; <xref ref-type="bibr" rid="bib61">Yamagishi et al., 2010</xref>). The recruitment of Ipl1 early in the cell cycle may instead be due to association with the Ndc10/Cbf3 kinetochore protein that is known to recruit the CPC to centromeres (<xref ref-type="bibr" rid="bib63">Yoon and Carbon, 1999</xref>; <xref ref-type="bibr" rid="bib4">Cho and Harrison, 2012</xref>). Sgo1-independent Ipl1 localization early in the cell cycle (<xref ref-type="fig" rid="fig3">Figure 3C</xref>) can explain why Ipl1, but not Sgo1, is essential for biorientation in an unperturbed cell cycle, though Ipl1 inhibition and deletion of <italic>SGO1</italic> similarly impair biorientation after microtubule depolymerization (<xref ref-type="fig" rid="fig1">Figure 1E</xref>, <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2</xref>; <xref ref-type="bibr" rid="bib2">Biggins et al., 1999</xref>; <xref ref-type="bibr" rid="bib53">Tanaka et al., 2002</xref>; <xref ref-type="bibr" rid="bib23">Indjeian et al., 2005</xref>; <xref ref-type="bibr" rid="bib22">Indjeian and Murray, 2007</xref>). The Ipl1-Sgo1 interaction (<xref ref-type="fig" rid="fig3">Figure 3A</xref>) and centromeric localization of Ipl1 (<xref ref-type="fig" rid="fig3">Figure 3E</xref>) were also similarly decreased in nocodazole-treated <italic>sgo1-100</italic>, <italic>sgo1-700</italic> and <italic>sgo1-3A</italic> mutants, which likely contributes to the biorientation defects of these mutants.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.006</object-id><label>Figure 3.</label><caption><title>Sgo1 is required for the maintenance of Ipl1 at centromeres, but is dispensable for its initial recruitment.</title><p>(<bold>A</bold>) Ipl1/aurora B co-immunoprecipitates with Sgo1. Cells producing SZZ(TAP)-tagged Sgo1 (AM8975), Sgo1-100 (AM8971), Sgo1-700 (AM8969), Sgo1-3A (AM8973) or no TAP (AM3513) and carrying <italic>IPL1-6HA</italic> were treated with nocodazole for 2 hr before cross-linking with DSP. Extracts were prepared as described in ‘Materials and methods’, incubated with IgG-coupled beads and immunoprecipitates were analyzed by immunoblot using the indicated antibodies. (<bold>B</bold>) Degradation of Sgo1 using the auxin-inducible degron system. Representative anti-Sgo1 immunoblot for the experiments in (<bold>C</bold> and <bold>D</bold>) showing that NAA treatment leads to Sgo1 degradation. Anti-Pgk1 immunoblot is shown as a loading control. See below for experimental conditions. (<bold>C</bold>) Ipl1 is initially recruited to centromeres in the absence of Sgo1. Wild-type (AM3513) and <italic>SGO1-aid</italic> (AM9619) cells carrying <italic>IPL1-6HA</italic> were released from a G1 block in the presence of auxin (NAA) and samples harvested at 15 min intervals for measurement of Ipl1-6HA levels by anti-HA ChIP-qPCR. Also shown is a G1 sample from cells lacking <italic>IPL1-6HA</italic> (AM1176; no tag). The percentages of metaphase and anaphase spindles after anti-tubulin immunoflurescence were scored as a marker of cell cycle progression and anti-Sgo1 immunoblot confirmed Sgo1-aid degradation (shown in <bold>B</bold>). A representative experiment is shown from a total of three repeats. (<bold>D</bold>) Wild-type (AM3513) and <italic>SGO1-aid</italic> (AM9619) cells carrying <italic>IPL1-6HA</italic> together with a no tag control were arrested in G1 by alpha factor treatment and then released into medium containing NAA and nocodazole for 3 hr before harvesting for ChIP. Levels of Ipl1-6HA were determined at <italic>CEN4</italic> and a pericentromeric site (<italic>PERICEN4</italic>) by qPCR and the mean of three experimental repeats is shown with bars representing standard error (*p<0.05, paired <italic>t</italic> test). (<bold>E</bold>) Ipl1-6HA levels at <italic>CEN4</italic> measured by anti-HA ChIP-qPCR in wild type (AM3513), <italic>sgo1-100</italic> (AM9090), <italic>sgo1-700</italic> (AM9082) and <italic>sgo1-3A</italic> (AM9076) after treating directly with nocodazole for 3 hr are shown, together with a no tag (AM1176) control, treated in the same way. The mean of three independent repeats is shown with bars representing standard error (*p<0.05, paired <italic>t</italic> test). Note that levels of Ipl1-6HA at <italic>CEN4</italic> were consistently higher in experiments where cells were directly treated with nocodazole, compared to those treated upon release from G1 (compare <bold>E</bold> with <bold>D</bold>). Presumably those cells in the population that are already in mitosis upon nocodazole addition experience an extended arrest during which Ipl1 is continually recruited.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.006">http://dx.doi.org/10.7554/eLife.01374.006</ext-link></p></caption><graphic xlink:href="elife01374f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.007</object-id><label>Figure 3—figure supplement 1.</label><caption><title>The Haspin homologs, Alk1 and Alk2, are not required for the initial recruitment of Ipl1 to centromeres.</title><p>Wild-type (AM3513), <italic>alk1Δ alk2Δ</italic> (AM10612) and <italic>alk1Δ alk2Δ SGO1-aid</italic> (AM10393) cells carrying <italic>IPl1-6HA</italic> as well as a no tag control (AM1176) were arrested in G1 by treatment with alpha factor. Samples were extracted for anti-HA ChIP at the indicated intervals after release from G1. The levels of Ipl1-6HA at <italic>CEN4</italic> were measured by ChIP-qPCR.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.007">http://dx.doi.org/10.7554/eLife.01374.007</ext-link></p></caption><graphic xlink:href="elife01374fs002"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.008</object-id><label>Figure 3—figure supplement 2.</label><caption><title>Defective biorientation in <italic>ipl1-as</italic> mutants.</title><p>Biorientation assay showing <italic>CEN4-GFP</italic> separation in wild-type (AM4643) and <italic>ipl1-as</italic> (AM10374) cells carrying SPB (<italic>SPC42-tdTomato</italic>) markers. Cells were released from G1 into nocodazole and NAPP1, before nocodazole was washed out and GFP foci were scored in the metaphase-arrested cells as in <xref ref-type="fig" rid="fig1">Figure 1C</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.008">http://dx.doi.org/10.7554/eLife.01374.008</ext-link></p></caption><graphic xlink:href="elife01374fs003"/></fig></fig-group></p></sec><sec id="s2-4"><title>Sgo1 associates with condensin and recruits it to the pericentromere</title><p>As an unbiased approach to isolate binding partners that might contribute to biorientation we purified Sgo1 from cycling cells or cells arrested in mitosis by microtubule perturbation (using a cold-sensitive tubulin mutant; <italic>tub2-401</italic> [<xref ref-type="bibr" rid="bib21">Huffaker et al., 1988</xref>]). To increase the probability of capturing transient interactions, we pre-treated cells with the cross-linking agent dithiobis(succunimidylpropionate) (DSP) before preparing extracts and immune-precipitating Sgo1-TAP. Associated proteins were identified by mass spectrometry (<xref ref-type="fig" rid="fig4">Figure 4A,B</xref>; <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>). Although subunits of PP2A co-purified with Sgo1, we did not detect peptides of the CPC. Interestingly, we identified four out of five subunits of the condensin complex co-purifying with Sgo1 (<xref ref-type="fig" rid="fig4">Figure 4A,B</xref>; <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>). Co-immunoprecipitation of the Ycs4 and Brn1 subunits of condensin (<xref ref-type="fig" rid="fig4">Figure 4C</xref>) with Sgo1-TAP confirmed the Sgo1-condensin interaction (<xref ref-type="fig" rid="fig4">Figure 4D,E</xref>). We confirmed that the Sgo1-Ycs4 interaction is not dependent on either DNA or the pre-treatment of cells with cross-linking agent (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). This suggests that Sgo1 and condensin form a complex independently of their association with the pericentromeric chromatin. Therefore, Sgo1 associates with three protein complexes during mitosis: PP2A, CPC, and condensin.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.009</object-id><label>Figure 4.</label><caption><title>Sgo1 interacts with condensin and recruits it to the pericentromere.</title><p>(<bold>A</bold> and <bold>B</bold>) Condensin and PP2A co-purify with Sgo1. Sgo1 was purified from wild-type or protease-deficient cells that were (<bold>A</bold>) cycling or (<bold>B</bold>) arrested in mitosis using the cold-sensitive tubulin allele <italic>tub2-401</italic> as described in ‘Materials and methods’. Comparable strains lacking TAP were used as a control for non-specific association with the beads. All cells were treated with the cross-linker, DSP, before harvesting and preparing extracts as described in ‘Materials and methods’. Extracts were incubated with IgG-coupled beads and immunoprecipitates were visualized on silver-stained SDS-PAGE gels. The table shows the number of peptides of subunits of the PP2A and condensin complexes that were identified in the Sgo1-TAP purifications after mass spectrometry. The full list of identified proteins is given in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>. Strains used in (<bold>A</bold>) were AM7509 (<italic>SGO1-SZZ(TAP)</italic>, AM1176 (no tag), AM8226 (protease-deficient, <italic>SGO1-SZZ(TAP</italic>) and AM8184 (protease-deficient, no tag). Strains used in (<bold>B</bold>) were AM8456 (<italic>tub2-401 SGO1-SZZ(TAP)</italic>, AM2730 (<italic>tub2-401,</italic> no tag), AM8455 (protease-deficient, <italic>tub2-401 SGO1-SZZ(TAP</italic>) and AM8259 (protease-deficient, <italic>tub401</italic> no tag. (<bold>C</bold>) A schematic diagram illustrating the composition of budding yeast condensin is shown. (<bold>D</bold> and <bold>E</bold>) Cells carrying either <italic>YCS4-6HA</italic> (AM9138) (<bold>D</bold>) or <italic>BRN1-6HA</italic> (AM9266) (<bold>E</bold>) and <italic>SGO1-SZZ(TAP)</italic> or no TAP (AM5705 and AM5708) were arrested in nocodazole for 2 hr before treating with DSP. Extracts were incubated with IgG-coupled beads and immunoprecipitates were analyzed with the indicated antibodies by immunoblot. (<bold>F</bold>–<bold>H</bold>) Sgo1 is required for Brn1 association with the pericentromere. The genome-wide localization of Brn1-6HA was determined in wild-type (AM5708) and <italic>sgo1Δ</italic> (AM8834) cells by anti-HA ChIP followed by high throughput sequencing (ChIP-Seq) after arresting in mitosis by treating with nocodazole for 3 hr. (<bold>F</bold>) Brn1 enrichment along chromosome V along with a magnification of a 50 kb region including the centromere is shown. The number of reads at each position was normalized to the total number of reads for each sample (RPM: reads per million) and shown in the Integrated Genome Viewer from the Broad Institute (<xref ref-type="bibr" rid="bib48">Robinson et al., 2011</xref>). (<bold>G</bold>) The number of reads at coordinates corresponding to the rDNA region on chromosome XII is shown for wild-type and <italic>sgo1Δ</italic> anti-HA ChIP samples normalized to the total number of reads for each sample. Brn1 enrichment at the rDNA is similar in wild-type and <italic>sgo1Δ</italic> cells. (<bold>H</bold>) Brn1 enrichment in a 50 kb domain surrounding all 16 budding yeast centromeres is shown for wild-type and <italic>sgo1Δ</italic> cells. For both wild type and <italic>sgo1Δ</italic>, the ratio of the local maximum in a 100 bp window for ChIP sample/input is calculated at the indicated distance from the centromere for all 16 chromosomes. Box plot of maximum count value for 100 bp windows for 25 kb on both sides of each centromere is shown to give a composite view of all 16 pericentromeres. (<bold>I</bold> and <bold>J</bold>) Recruitment of Brn1 to centromeres occurs coincident with, and is dependent on, Sgo1. Wild-type cells carrying <italic>SGO1-9MYC</italic> and <italic>BRN1-6HA</italic> (AM9622) as well as <italic>sgo1Δ</italic> cells (AM8834) carrying <italic>BRN1-6HA</italic> were arrested in G1 using alpha factor. Samples were extracted at 15 min intervals after release from G1 for anti-HA and anti-Myc ChIP and tubulin immunofluorescence. (<bold>I</bold>) The levels of Brn1-6HA and Sgo1-9Myc at <italic>CEN4</italic> were measured at the indicated timepoints by anti-HA and anti-Myc ChIP-qPCR, respectively. Also shown is a G1 sample from cells lacking <italic>BRN1-6HA</italic> (no tag; AM1176). (<bold>J</bold>) The percentages of metaphase and anaphase spindles after anti-tubulin immunofluorescence were scored as a marker of cell cycle progression. Shown is a representative experiment from three repeats. (<bold>K</bold>) Schematic diagram illustrating the protein complexes (PP2A, condensin, CPC) recruited to the pericentromere by shugoshin.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.009">http://dx.doi.org/10.7554/eLife.01374.009</ext-link></p></caption><graphic xlink:href="elife01374f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.010</object-id><label>Figure 4—figure supplement 1.</label><caption><title>The Sgo1-condensin interaction is not dependent on DNA or treatment with the cross-linking agent, DSP.</title><p>Cells carrying <italic>YCS4-6HA</italic> and <italic>SGO1-SZZ(TAP)</italic> (AM9138) or no TAP (AM5705) were arrested in mitosis by treatment with nocodazole for 2 hr. Cultures were either harvested and directly drop-frozen (−DSP) or treated with DSP prior to drop freezing (+DSP) as described in ‘Materials and methods’. Extracts were either treated with 25 U of the DNA degrading agent, benzonase (+benzonase) and rotated at room temperature or held on ice (−benzonase) for 30 min. All samples were incubated with IgG-coupled beads and analyzed by immunoblot with the indicated antibodies.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.010">http://dx.doi.org/10.7554/eLife.01374.010</ext-link></p></caption><graphic xlink:href="elife01374fs004"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.011</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Removal of PCR duplicates does not alter the conclusion that Sgo1 is important for Brn1 enrichment in the pericentromere.</title><p>This is the same analysis as in <xref ref-type="fig" rid="fig3">Figure 3H</xref>, except that only unique reads are included (therefore eliminating duplicate samples generated during the PCR amplification step). Both methods of analysis lead to the conclusion that Brn1 levels in the pericentromere are greatly reduced in <italic>sgo1Δ</italic> cells. Note that the centromeric peak may be an artifact as centromeric sequences were over-represented in the <italic>sgo1Δ</italic> sample compared to wild type (where they were under-represented compared to the rest of the genome). Potentially, altered pericentromeric structure in <italic>sgo1Δ</italic> cells could enable more efficient recovery of these sequences during the purification procedure.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.011">http://dx.doi.org/10.7554/eLife.01374.011</ext-link></p></caption><graphic xlink:href="elife01374fs005"/></fig><fig id="fig4s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.012</object-id><label>Figure 4—figure supplement 3.</label><caption><title>Brn1 is reduced around all 16 individual centromeres in <italic>sgo1Δ</italic> cells.</title><p>Brn1 enrichment in a 20 kb region surrounding all 16 individual centromeres in wild type and <italic>sgo1Δ</italic> cells is shown for the experiment described in <xref ref-type="fig" rid="fig4">Figure 4F–H</xref>. All reads were included in this analysis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.012">http://dx.doi.org/10.7554/eLife.01374.012</ext-link></p></caption><graphic xlink:href="elife01374fs006"/></fig></fig-group></p><p>Condensin complexes structurally organize chromosomes and enable their efficient segregation, though how they do so remains unclear (<xref ref-type="bibr" rid="bib8">Cuylen and Haering, 2011</xref>; <xref ref-type="bibr" rid="bib19">Hirano, 2012</xref>). In budding yeast, condensin is most highly enriched in the rDNA and at each pericentromere (<xref ref-type="bibr" rid="bib10">D’Ambrosio et al., 2008</xref>). Condensin recruitment to the rDNA depends on monopolin (Csm1/Lrs4) (<xref ref-type="bibr" rid="bib10">D’Ambrosio et al., 2008</xref>). Fission yeast monopolin (Pcs1/Mde4) recruits condensin to centromeres where it prevents merotely (attachment of a single kinetochore to microtubules from both poles) (<xref ref-type="bibr" rid="bib15">Gregan et al., 2007</xref>; <xref ref-type="bibr" rid="bib51">Tada et al., 2011</xref>), unlike budding yeast condensin which is recruited to centromeres independently of monopolin subunit Lrs4 (<xref ref-type="bibr" rid="bib3">Brito et al., 2010</xref>). How condensin is recruited to the pericentromere remains unknown. To test whether the pericentromeric localization of condensin depends on Sgo1 we examined the association of the Brn1 condensin subunit genome wide using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) in wild-type and <italic>sgo1Δ</italic> cells arrested in mitosis by treatment with nocodazole. Although the pattern of reads along chromosome arms and mapping to the rDNA was similar in wild-type and <italic>sgo1Δ</italic> cells (<xref ref-type="fig" rid="fig4">Figure 4F,G</xref>), we observed a clear reduction in pericentromeric levels of Brn1 in <italic>sgo1Δ</italic> cells, although peaks of variable height remained at some, but not all core centromeres (<xref ref-type="fig" rid="fig4">Figure 4H</xref>, <xref ref-type="fig" rid="fig4s2 fig4s3">Figure 4—figure supplement 2, 3</xref>). Consideration of all 16 centromeres collectively revealed that in wild-type cells, condensin is enriched on average throughout an approximately 15 kb domain on either side of the centromere and that this enrichment is lost in <italic>sgo1Δ</italic> cells (<xref ref-type="fig" rid="fig4">Figure 4H</xref>). We conclude that Sgo1 is required for condensin association throughout the pericentromere.</p><p>Sgo1 is absent in G1 and produced only upon cell cycle entry (<xref ref-type="bibr" rid="bib37">Marston et al., 2004</xref>). Although condensin is present in G1 cells and localized to the nucleolus, it begins to co-localize with kinetochores only upon cell cycle entry (<xref ref-type="bibr" rid="bib1">Bachellier-Bassi et al., 2008</xref>). We found that recruitment of condensin to a centromere-proximal site occurs coincidently with, and depends on, Sgo1 (<xref ref-type="fig" rid="fig4">Figure 4I,J</xref>). Therefore, in addition to controlling the centromere localization of PP2A-Rts1 and the CPC, Sgo1 recruits condensin to the pericentromere (<xref ref-type="fig" rid="fig4">Figure 4K</xref>).</p></sec><sec id="s2-5"><title>Hierarchical assembly of pericentromeric factors</title><p>Like condensin and shugoshin (<xref ref-type="bibr" rid="bib10">D′Ambrosio et al., 2008</xref>; <xref ref-type="bibr" rid="bib29">Kiburz et al., 2005</xref>; <xref ref-type="fig" rid="fig4">Figure 4</xref>), cohesin is highly enriched throughout the pericentromere (<xref ref-type="bibr" rid="bib14">Glynn et al., 2004</xref>; <xref ref-type="bibr" rid="bib33">Lengronne et al., 2004</xref>; <xref ref-type="bibr" rid="bib58">Weber et al., 2004</xref>). What is the relationship between cohesin, condensin, and shugoshin? Although shugoshins play important roles in regulating the timing of cohesion loss during meiosis and mammalian mitosis (see <xref ref-type="bibr" rid="bib7">Clift and Marston, 2011</xref>; <xref ref-type="bibr" rid="bib16">Gutiérrez-Caballero et al., 2012</xref> for reviews), this is not the case in budding yeast mitosis. Budding yeast <italic>sgo1</italic> mutants are not defective in cohesion (<xref ref-type="bibr" rid="bib23">Indjeian and Murray, 2005</xref>; <xref ref-type="bibr" rid="bib37">Marston et al., 2004</xref>) and cohesin is normally localized to chromosomes (<xref ref-type="bibr" rid="bib29">Kiburz et al., 2005</xref>). We confirmed the proper association of cohesin in <italic>sgo1Δ</italic> cells arrested in mitosis by ChIP-Seq of its HA-tagged Scc1 subunit (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>). The profile of Scc1 association along chromosome V (<xref ref-type="fig" rid="fig5">Figure 5A</xref>) and surrounding all 16 budding yeast centromeres in <italic>sgo1Δ</italic> cells (<xref ref-type="fig" rid="fig5">Figure 5B</xref>, <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>) is indistinguishable from that of wild-type cells. ChIP-qPCR analysis confirmed that the levels of Scc1 cohesin subunit are similar at two tested centromeres in wild-type and <italic>sgo1Δ</italic> cells (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>). We conclude that Sgo1 is not required for cohesin localization at centromeres, pericentromeres or along chromosomes.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.013</object-id><label>Figure 5.</label><caption><title>Sgo1 is not required for cohesin association with chromosomes.</title><p>Wild-type (AM1145) and <italic>sgo1Δ</italic> (AM1474) cells carrying <italic>SCC1-6HA</italic> were arrested in mitosis by treatment with nocodazole for 3 hr. Samples were harvested, anti-HA ChIP was performed and both input and IP samples were sequenced for both strains. (<bold>A</bold>) Scc1-6HA enrichment along chromosome V along with a magnification of a 50 kb region including the centromere is shown. The number of reads at each position were normalized to the total number of reads for each sample and displayed using the Integrated Genome Viewer from the Broad Institute (<xref ref-type="bibr" rid="bib48">Robinson et al., 2011</xref>). (<bold>B</bold>) Box plot of maximum count value for 100 bp windows for 25 kb on both sides of each centromere is shown to give a composite view of all 16 pericentromeres. All reads are included. (<bold>C</bold>) Schematic diagram indicating hierarchy of factors required for condensin association with the pericentromere.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.013">http://dx.doi.org/10.7554/eLife.01374.013</ext-link></p></caption><graphic xlink:href="elife01374f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.014</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Scc1 association with all 16 centromeres is unaffected by <italic>SGO1</italic> deletion.</title><p>Scc1 enrichment in a 20 kb region surrounding all 16 individual centromeres in wild type and <italic>sgo1Δ</italic> cells is shown from the experiment in <xref ref-type="fig" rid="fig5">Figure 5</xref>. All reads were included in this analysis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.014">http://dx.doi.org/10.7554/eLife.01374.014</ext-link></p></caption><graphic xlink:href="elife01374fs007"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.015</object-id><label>Figure 5—figure supplement 2.</label><caption><title>ChIP-qPCR analysis showing Scc1-6HA levels at the centromere and pericentromere.</title><p>Wild type and <italic>sgo1Δ</italic> cells were treated as described in <xref ref-type="fig" rid="fig5">Figure 5</xref> except that ChIP samples were analyzed by qPCR using primer sets at <italic>CEN4</italic>, <italic>CEN5</italic> and a site in the pericentromere of chromosome IV. The mean of three independent experiments is shown with bars indicating standard error.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.015">http://dx.doi.org/10.7554/eLife.01374.015</ext-link></p></caption><graphic xlink:href="elife01374fs008"/></fig><fig id="fig5s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.016</object-id><label>Figure 5—figure supplement 3.</label><caption><title>Cohesin is required for normal Sgo1 association with the pericentromere.</title><p>Wild type (AM906) and <italic>pMET-</italic>SCC1 (AM6673) strains carrying <italic>SGO1-6HA</italic> together with no tag wild type (AM1176) and <italic>pMET-SCC1</italic> controls (AM1599) were arrested in G1 using alpha factor in the presence of methionine (to deplete Scc1). Strains were released into medium containing nocodazole and methionine for 3 hr and levels of Sgo1-6HA at the indicated sites were measured by ChIP-qPCR.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.016">http://dx.doi.org/10.7554/eLife.01374.016</ext-link></p></caption><graphic xlink:href="elife01374fs009"/></fig><fig id="fig5s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.017</object-id><label>Figure 5—figure supplement 4.</label><caption><title>Cohesin loading factors, but not monopolin, are important for proper condensin association with the centromere.</title><p>Wild type (AM5708), <italic>SCC2-aid</italic> (AM8918), <italic>chl4Δ</italic> (AM8885), <italic>iml3Δ</italic> (AM5710) and <italic>lrs4Δ</italic> (AM9766) strains carrying <italic>BRN1-6HA</italic>, as well as a no tag control (AM1176) were treated with nocodazole and NAA (to degrade Scc2-aid) for 3 hr before harvesting for ChIP and measuring the levels of Brn1-6HA at <italic>CEN4</italic> by ChIP-qPCR.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.017">http://dx.doi.org/10.7554/eLife.01374.017</ext-link></p></caption><graphic xlink:href="elife01374fs010"/></fig></fig-group></p><p>Conversely, we found that cohesin, and factors required for its loading, are required for the proper association of Sgo1 with the centromere and pericentromere (<xref ref-type="fig" rid="fig5">Figure 5</xref>, <xref ref-type="fig" rid="fig5s3 fig5s4">Figure 5—figure supplements 3 and 4</xref>). Depletion of the Scc1 subunit of cohesin led to a great reduction in the pericentromeric levels of Sgo1 with only low levels remaining at the centromere itself (<xref ref-type="fig" rid="fig5s3">Figure 5—figure supplement 3</xref>). These findings suggest that cohesin promotes Sgo1 association with the pericentromere, which, in turn, recruits condensin, implying an indirect role for cohesin in localizing condensin (through Sgo1) (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). Consistent with this idea, proper Brn1 association with the centromere in mitosis requires both the Scc2 protein that is required for cohesin loading onto chromosomes and subunits of the kinetochore (Iml3/Chl4) that target Scc2 to centromeres (<xref ref-type="bibr" rid="bib5">Ciosk et al., 2000</xref>; <xref ref-type="bibr" rid="bib13">Fernius et al., 2013</xref>; <xref ref-type="bibr" rid="bib10">D′Ambrosio et al., 2008</xref>; <xref ref-type="bibr" rid="bib40">Ng et al., 2009</xref>; <xref ref-type="fig" rid="fig5s4">Figure 5—figure supplement 4</xref>), However, unlike in fission yeast, the monopolin subunit Lrs4 is not required for Brn1 association with the centromere (<xref ref-type="bibr" rid="bib3">Brito et al., 2010</xref>; <xref ref-type="fig" rid="fig5s4">Figure 5—figure supplement 4</xref>). Overall, we suggest a hierarchy of assembly in which the combined effects of Bub1 kinase and cohesin concentrate shugoshin in the pericentromere, which in turn recruits condensin (<xref ref-type="bibr" rid="bib11">Fernius and Hardwick, 2007</xref>; <xref ref-type="bibr" rid="bib26">Kawashima et al., 2010</xref>; <xref ref-type="bibr" rid="bib61">Yamagishi et al., 2010</xref>; <xref ref-type="bibr" rid="bib62">2008</xref>) (<xref ref-type="fig" rid="fig5">Figure 5C</xref>).</p></sec><sec id="s2-6"><title>Sgo1 is sufficient for condensin recruitment</title><p>Next, we asked whether Sgo1 was sufficient to recruit condensin to chromosomes. Overproduction of Sgo1, which is known to enable its association with chromosome arms and delay cells in metaphase (<xref ref-type="bibr" rid="bib6">Clift et al., 2009</xref>; <xref ref-type="fig" rid="fig6">Figure 6A</xref>) led to increased levels of condensin at centromere, pericentromere and chromosome arm sites (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). <italic>SGO1</italic> overexpression also increased Brn1 association with the centromere and pericentromere in cells arrested in mitosis by nocodazole treatment (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>), indicating that increased enrichment of Brn1 in <italic>SGO1</italic>-overexpressing cells was not purely a consequence of the metaphase arrest. As a more direct test of the ability of Sgo1 to bring condensin to chromosomes, we produced a Sgo1-GFP-TetR fusion protein in cells carrying <italic>tetO</italic> repeats integrated on a chromosomal arm. In the absence, but not the presence of doxycycline, Sgo1-GFP-TetR is expected to bind to the ectopic site and recruit its binding partners (<xref ref-type="fig" rid="fig6">Figure 6C</xref>). Indeed we found that tethered Sgo1-GFP-TetR efficiently recruited the condensin subunit Brn1, the PP2A subunit, Rts1, and to a lesser extent the CPC subunit, Ipl1 to a site directly adjacent to the <italic>tetOs</italic> (<xref ref-type="fig" rid="fig6">Figure 6D,F</xref>; ∼50 bp R ectopic site), although centromeric levels were not affected (<xref ref-type="fig" rid="fig6">Figure 6E,G</xref>). The recruitment of these proteins to a site ∼800 bp to the left of the tethering site was much less efficient (<xref ref-type="fig" rid="fig6">Figure 6D,F</xref>), suggesting that recruitment occurs through direct binding to Sgo1, rather than an effect of Sgo1 on the surrounding chromatin. Taken together, these results show that Sgo1 is both necessary and sufficient for condensin recruitment.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.018</object-id><label>Figure 6.</label><caption><title>Sgo1 is sufficient for condensin recruitment.</title><p>(<bold>A</bold> and <bold>B</bold>) Sgo1 overproduction leads to increased levels of Brn1 on chromosomes. Cells carrying <italic>BRN1-6HA</italic> and that were otherwise wild type (AM5708) or carrying <italic>pGAL-SGO1</italic> (AM10859) integrated an ectopic locus<italic>,</italic> were arrested in G1 in rich medium containing raffinose and adenine using alpha factor (YEP + R + A). After 2 hr 30 min, galactose (2%) was added to induce <italic>SGO1</italic> overexpression and 30 min later, cells were released from G1. Samples were collected at the indicated times after release from G1 for analysis of cell cycle progression by scoring spindle morphology after anti-tubulin immunofluorescence (<bold>A</bold>) or for measurement of Brn1 levels by anti-HA ChIP-qPCR (<bold>B</bold>). Sites analyzed were at <italic>CEN4</italic>, a pericentromeric site or a chromosomal arm site on chromosome IV. A representative experiment from a total of three independent repeats is shown. (<bold>C</bold>–<bold>G</bold>) Tethered Sgo1 at an ectopic site recruits Brn1, Rts1 and Ipl1. (<bold>C</bold>) Schematic diagram showing the expected effects of doxycycline at the ectopic site and at <italic>CEN4</italic>, as well as the locations of primer sets used for qPCR (yellow stars). Primer sets used were ∼800 bp left of the tethering site, ∼50 bp right of the tethering site and at <italic>CEN4</italic>. (<bold>D</bold>–<bold>G</bold>) Strains carrying Sgo1-TetR-GFP and <italic>tetOs</italic> integrated at the <italic>HIS3</italic> locus were arrested in nocodazole for 3 hr either in the presence (+DOX) or absence (−DOX) of doxycycline and harvested for ChIP-qPCR. (<bold>D</bold> and <bold>E</bold>) Anti-HA ChIP was performed on <italic>SGO1-TetR-GFP HIS3::tetOs</italic> strains carrying either <italic>BRN1-6HA</italic> (AM9847), <italic>IPL1-6HA</italic> (AM9940) or no tag (AM9655) and levels of Brn1-6HA and Ipl1-6HA were measured by qPCR at the indicated sites adjacent to the ectopic site (<bold>D</bold>) or at <italic>CEN4</italic> (<bold>E</bold>). (<bold>F</bold> and <bold>G</bold>) Anti-PK ChIP was performed on <italic>SGO1-TetR-GFP HIS3::tetOs</italic> strains carrying Rts1-3PK (AM9783) or no tag (AM9655) and levels of Rts1-3PK were measured by qPCR at the indicated sites adjacent to the ectopic site (<bold>F</bold>) or at <italic>CEN4</italic> (<bold>G</bold>). In (<bold>D</bold>–<bold>G)</bold>, the mean of three or four experimental repeats is shown with bars representing standard error (*p<0.05, unpaired <italic>t</italic> test).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.018">http://dx.doi.org/10.7554/eLife.01374.018</ext-link></p></caption><graphic xlink:href="elife01374f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.019</object-id><label>Figure 6—figure supplement 1.</label><caption><title><italic>SGO1</italic> overexpression in metaphase-arrested cells increases Brn1 association with the centromere.</title><p>Strains AM5708 (<italic>BRN1-6HA</italic>), AM10859 (<italic>BRN1-6HA pGAL-SGO1</italic>) were pre-cultured in YEP + R + Ade medium before supplementing with nocodazole and galactose (2 hr) for 3 hr and then harvesting for anti-HA ChIP. The levels of Brn1-6HA were measured at the indicated sites by qPCR.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.019">http://dx.doi.org/10.7554/eLife.01374.019</ext-link></p></caption><graphic xlink:href="elife01374fs011"/></fig></fig-group></p></sec><sec id="s2-7"><title>Pericentromeric condensin contributes to error correction, independently of aurora B recruitment</title><p>Biorientation is achieved both because of a bias for sister kinetochores to be captured by microtubules from opposite poles and owing to error correction which destabilizes mono-oriented kinetochores, allowing a further opportunity for biorientation to occur (<xref ref-type="bibr" rid="bib52">Tanaka, 2010</xref>). We tested the requirement of condensin for error correction using a conditional degron version of its Ycs5 subunit by monitoring <italic>CEN4-GFP</italic> separation in metaphase-arrested cells after microtubule depolymerization (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). In cells where degradation of condensin’s Ycs5 subunit (<italic>YCS5-aid</italic>) was induced, <italic>CEN4-GFP</italic> separation was delayed compared to wild-type cells, albeit not to the extent of <italic>sgo1Δ</italic> cells (<xref ref-type="fig" rid="fig7">Figure 7A</xref>), suggesting that condensin facilitates biorientation. To further test whether the error correction process operates normally we developed a live cell microfluidics assay to allow biorientation to be observed directly as microtubules were allowed to reform (<xref ref-type="fig" rid="fig7">Figure 7B,C</xref>; <xref ref-type="other" rid="video1 video2">Videos 1 and 2</xref>). Overall, as expected, and consistent with a biorientation defect, the number of frames in which cells with split <italic>CEN4-GFP</italic> foci were observed was reduced in cells lacking <italic>SGO1</italic>, where <italic>IPL1</italic> was inhibited (<italic>ipl1-as</italic> with NAPP1) or Ycs5 was degraded (Ycs5-aid with NAA) compared to wild-type cells (<xref ref-type="fig" rid="fig7">Figure 7D</xref>). However, the distance between separated <italic>CEN4-GFP</italic> foci was comparable in all strains, suggesting that kinetochore-microtubule attachments, spindle tension, and cohesion are all functional and only the orientation of attachment is defective in <italic>sgo1Δ</italic>, <italic>YCS5-aid</italic> and <italic>ipl1-as</italic> cells (<xref ref-type="fig" rid="fig7">Figure 7E</xref>). Furthermore, consistent with a failure to properly biorient chromosomes, unseparated <italic>CEN4-GFP</italic> tended to be closer to the SPB in <italic>sgo1Δ</italic>, <italic>YCS5-aid</italic> and <italic>ipl1-</italic>as cells than in wild-type cells (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). We used the switch between one or two <italic>CEN4-GFP</italic> foci as a measure of kinetochore reorientation during error correction (<xref ref-type="fig" rid="fig7">Figure 7F</xref>). The average frequency of switching between 1 and 2 GFP foci was significantly reduced in <italic>sgo1Δ</italic> and <italic>ipl1-as</italic> cells, as expected (<xref ref-type="fig" rid="fig7">Figure 7F</xref>). We further observed a more modest reduction in switching in <italic>YCS5-aid</italic> cells, indicating that condensin contributes to proper error correction (<xref ref-type="fig" rid="fig7">Figure 7F</xref>). The role of condensin in error correction cannot be to localize Ipl1, as we found that Ipl1 maintenance at kinetochores requires Sgo1 (<xref ref-type="fig" rid="fig3">Figure 3D</xref>), but not Ycs5 (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). Rather, we speculate that condensin shapes the pericentromere to place sister kinetochores in a rigid back-to-back orientation that provides the framework for tension-sensing.<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.020</object-id><label>Figure 7.</label><caption><title>Condensin facilitates effective error correction.</title><p>(<bold>A</bold>) Sister kinetochore biorientation is defective after nocodazole washout in cells lacking condensin. Strains carrying SPB (Spc42-tdTomato) and <italic>CEN4</italic> (<italic>CEN4-GFP</italic>) markers were released from a G1 arrest into nocodazole and arrested in metaphase by <italic>CDC20</italic> depletion. After 3 hr, nocodazole was washed out (<italic>t =</italic> 0) and <italic>CEN4-</italic>GFP separation scored at the indicated intervals as shown in <xref ref-type="fig" rid="fig1">Figure 1C</xref>. Error bars represent standard deviation (wild type and <italic>YCS5-aid</italic>; <italic>n =</italic> 3) or range (<italic>sgo1Δ</italic>; <italic>n =</italic> 2; reproduced from <xref ref-type="fig" rid="fig1">Figure 1E</xref>). A representative anti-aid immunoblot is shown to confirm Ycs5 degradation upon NAA addition. Samples were taken in G1 (−NAA) and 120 min after release (+NAA). Anti-Kar2 immunoblot is shown as a loading control. (<bold>B</bold>–<bold>F</bold>) Condensin contributes to efficient error correction. (<bold>B</bold>) Scheme of the live single-cell microfluidics experiment. Wild-type (AM4643), <italic>sgo1Δ</italic> (AM6117), <italic>YCS5-aid</italic> (AM9038) and <italic>ipl1-as</italic> (AM10374) cells carrying <italic>CEN4</italic> (<italic>CEN4-GFP</italic>) and SPB markers (<italic>SPC42-tdTomato</italic>) were released from a G1 arrest into nocodazole, NAA and NAPP1 and arrested in metaphase by <italic>CDC20</italic> depletion. After 30 min, nocodazole was washed out. When the majority of cells had 2 SPBs (∼1 hr 30 min later), we began imaging and a total of 21 frames were grabbed at approximately 74 s intervals. (<bold>C</bold>) Representative images for wild type and <italic>ipl1-as</italic> are shown. Numbers indicate time (s) each frame was grabbed and asterisks indicate a change in GFP dot number compared to the previous frame. (<bold>D</bold>) The overall percentage of separated <italic>CEN4-GFP</italic> foci was determined for cells with two visible SPBs from all frames combined. (<bold>E</bold>) The distance between <italic>CEN4-GFP</italic> foci was measured in cells with separated foci. Box boundaries represent the upper and lower quartiles, respectively. The red cross indicates the mean, the horizontal line indicates the median and error bars show the maximum and minimum values observed. <italic>n =</italic> 396 (wild type), 108 (<italic>sgo1Δ</italic>), 267 (<italic>YCS5-aid</italic>) and 154 (<italic>ipl1-as</italic>). (<bold>F</bold>) The observed frequency of switching between one and two GFP foci was calculated for cells with 2 SPBs. A student <italic>t</italic> test was used to obtain p values. (<bold>G</bold>) Ycs5 is not required for Ipl1 association with the centromere. The levels of 6HA-tagged Ipl1 in wild-type (AM3513) and <italic>YCS5-aid</italic> (AM10334) cells, grown in the presence of NAA and nocodazole for 3 hr, were measured at <italic>CEN4</italic> by anti-HA ChIP-qPCR and compared to a no tag control (AM1176). The mean of three independent experiments is shown with bars representing standard error. This is the same experiment as shown in <xref ref-type="fig" rid="fig3">Figure 3D</xref> and the wild-type data is reproduced for comparison.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.020">http://dx.doi.org/10.7554/eLife.01374.020</ext-link></p></caption><graphic xlink:href="elife01374f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.021</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Deletion of <italic>SGO1</italic> impairs biorientation rather than centromere cohesion.</title><p>The distance from <italic>CEN4-GFP</italic> to the nearest <italic>SPC42-tdTomato</italic> focus was measured for cells with just one visible <italic>CEN4-GFP</italic> foci from the experiment shown in <xref ref-type="fig" rid="fig7">Figure 7C–F</xref>. The fraction of cells with a <italic>CEN4-GFP</italic> to SPB distance greater or less than the median value for wild type (0.865 mm) is plotted for wild type, <italic>sgo1Δ</italic>, <italic>YCS5-aid</italic> and <italic>ipl1-as</italic> cells. p values were obtained using a chi square test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.021">http://dx.doi.org/10.7554/eLife.01374.021</ext-link></p></caption><graphic xlink:href="elife01374fs012"/></fig></fig-group><media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="avi" mimetype="video" xlink:href="elife01374v001.avi"><object-id pub-id-type="doi">10.7554/eLife.01374.022</object-id><label>Video 1.</label><caption><title>Example video of a wild-type cell in the error correction assay.</title><p>The video corresponds to the image gallery in <xref ref-type="fig" rid="fig5">Figure 5C</xref> (upper panel).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.022">http://dx.doi.org/10.7554/eLife.01374.022</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video2" mime-subtype="avi" mimetype="video" xlink:href="elife01374v002.avi"><object-id pub-id-type="doi">10.7554/eLife.01374.023</object-id><label>Video 2.</label><caption><title>Example video of an ipl1-as cell in the error correction assay.</title><p>The video corresponds to the image gallery in <xref ref-type="fig" rid="fig5">Figure 5C</xref> (lower panel).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.023">http://dx.doi.org/10.7554/eLife.01374.023</ext-link></p></caption></media></p></sec><sec id="s2-8"><title>Shugoshin and pericentromeric condensin bias sister kinetochores to capture by microtubules from opposite poles</title><p>Although it has been recognized that sister kinetochores are intrinsically biased towards capture by microtubules from opposite poles (<xref ref-type="bibr" rid="bib22">Indjeian and Murray, 2007</xref>), the factors required were not identified. The molecular basis of the bias towards sister kinetochore biorientation has therefore remained unknown. If our hypothesis that condensin creates a preferred pericentromeric framework upon which the error correction machinery can act is correct, we reasoned that condensin could also impose a bias on kinetochores to biorient. When SPBs are allowed to separate before microtubules attach to sister kinetochores, they tend to biorient normally, even when error correction is impaired (<xref ref-type="bibr" rid="bib22">Indjeian and Murray, 2007</xref>). To test the requirement of shugoshin and condensin for sister kinetochore bias, we allowed cells to progress from G1 into the cell cycle for 1.5 hr to allow SPB separation, before treating the cells with nocodazole for 30 min. Subsequently, nocodazole was washed out and we simultaneously began filming (<xref ref-type="fig" rid="fig8">Figure 8A</xref>). We recorded the percentage of cells with separated SPBs at the start of filming that separated <italic>CEN4-GFP</italic> foci at least once during the observation period (approximately 30 min) (<xref ref-type="fig" rid="fig8">Figure 8B,C</xref>; <xref ref-type="other" rid="video3 video4">Videos 3 and 4</xref>). Similar numbers of wild-type and <italic>ipl1-as</italic> cells achieved sister centromere separation, indicating that the error correction process is not required to bias sister kinetochores towards biorientation (<xref ref-type="fig" rid="fig8">Figure 8B</xref>). However, remarkably, the frequency of separated <italic>CEN4-GFP</italic> foci was reduced about two-fold in <italic>sgo1Δ</italic> and <italic>YCS5-aid</italic> cells, as compared to wild-type cells (<xref ref-type="fig" rid="fig8">Figure 8B</xref>). This indicates that in the absence of shugoshin or pericentromeric condensin, the bias to sister kinetochore biorientation is lost. We conclude that both shugoshin and condensin impose a bias on sister kinetochores to biorient and that this is independent of error correction by Aurora B (Ipl1).<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.024</object-id><label>Figure 8.</label><caption><title>Condensin biases chromosomes to biorient.</title><p>(<bold>A</bold>–<bold>C</bold>) Condensin and Sgo1, but not Ipl1, are required to bias sister kinetochores towards biorientation. (<bold>A</bold>) Scheme of the microfluidics assay to test sister kinetochore bias. Wild-type, <italic>sgo1Δ</italic>, <italic>YCS5-aid</italic> and <italic>ipl1-as</italic> cells as in <xref ref-type="fig" rid="fig5">Figure 5B</xref> were released from a G1 arrest into NAA and NAPP1 and arrested in metaphase by <italic>CDC20</italic> depletion. SPBs were allowed to separate for 1 hr 30 min before cells were treated with nocodazole for an additional 30 min. After 2 hr total, nocodazole was washed out and frames were grabbed at approximately 94 s intervals for a total of 21 frames. (<bold>B</bold>) The percentage of cells that separated <italic>CEN4-GFP</italic> foci at least once during the observation period is shown for the indicated strains. p values indicate significance (chi-square test). (<bold>C</bold>) Representative images of wild-type and <italic>YCS5-aid</italic> cells are shown. Time of image acquisition (s) is shown. The asterisk indicates the first time GFP foci are separated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.024">http://dx.doi.org/10.7554/eLife.01374.024</ext-link></p></caption><graphic xlink:href="elife01374f008"/></fig><media content-type="glencoe play-in-place height-250 width-310" id="video3" mime-subtype="avi" mimetype="video" xlink:href="elife01374v003.avi"><object-id pub-id-type="doi">10.7554/eLife.01374.025</object-id><label>Video 3.</label><caption><title>Example video of a wild type cell in the assay to test for a bias towards sister kinetochore biorientation.</title><p>The video corresponds to the image gallery in <xref ref-type="fig" rid="fig6">Figure 6C</xref> (upper panel).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.025">http://dx.doi.org/10.7554/eLife.01374.025</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video4" mime-subtype="avi" mimetype="video" xlink:href="elife01374v004.avi"><object-id pub-id-type="doi">10.7554/eLife.01374.026</object-id><label>Video 4.</label><caption><title>Example video of a YCS5-aid cell in the assay to test for a bias towards sister kinetochore biorientation.</title><p>The video corresponds to the image gallery in <xref ref-type="fig" rid="fig6">Figure 6C</xref> (lower panel).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.026">http://dx.doi.org/10.7554/eLife.01374.026</ext-link></p></caption></media></p></sec><sec id="s2-9"><title>The bias towards sister kinetochore biorientation relies on the shugoshin-condensin interaction</title><p>Shugoshins are emerging as factors that define a hub at the pericentromere that integrates the functions of multiple protein complexes to ensure the accuracy of chromosome segregation (<xref ref-type="fig" rid="fig9">Figure 9A</xref>) (<xref ref-type="bibr" rid="bib65">Rattani et al., 2013</xref>). To explore the relationship between shugoshin and its binding partners further, we asked whether the Sgo1-100, Sgo1-700 and Sgo1-3A proteins retain their interaction with condensin. We found that Sgo1-700 failed to associate with Brn1, whereas Sgo1-100 and Sgo1-3A retained Brn1 association (<xref ref-type="fig" rid="fig9">Figure 9B</xref>). Analysis of Brn1 association with representative centromere-proximal and chromosomal arm sites by ChIP-qPCR revealed that only the <italic>sgo1-3A</italic> mutant, and not the <italic>sgo1-100</italic> or <italic>sgo1-700</italic> mutants maintained Brn1 localization at the pericentromere in cells arrested in mitosis by nocodazole treatment (<xref ref-type="fig" rid="fig9">Figure 9C</xref>). However, in both <italic>sgo1-100</italic> and <italic>sgo1-700</italic> mutant cells progressing from G1 into the cell cycle, Brn1-6HA retained a partial ability to associate with the centromere and pericentromere, though only in the case of the <italic>sgo1-100</italic> did this occur in a timely manner (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>). As both the Sgo1-100 and Sgo1-700 proteins themselves fail to be maintained at the centromere (<xref ref-type="fig" rid="fig1">Figure 1B</xref>), these observations are difficult to interpret. As a more direct test of the ability of the mutant proteins to recruit condensin, we fused Sgo1-100, Sgo1-700, and Sgo1-3A to tetR and artificially tethered them to <italic>tetO</italic> arrays located adjacent to <italic>CEN4</italic> in cells that otherwise lacked <italic>SGO1</italic> (<xref ref-type="fig" rid="fig9">Figure 9D–F</xref>)<italic>.</italic> Tethering of Sgo1-tetR-GFP, Sgo1-100-tetR-GFP, or Sgo1-3A-tetR-GFP, all significantly increased Brn1-6HA levels at <italic>CEN4</italic> compared to a no tag control (<xref ref-type="fig" rid="fig9">Figure 9E</xref>). In contrast, and consistent with our finding that Brn1 fails to co-immunoprecipitate with Sgo1-700 (<xref ref-type="fig" rid="fig9">Figure 9B</xref>), tethering of Sgo1-700-tetR-GFP did not significantly enrich Brn1-6HA at the same site, though it was produced to similar levels as the Sgo1-100-tetR-GFP protein (<xref ref-type="fig" rid="fig9">Figure 9D,E</xref>). Despite the ability of Sgo1-100-tetR-GFP and Sgo1-3A-tetR-GFP fusion proteins to recruit Brn1 to the tethering site at <italic>CEN4</italic>, only the wild-type Sgo1-tetR-GFP fusion protein was able to partially rescue the segregation of chromosome IV after release from a nocodazole arrest (<xref ref-type="fig" rid="fig9s2">Figure 9—figure supplement 2</xref>). This is anticipated as none of the mutant proteins are expected to enable proper Ipl1 association with the centromere (<xref ref-type="fig" rid="fig3">Figure 3E</xref>). We conclude that Sgo1-100 and Sgo1-3A are able to associate with, and recruit, condensin, whereas Sgo1-700 loses this interaction.<fig-group><fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.027</object-id><label>Figure 9.</label><caption><title>Shugoshin enables the bias towards sister kinetochore biorientation by condensin recruitment.</title><p>(<bold>A</bold>) Schematic diagram showing Sgo1-associated complexes and their functions at the pericentromere. (<bold>B</bold>) Sgo1-100 and Sgo1-3A, but not Sgo1-700, retain association with Brn1. Cells carrying <italic>BRN1-6HA</italic> and <italic>SGO1-SZZ(TAP)</italic> (AM9266)<italic>, SGO1-100-SZZ(TAP)</italic> (AM9149), <italic>SGO1-700-SZZ(TAP)</italic> (AM9264), <italic>SGO1-3A-SZZ(TAP)</italic> (AM9262) or no TAP (AM5708) were arrested in nocodazole for 2 hr before treating with the cross-linker DSP. Prepared extracts were incubated with IgG-coupled beads and immunoprecipitates analyzed by immunoblotting with the indicated antibodies. (<bold>C</bold>) Brn1 is maintained at the centromere in metaphase-arrested <italic>sgo1-3A,</italic> but not <italic>sgo1-100</italic> or <italic>sgo1-700</italic> cells. Wild-type (AM5708), <italic>sgo1Δ</italic> (AM8834), <italic>sgo1-100</italic> (AM9442), <italic>sgo1-700</italic> (AM9291) and <italic>sgo1-3A</italic> (AM9276) cells carrying <italic>BRN1-6HA</italic> as well as a no tag control (AM1176) were arrested in nocodazole for 2 hr before harvesting for anti-HA ChIP. The levels of Brn1-6HA were measured at the indicated sites by qPCR. (<bold>D</bold>–<bold>F</bold>) Tethered Sgo1, Sgo1-100 or Sgo1-3A, but not Sgo1-700 can enrich Brn1-6HA at <italic>CEN4</italic> in otherwise <italic>sgo1Δ</italic> cells. <italic>SGO1-tetR-GFP</italic> (AM14012), <italic>sgo1-100-tetR-GFP</italic> (AM13902), <italic>sgo1-700-tetR-GFP</italic> (AM13907), and <italic>sgo1-3A-tetR-GFP</italic> (AM13904) were introduced into cells carrying <italic>tetOs</italic> integrated at <italic>CEN4</italic>, producing Brn1-6HA and with <italic>SGO1</italic> deleted from its endogenous locus. A strain carrying just <italic>tetOs</italic> integrated at <italic>CEN4</italic> but otherwise wild type was used as a no tag control (AM11060). All strains were arrested in mitosis by treatment with nocodazole for 3 hr before harvesting for ChIP and immunoblotting. (<bold>D</bold>) Schematic diagram of the tethering locus. (<bold>E</bold>) Levels of Brn1 recruited adjacent to the tethering site (<italic>CEN4</italic>) when the indicated proteins are fused to TetR-GFP, as measured by ChIP-qPCR. The mean of four independent repeats is shown except for <italic>sgo1-3A-tetR-GFP</italic> where six repeats are included. Error bars are standard error, significance was calculated using the student <italic>t</italic> test (*p<0.05). (<bold>F</bold>) Total cellular levels of the Sgo1-tetR-GFP fusion proteins, Brn1-6HA and Pgk1 (loading control) were analyzed by immunoblot using the indicated antibodies. (<bold>G</bold>) The bias to sister kinetochore biorientation is absent in <italic>sgo1-700</italic> cells. Wild-type (AM4643), <italic>sgo1-100</italic> (AM8924), <italic>sgo1-700</italic> (AM8925) and <italic>sgo1-3A</italic> (AM8923) cells were released from G1 and treated with nocodazole after SPB separation as in <xref ref-type="fig" rid="fig7">Figure 7A</xref>. The percentage of cells that separated <italic>CEN4-GFP</italic> foci at least once during the observation period is shown for the indicated strains. p values indicate significance (chi-square test).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.027">http://dx.doi.org/10.7554/eLife.01374.027</ext-link></p></caption><graphic xlink:href="elife01374f009"/></fig><fig id="fig9s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.028</object-id><label>Figure 9—figure supplement 1.</label><caption><title>The Sgo1-100 protein can recruit condensin to kinetochores.</title><p>(<bold>A</bold> and <bold>B</bold>) Condensin is at least partially recruited to the centromere after release from G1 in <italic>sgo1-100</italic> cells. Wild-type (AM5708), <italic>sgo1-100</italic> (AM9442) and <italic>sgo1-700</italic> (AM9291) cells carrying <italic>BRN1-6HA</italic> were arrested in G1 using alpha factor. Samples were extracted for anti-HA ChIP-qPCR at the indicated levels for analysis of Brn1 association with <italic>CEN4</italic> (<bold>A</bold>) and the pericentromere of chromosome IV (<bold>B</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.028">http://dx.doi.org/10.7554/eLife.01374.028</ext-link></p></caption><graphic xlink:href="elife01374fs013"/></fig><fig id="fig9s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01374.029</object-id><label>Figure 9—figure supplement 2.</label><caption><title>Sgo1-tetR-GFP, but not Sgo1-100-tetR-GFP, Sgo1-700-tetR-GFP or Sgo1-3A-tetR-GFP tethered to <italic>CEN4</italic> can partially rescue the mis-segregation of chromosome IV after nocodazole washout in otherwise <italic>sgo1Δ</italic> cells.</title><p>Diploid cells carrying <italic>tetR-tdTomato</italic> and <italic>SGO1-tetR-GFP</italic> (AM14005), <italic>sgo1-100-tetR-GFP</italic> (AM14006), <italic>sgo1-700-tetR-GFP</italic> (AM14007), <italic>sgo1-3A-tetR-GFP</italic> (AM14008) or no Sgo1-TetR fusion (AM14009), with <italic>tetOs</italic> integrated at <italic>CEN4</italic> and with <italic>SGO1</italic> deleted from its endogenous locus were treated with nocodazole to depolymerize microtubules and arrest cells in mitosis. Nocodazole was washed out, allowing microtubules to reform and, 60 min later, the position of <italic>CEN4-tdTomato</italic> foci was scored in the anaphase cells after chromosome segregation. At least 100 cells were scored from each of two experimental repeats with error bars representing the range, except for the <italic>sgo1-3A-tetR-GFP</italic> strain, where results are shown from a single experiment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.029">http://dx.doi.org/10.7554/eLife.01374.029</ext-link></p></caption><graphic xlink:href="elife01374fs014"/></fig></fig-group></p><p>This finding allowed us to test a prediction: if Sgo1-dependent deposition of condensin at the pericentromere is critical for biasing sister kinetochores towards biorientation, then Sgo1-700, which fails to bind to or recruit condensin to the pericentromere (<xref ref-type="fig" rid="fig9">Figure 9B–E</xref>) should lack the sister kinetochore bias. In contrast, Sgo1-3A, which recruits condensin to the pericentromere (<xref ref-type="fig" rid="fig9">Figure 9B,C</xref>), and Sgo1-100, which retains at least a partial ability to deposit condensin at the pericentromere (<xref ref-type="fig" rid="fig9">Figure 9F</xref>, <xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>), should enable some degree of sister kinetochore bias. In accordance with these predictions, our live cell assay revealed that only the <italic>sgo1-</italic>700 mutant was significantly defective in the sister kinetochore bias (<xref ref-type="fig" rid="fig9">Figure 9G</xref>). We conclude that condensin recruitment by Sgo1 biases sister kinetochores towards biorientation.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Our findings have demonstrated that Sgo1 plays a central role in promoting biorientation, through at least two separate mechanisms. Sgo1 enables error correction by retention of Ipl1 (aurora B) at the centromere. Sgo1 separately recruits condensin to the pericentromere, which both imposes a bias on sister kinetochores to biorient and enables effective error correction.</p><p>Our findings provide the first molecular explanation for the bias to sister kinetochore biorientation. Interestingly, <xref ref-type="bibr" rid="bib22">Indjeian and Murray (2007)</xref> demonstrated the existence of a bias to sister kinetochore biorientation in the <italic>sgo1-100</italic> mutant, which is defective in the error correction process (<xref ref-type="bibr" rid="bib23">Indjeian et al., 2005</xref>). Consistent with this study, we confirmed that the <italic>sgo1-100</italic> mutant retains this bias to sister kinetochore biorientation (<xref ref-type="fig" rid="fig9">Figure 9G</xref>). Through analysis of both the <italic>sgo1-700</italic> and <italic>sgo1Δ</italic> mutants we have, nevertheless uncovered a role for Sgo1 in biasing sister kinetochores towards biorientation. Moreover, our data provide a molecular explanation for the loss of sister kinetochore bias in <italic>sgo1-700</italic> and <italic>sgo1Δ</italic> cells: that is the inability to associate with and recruit condensin to centromeres. In contrast, <italic>sgo1-100</italic> and <italic>sgo1-3A</italic> cells, which can enable condensin recruitment to centromeres are proficient in the bias to sister kinetochore biorientation.</p><p>Condensin is a cohesin-related complex that can form a ring-like structure and has been proposed to organize chromosomes by bringing distant chromosomal sequences together (<xref ref-type="bibr" rid="bib8">Cuylen and Haering, 2011</xref>; <xref ref-type="bibr" rid="bib9">Cuylen et al., 2011</xref>; <xref ref-type="bibr" rid="bib19">Hirano, 2012</xref>). The mechanism by which condensin is loaded onto and subsequently maintained on chromosomes is largely unknown. Here, we have identified shugoshin as an important determinant of condensin association with the pericentromere. Indeed, the pericentromeres and rDNA appear to be the predominant, if not the only sites of condensin association with chromosomes in mitosis. Our findings have also suggested a hierarchy of assembly at the pericentromere. In contrast to the idea that the Scc2/4 cohesin-loader complex loads condensin directly (<xref ref-type="bibr" rid="bib10">D’Ambrosio et al., 2006</xref>), we propose that Scc2/4 indirectly affects condensin localization through loading cohesin, which we show is required for the pericentromeric association of Sgo1. In support of this idea, only cohesin and not condensin subunits were identified in Scc2/4 immunoprecipiates (<xref ref-type="bibr" rid="bib13">Fernius et al., 2013</xref>). Furthermore, since Shugoshin relies on cohesin for its association with the pericentromere (<xref ref-type="bibr" rid="bib29">Kiburz et al., 2005</xref>; <xref ref-type="bibr" rid="bib62">Yamagishi et al., 2008</xref>; <xref ref-type="fig" rid="fig5">Figure 5</xref>), the importance of cohesin in defining kinetochore geometry (<xref ref-type="bibr" rid="bib49">Sakuno et al., 2009</xref>) could be to enable proper condensin association with chromosomes by recruiting shugoshin to the pericentromere. How might condensin shape a favorable pericentromeric geometry? While the molecular function of condensin is not well understood, condensin is known to alter the structural properties of centromeres that affect their dynamic behavior (<xref ref-type="bibr" rid="bib45">Ribeiro et al., 2009</xref>; <xref ref-type="bibr" rid="bib50">Stephens et al., 2011</xref>). We propose that condensin organizes the pericentromeric chromatin to provide a structural integrity to the pericentromere that enables it to adopt a ‘back-to-back’ geometry that orients sister kinetochores in opposite directions, thereby favoring their biorientation (<xref ref-type="fig" rid="fig10">Figure 10</xref>). Although our data indicate that condensin is important to increase the efficiency of biorientation, we do not believe that it is essential for this process in budding yeast, provided that error correction machinery is intact. However, budding yeast centromeres are relatively simple and only a single microtubule contacts each kinetochore (<xref ref-type="bibr" rid="bib59">Winey et al., 1995</xref>) increasing the probability that correct attachments can be made by chance. This is not the case for organisms with more complex centromeres where it is likely that kinetochore geometry plays a major role in ensuring sister kinetochore biorientation is achieved.<fig id="fig10" position="float"><object-id pub-id-type="doi">10.7554/eLife.01374.030</object-id><label>Figure 10.</label><caption><title>Model for dual role of Sgo1 in biorientation is shown.</title><p>Shugoshin ensures sister kinetochore biorientation through two mechanisms. First, early in the cell cycle, shugoshin mediates the enrichment of condensin within the pericentromere. We propose that condensin enables the pericentromere to adopt a geometry that favors the capture of sister kinetochores by microtubules from opposite poles, thereby biasing them to biorient. Second, shugoshin maintains aurora B at the pericentromere for those kinetochores that fail to biorient and come under tension. Aurora B destabillizes these tension-less attachments, thereby providing a further chance to for sister kinetochores to make the appropriate, tension-generating attachments. We suggest that condensin facilitates this ‘error correction’ process by conferring a rigid structure to the pericentromere upon which aurora B can act.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.030">http://dx.doi.org/10.7554/eLife.01374.030</ext-link></p></caption><graphic xlink:href="elife01374f010"/></fig></p><p>In addition to influencing pericentromeric structure through condensin, shugoshins also confer distinct properties to pericentromeric cohesin through PP2A, both in meiosis and mammalian mitosis, as well as influence kinetochore–microtubule interactions through aurora B. Shugoshins are therefore emerging as functional hubs that define the pericentromere, allowing it to perform specialized functions that are key for the fidelity of chromosome segregation.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Yeast strains</title><p>Strains used in this work are listed in <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2A</xref>. All yeast strains were derivatives of W303 except the protease-deficient strain, JB811, used for TAP pulldowns. The <italic>sgo1-100</italic> and <italic>sgo1-700</italic> alleles were described in <xref ref-type="bibr" rid="bib23">Indjeian et al. (2005)</xref>, the <italic>sgo1-3A</italic> allele was described in <xref ref-type="bibr" rid="bib60">Xu et al. (2009)</xref> and <italic>sgo1Δ</italic> was described in <xref ref-type="bibr" rid="bib6">Clift et al. (2009)</xref>. A PCR-based approach was used to tag Sgo1 with SZZ(TAP); Ipl1, Brn1 and Ycs4 with 6HA; and Rts1 with 3Pk or 9Myc (<xref ref-type="bibr" rid="bib36">Longtine et al., 1998</xref>; <xref ref-type="bibr" rid="bib32">Knop et al., 1999</xref>). Auxin-inducible degron versions of Sgo1 and Ycs5 were constructed as described by <xref ref-type="bibr" rid="bib41">Nishimura et al. (2009)</xref>. To generate a strain carrying Sgo1-TetR-GFP, <italic>SGO1</italic> was cloned upstream of <italic>tetR-GFP</italic> in p128(TetR-GFP) (<xref ref-type="bibr" rid="bib38">Michaelis et al., 1997</xref>) to generate plasmid AMp769 which was integrated at the <italic>LEU2</italic> locus after <italic>EcoRV</italic> digestion. TetR-GFP fusions to Sgo1-100, Sgo1-700 or Sgo1-3A were generated by PCR amplication of these alleles from the genomic locus and replacement of <italic>SGO1</italic> in AMp769 (<italic>sgo1-700, sgo1-3A</italic>) or by site-directed mutagenesis using the Quikchange kit (Agilent Technologies, Santa Clara, CA). <italic>Ipl1-as5</italic> was described in <xref ref-type="bibr" rid="bib42">Pinsky et al. (2006)</xref>. <italic>pMET-CDC20</italic> was described in <xref ref-type="bibr" rid="bib12">Fernius and Marston (2009)</xref>. The <italic>CEN4-GFP</italic> label (<italic>CEN4-tetOs::URA3 leu2::TetR-GFP::LEU2</italic>) was described in <xref ref-type="bibr" rid="bib18">He et al. (2000)</xref>; <xref ref-type="bibr" rid="bib52">Tanaka (2010)</xref> and <italic>SPC42-tdTomato</italic> was described in <xref ref-type="bibr" rid="bib11">Fernius and Hardwick (2007)</xref>.</p></sec><sec id="s4-2"><title>Growth conditions</title><p>Nocodazole was used at 15 μg/ml and re-added to 7.5 μg/ml every 1 hr. NAA (synthetic auxin) was used at 500 μM and readded to 250 μM every 45 min. Methionine was used at 8 mM and re-added to 4 mM every 45 min. NAPP1 (to inhibit <italic>ipl1-as</italic>) was used at 50 μm and doxycycline was used at 5 μg/ml.</p></sec><sec id="s4-3"><title>Chromatin immunoprecipitation</title><p>ChIP was performed as described using anti-HA 12CA5, anti-Myc 9E10 or anti-Pk(V5) antibody (<xref ref-type="bibr" rid="bib13">Fernius et al., 2013</xref>). Primers used for qPCR analysis are given in <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2B</xref>. qPCR was performed in a 20 μl Express SYBR GreenER (Life Technologies, Carlsbad, CA) reaction using a Lightcycler machine (Roche, Switzerland). To calculate ChIP enrichment/input, ΔCT was calculated according to: ΔCT = (CT<sub>(ChIP)</sub> − [CT<sub>(Input)</sub>− logE (Input dilution factor)]) where E represents the specific primer efficiency value. Enrichment/ input value was obtained from the following formula: E^<sup>−ΔCT</sup>. qPCR was performed in triplicate, typically for each of three or more independent experimental repeats. Error bars represent standard error. For ChIP-Seq, purified chromatin was recovered using a PCR purification kit (Qiagen, Netherlands) followed by drying in a speedvac. Samples were sequenced on a HiSeq2000 instrument (Illumina, San Diego, CA) by the EMBL Core Genomics Facility (Heidelberg, Germany). The summary of data obtained is given in <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2C</xref>.</p></sec><sec id="s4-4"><title>Analysis of ChIP-Seq data</title><p>Scripts, data files and workflows used to create the ChIP-Seq data can be found on the github repository: <ext-link ext-link-type="uri" xlink:href="https://github.com/AlastairKerr/Marston2013">https://github.com/AlastairKerr/Marston2013</ext-link>. Single reads were mapped using BWA (Version: 0.6.1-r104) (<xref ref-type="bibr" rid="bib34">Li and Durbin, 2010</xref>) to the sacCer3 reference genome and were processed with samtools (<xref ref-type="bibr" rid="bib35">Li et al., 2009</xref>) to remove duplicated reads for parallel analysis. The region of chrXII containing rDNA (451400 bp to 490600 bp) was removed and studied separately using bedtools (version v2.16.2) (<xref ref-type="bibr" rid="bib43">Quinlan and Hall, 2010</xref>). As this region is highly repetitive and contained a high density of reads, duplicate reads were not removed for this analysis as it would not be possible to differentiate duplicate reads from independent fragments. All data shown are normalized to the number of mapped reads per million total mapped reads [RPM]. Total mapped reads were calculated after any processing was done for rDNA or duplicate read removal.</p><p>During the amplification step prior to sequencing, multiple identical reads will be generated. Due to the low yield of total DNA precipitating with Brn1-6HA in <italic>sgo1Δ</italic> cells, we were concerned that the small number of precipitating sequences would be biased for amplification. To avoid this problem, we removed PCR replicates from our analysis. However, since this would also remove bona fide identical sequences from our dataset, we simultaneously analyzed the data with these reads present and compared the two data sets where all 16 centromeres were considered together. To do this, 100 bp windows were examined that extended 25 kb in each direction of every centromere, and a local maximum of the number of mapped reads was taken. The comparison of this data with and without PCR replicates included can be seen in <xref ref-type="fig" rid="fig3">Figure 3H</xref> (unfiltered data) and <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref> (PCR replicates removed). Both approaches led to very similar conclusions, therefore for all other data presented we used the unfiltered data.</p></sec><sec id="s4-5"><title>TAP pulldowns and mass spectrometry</title><p>To grow cells for purification of Sgo1 from cycling cells, 4 L of YPDA culture were inoculated to OD<sub>600</sub> = 0.2 and grown at room temperature for 6 hr. Cells were harvested by centrifugation and the cell pellet was resuspended in 2 μM of dithiobis(succunimidylpropionate) (DSP; Proteochem, Loves Park, IL) crosslinker for 30 min at room temperature before cell pellets were drop-frozen in liquid nitrogen as described by <xref ref-type="bibr" rid="bib13">Fernius et al. (2013)</xref>. To purify Sgo1 from cells arrested in mitosis using the cold-sensitive <italic>tub2-401</italic> allele, 4 L of media was inoculated to OD<sub>600</sub> = 0.02, grown at 30°C for 8 hr with shaking and then the temperature was reduced to 18°C for 7 hr to induce the arrest before holding the cells at 4°C for up to 5 hr before harvesting, crosslinking, and freezing as described above. Pulldowns and mass spectrometry were performed as described in <xref ref-type="bibr" rid="bib13">Fernius et al. (2013)</xref>.</p></sec><sec id="s4-6"><title>Co-immunoprecipitation, western blotting, immunofluorescence, and FACS</title><p>For co-immunoprecipitation of TAP-tagged Sgo1, 200 ml of nocodazole-treated culture was harvested and either firstly cross-linked using DSP as described in <xref ref-type="bibr" rid="bib13">Fernius et al. (2013)</xref>, or directly drop-frozen in liquid nitrogen. Frozen pellets were ground in a pestle and mortar for 5 min. Ground lysates, prepared as in <xref ref-type="bibr" rid="bib13">Fernius et al. (2013)</xref>, were incubated with 1 mg of IgG-coupled dynabeads for 1.5 hr at 4°C. For the DNAase treated samples in <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>, benzonase (25U) was added to the extracts before incubating at room temperature for 30 min before proceeding with immunoprecipitation. Protein complexes were eluted from the beads by addition of 30 μl of sample buffer and loaded onto polyacrylamide gels.</p><p>Western immunoblot was performed as described in <xref ref-type="bibr" rid="bib6">Clift et al. (2009)</xref> and visualized by detection of chemiluminesence on autoradiograms except for the data shown in <xref ref-type="fig" rid="fig9">Figure 9E</xref> where proteins were visualized using a fluorophore-conjugated antibody and the Odyssey system (Li-Cor, Lincoln, NE). Mouse anti-aid (CosmoBio, Japan), anti-HA11, and anti-Myc antibodies were all used at a dilution of 1:1000. Mouse anti-Pgk1 antibodies were used at a dilution of 1:5000. Rabbit anti-Sgo1 (a kind gift of Adam Rudner, Ottawa Institute of Systems Biology, Canada), anti-GFP (a kind gift of Eric Schirmer, University of Edinburgh) and anti-Pgk1 antibodies were used at a dilution of 1:5000. Rabbit anti-Kar2 antibodies (laboratory stock) were used at a dilution of 1:5000.</p><p>Indirect immunofluorescence and FACS were performed as described in <xref ref-type="bibr" rid="bib6">Clift et al. (2009)</xref> and <xref ref-type="bibr" rid="bib12">Fernius and Marston (2009)</xref>, respectively.</p></sec><sec id="s4-7"><title>Biorientation assay</title><p>Cells carrying <italic>pMET-CDC20</italic>, <italic>CEN4-GFP</italic> and <italic>SPC42-tdTomato</italic> were arrested in G1 using alpha factor (4 μg/ml) in minimal medium lacking methionine (SD/-met) at room temperature for 3 hr. Alpha factor was washed out and cells were released from the G1 arrest into rich medium containing methionine and nocodazole (YPDA + Met + NOC) plus the appropriate drugs (NAPP1, NAA). Methionine represses <italic>pMET-CDC20</italic> expression, resulting in a metaphase arrest and nocodazole depolymerizes microtubules. After 3 hr, a sample was extracted (t = 0) and nocodazole was washed out by filtering in the presence of methionine and cultures were released into YPDA + Met to allow spindles to reform while maintaining the metaphase arrest. Samples were taken at 20 min intervals. Samples were fixed in 3.7% formaldehyde for 10 min, before washing in PBS and resuspending in DAPI for microscopy. Samples with two separated SPC42-tdTomato foci were scored as containing one or two <italic>CEN4-GFP</italic> foci. Typically 200, and at least 100, cells were scored for each timepoint.</p></sec><sec id="s4-8"><title>Microfluidics and image analysis</title><p>For live cell imaging, cells were loaded onto the Onix Microfluidic Perfusion system (CellAsic, Hayward, CA) and visualized using a Deltavision Elite (Applied Precision, Issaquah, WA) coupled to a Cascade 2 EMCCD camera with temperature control to 30°C. Frames were grabbed at the indicated intervals and images were processed in ImagePro software (Media Cybernetics, Rockville, MD).</p><p>For image analysis, a custom-written macro was developed in ImagePro software (Media Cybernetics), details of which are available upon request. In brief, yeast are selected from a reference DIC image taken at each time point. The yeast are automatically thresholded and the centre pixel from each red and green spot is then calculated. The distances between the spots can then be calculated and the measurements output to a text file for analysis using microsoft excel. Measurements of inter-centromere distance and distance from <italic>CEN4-</italic>GFP dot to nearest Spc42-tdTomato were obtained using this automated system.</p></sec><sec id="s4-9"><title>Error correction assay using microfludics</title><p>To test the efficiency of error correction, cells carrying <italic>CEN4</italic> (<italic>CEN4-GFP</italic>) and SPB (<italic>SPC42-tdTomato</italic>) markers together with <italic>CDC20</italic> under control of a methionine-repressible promoter (<italic>pMET-CDC20</italic>) were arrested in G1 using alpha factor and then loaded onto the microfluidic chamber. Cells were released in the chamber into medium containing nocodazole (to depolymerize microtubules) and methionine (to deplete <italic>CDC20</italic> and induce a metaphase arrest) for 30 min. The addition of nocodazole to G1 cells prevents SPB separation because this requires microtubules. Therefore, under these conditions, microtubules do not separate which leads to a high rate of monoorientation once microtubules are allowed to reform. This leads to a strong reliance on the error correction machinery to establish biorientation (<xref ref-type="bibr" rid="bib22">Indjeian and Murray, 2007</xref>). After 30 min, nocodazole was washed out, all the time maintaining the <italic>CDC20</italic> arrest by inclusion of methionine in the media. After 90% of cells had 2 SPBs (typically 1.5 hr), we began imaging at ∼74s intervals for a total of 21 frames.</p><p>For determination of the overall ability of strains to biorient in <xref ref-type="fig" rid="fig2">Figure 2D</xref> all cells in each frame where two SPB foci were detected by the software were scored for the presence of either one or two <italic>CEN4-GFP</italic> foci. The total percentage of cells with two SPB foci that contained two GFP foci was calculated for all frames for each strain. The number of cell images analyzed was 1116 (wild type), 697 (<italic>sgo1Δ</italic>), 1251 (<italic>YCS5-aid</italic>) and 968 (<italic>ipl1-as</italic>). To m</p><p>As a measure of the efficacy of the error correction machinery, we determined the ability of cells to switch between one and two visible <italic>CEN4-GFP</italic> foci. Cells in which two SPB foci were detected for at least four consecutive frames were scored for the number of times that the number of <italic>CEN4-GFP</italic> foci changed from one to two or vice-versa for the frames in which two SPBs were consecutively visible. The ‘switching rate’ was calculated by dividing the number of times a cell alternated between one and two <italic>CEN4-GFP</italic> foci by the total time in which two SPB foci were consecutively visible. The average switching rate was determined for all cells in which two SPB foci were detected for at least four consecutive frames. In <xref ref-type="fig" rid="fig2">Figure 2F</xref>, we analyzed 92 wild-type cells (971 frames), 55 <italic>sgo1Δ</italic> cells (551 frames), 102 <italic>YCS5-aid</italic> cells (1046 frames) and 86 <italic>ipl1-as</italic> cells (748 frames).</p></sec><sec id="s4-10"><title>Sister kinetochore bias assay using microfludics</title><p>To test the bias on sister kinetochores to biorient, cells carrying <italic>CEN4</italic> (<italic>CEN4-GFP</italic>) and SPB (<italic>SPC42-tdTomato</italic>) markers together with <italic>CDC20</italic> under control of a methionine-repressible promoter (<italic>pMET-CDC20</italic>) were arrested in G1 using alpha factor and then released into medium containing methionine to deplete <italic>CDC20</italic> and induce a metaphase arrest. Cells were loaded onto the microfluidics chamber and after approximately 1.5 hr, when around 90% of cells had 2 SPBs, cells were treated with YPDA containing nocodazole for 30 min. After 30 min, nocodazole was washed out after which cells were immediately imaged every ∼94 s for a total of 21 frames. We scored the percentage of cells in which separated GFP dots were observed at least once during the observation period.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We are grateful to Adam Rudner for the kind gift of anti-Sgo1 antibody, to Eric Schirmer for the anti-GFP antibody and Andrew Murray and Kim Nasmyth for yeast strains. We thank Bianka Baying at Genecore, EMBL for library preparation and sequencing. We appreciate help from Claudia Schaffner with FACS. We are grateful to Robin Allshire, Kevin Hardwick, and Vasso Makrantoni for helpful comments on the manuscript.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>KFV, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>ALM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>OON, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con4"><p>DK, Wrote a macro for analysis of microscopy data, Assisted with acquisition and analysis of live cell imaging data</p></fn><fn fn-type="con" id="con5"><p>AK, Bioinformatics associated with ChIP-Seq data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>DC, Acquisition of data</p></fn><fn fn-type="con" id="con7"><p>FLA, Acquisition of data</p></fn><fn fn-type="con" id="con8"><p>JR, Acquisition of data</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.01374.031</object-id><label>Supplementary file 1.</label><caption><p>Complete list of peptides identified in the experiments shown in <xref ref-type="fig" rid="fig4">Figure 4A,B</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.031">http://dx.doi.org/10.7554/eLife.01374.031</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01374s001.xlsx"/></supplementary-material><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.01374.032</object-id><label>Supplementary file 2.</label><caption><p>(<bold>A</bold>) Yeast strains used in this study. (<bold>B</bold>) qPCR primers used in this study. (<bold>C</bold>) Genome summary table for Brn1-6HA ChIP-seq.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01374.032">http://dx.doi.org/10.7554/eLife.01374.032</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01374s002.xlsx"/></supplementary-material><sec sec-type="datasets"><title>Major dataset</title><p>The following dataset was generated:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><name><surname>Verzijlbergen</surname><given-names>K</given-names></name>, <name><surname>Kerr</surname><given-names>A</given-names></name>, <name><surname>Marston</surname><given-names>A</given-names></name>, <year>2013</year><x>, </x><source>Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere</source><x>, </x><object-id pub-id-type="art-access-id">GSE53856</object-id><x>; 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pub-id-type="doi">10.7554/eLife.01374.033</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Pines</surname><given-names>Jon</given-names></name><role>Reviewing editor</role><aff><institution>The Gurdon Institute</institution>, <country>United Kingdom</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elife.elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>In this study the authors have investigated how sister kinetochores in budding yeast are biased towards biorientation. The authors have found that the Shuoshin protein (Sgo1) is important to promote biorientation through two mechanisms: through binding to the chromosome passenger complex, and through a newly identified association with the condensin complex. The authors show that Sgo1 is able to recruit condensin to centromeres or to ectopic sites. The authors identify a mutation in Sgo1 that perturbs binding to condensin and that this association is important for subsequent biorientation. The authors conclude that condensin is required to form a rigid back-to-back structure such that sister kinetochores are biased towards microtubule capture from opposite spindle poles.</p><p>Overall this is an interesting study that reveals new insights into how budding yeast chromosomes biorent, and uncover a new role for condensin in centromere and kinetochore behaviour.</p><p>There are, however, a number of substantive concerns that preclude publication in its present form. In particular, the conclusion that condensin recruitment alters kinetochore geometry to promote orientation towards opposite poles requires further supporting evidence. Moreover, the authors themselves do not appear to be certain whether there is a direct link between Sgo1 and condensin recruitment. They argue that Sgo1 is sufficient for condensin targeting to the pericentromere by over-expressing Sgo1 and measuring Brn1 accumulation. However, they previously state that Sgo1's effect on condensin is indirect, mediated through Scc2/4 (cohesin). There are also concerns regarding the validity of the approach the authors use to measure bi-orientation and the presentation of the ChIP analyses. The latter prevents a critical evaluation of the data quality and missing information will need to be added.</p><p>Concerns to be addressed:</p><p>1) The evidence for a direct interaction between Sgo1 and condensin is limited. Only a small number (especially considering the large size of all five condensin subunits) of condensin peptides can be identified in the mass-spec analysis of Sgo1 purifications. In fact, the highest scoring condensin subunit (by numbers of found peptides; Ycg1) ranks number 137 in the list of proteins identified (see supplemental Excel file). Moreover, only a minute fraction of condensin (less than 0.01%? See <xref ref-type="fig" rid="fig3">Figures 3 D and E</xref>) can be detected by Western blotting in Sgo1 immunoprecipitations. Since both Sgo1 and condensin are enriched at centromeres, it is very likely that a small amount of condensin co-precipitates with Sgo1 because both proteins are bound to the same stretch of chromatin. In addition, the authors add amine-reactive crosslinkers to the cell extracts in both experiments (a fact they should mention at least in the figure legends and not only in the methods section), which will result in the formation of protein-chromatin crosslinks. The authors will need to repeat the pull-down experiments after DNase digestion and in the absence of crosslinker in order to rule out these concerns.</p><p>A second argument for a role of Sgo1 in condensin loading is that centromeric condensin levels increase upon Sgo1 overexpression. The increase in condensin binding could, however, result from the delay in anaphase onset caused by Sgo1 overexpression. Would condensin levels remain unchanged if anaphase onset were delayed by other means?</p><p>Ectopic recruitment of condensin by Sgo1 fused to TetR seems rather inefficient, with a two-fold enrichment (and considerable variation?). The authors should at least test whether ectopic recruitment can be measured at both sides of the TetO repeats. Yet a much better test whether condensin recruitment by Sgo1 is significant would be to employ the TetO-TetR approach to tether to centromeres an Sgo1 mutant that can otherwise not bind (e.g., Sgo1-100 or Sgo1-700) and assay whether this restores condensin levels and chromosome bi-orientation.</p><p>An explanation for the altered binding profiles of condensin measured by ChIP-seq might be changes in cohesin accumulation at centromeres in the Sgo1 mutants. The authors need to check by ChIP-qPCR (and ideally also ChIP-seq) whether cohesin levels at centromeres are affected in the Sgo1 mutant.</p><p>2) The authors monitor the splitting of a sister centromere pair as a measure for sister chromatid bi-orientation (<xref ref-type="fig" rid="fig1 fig5">Figures 1A-C,F, 5A</xref>, <xref ref-type="fig" rid="fig3s2">Figure 3–figure supplement 2</xref>). The reduction of sister centromere splitting observed in the Sgo1 and condensin mutants could, however, equally well result from an increase in centromeric sister chromatid cohesion. An increase in centromeric cohesion would prevent splitting of bi-oriented sister chromatids.</p><p>One possibility to distinguish whether chromatid pairs have attached to spindle microtubules from only one spindle pole or whether chromatids have bi-oriented but their centromeres cannot separate would be to score centromere-spindle pole body (SPB) distances in the live cell microscopy assay (<xref ref-type="fig" rid="fig5">Figures 5B-F</xref>). If centromeres were bi-oriented but merely failed to split, they should be positioned at equal distances to both SPBs. If sister centromeres failed to bi-orient, they should frequently be found close to one SPB. Alternatively, the authors could release cells from the Cdc20 arrest and allow them to enter anaphase, then score whether sister centromeres segregate to one pole or to opposite poles (see Indjeian and Murray, CurBiol 2007).</p><p>The data in <xref ref-type="fig" rid="fig6 fig7">Figures 6 and 7</xref> show a mild, real defect in biorientation but don't specifically address the of kineteochore geometry. The authors should either perform further experiments to support their hypothesis or confine their “geometric bias” to speculation in the Discussion.</p><p>3) Most of the graphs that represent the results from ChIP-qPCR experiments (<xref ref-type="fig" rid="fig1 fig2 fig5 fig7">Figures 1A, E, 2C, D, 5G, 7C</xref>, <xref ref-type="fig" rid="fig5s3">Figure 5–figure supplement 3</xref>) lack information that is essential to evaluate the data. What do the column plots represent? Do they show mean values, and if yes, from how many PCR runs and how many independent immunoprecipitation experiments? What do the error bars represent? The legend for <xref ref-type="fig" rid="fig4">Figures 4D-F</xref> is missing completely.</p><p>Taking into account the possible variability between individual ChIP-qPCR experiments, careful statistical analysis will be required to evaluate the significance of the observed differences. For example, the values measured at CEN4 for the same wild-type strain (AM3513) in two independent Ipl1 HA-ChIP experiments vary 4-fold (compare <xref ref-type="fig" rid="fig2">Figures 2C and D</xref>), calling into question whether the 2-fold reduction in ChIP signals in the Sgo1 mutants (<xref ref-type="fig" rid="fig2">Figure 2D</xref>) is significant. Similarly, the ChIP signal at CEN4 measured for the Sgo1-100 mutant in G1 phase is more than 2-fold higher than the signal for wildtype Sgo1 (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). Is the 4-fold increase in Sgo1 ChIP signals during mitosis (e.g., 2-fold when compared to the Sgo1-100 mutant) then still significant?</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01374.034</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The evidence for a direct interaction between Sgo1 and condensin is limited. Only a small number (especially considering the large size of all five condensin subunits) of condensin peptides can be identified in the mass-spec analysis of Sgo1 purifications. In fact, the highest scoring condensin subunit (by numbers of found peptides; Ycg1) ranks number 137 in the list of proteins identified (see supplemental Excel file)</italic>.</p><p>The Excel file submitted with the original manuscript was unsorted and did not take the contaminant proteins that also co-purify with the “no tag” control into account. When contaminants are taken into account, the condensin subunits rank number 8 and 26 in the list of proteins identified, in cycling and arrested cells, respectively. We have revised the file with the list of proteins sorted to exclude contaminant proteins.</p><p><italic>Moreover, only a minute fraction of condensin (less than 0.01%? See</italic> <xref ref-type="fig" rid="fig3"><italic>Figures 3 D and E</italic></xref><italic>) can be detected by Western blotting in Sgo1 immunoprecipitations. Since both Sgo1 and condensin are enriched at centromeres, it is very likely that a small amount of condensin co-precipitates with Sgo1 because both proteins are bound to the same stretch of chromatin. In addition, the authors add amine-reactive crosslinkers to the cell extracts in both experiments (a fact they should mention at least in the figure legends and not only in the methods section), which will result in the formation of protein-chromatin crosslinks. The authors will need to repeat the pull-down experiments after DNase digestion and in the absence of crosslinker in order to rule out these concerns</italic>.</p><p>We thank the reviewers for raising this important point. We performed the experiment suggested by the reviewers and found that the amount of the Ycs4 condensin subunit co-purifying with Sgo1 was independent of treatment with cross-linker or DNase. This shows that the interaction between Sgo1 and condensin is not dependent on DNA. This data is now presented in <xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1</xref>. We agree that the amount of condensin that purifies with Sgo1 is a small fraction of the total cellular condensin. However, this is expected as our Brn1 ChIP-Seq showed that the bulk of condensin is associated with the rDNA and would not be expected to bind to Sgo1.</p><p><italic>A second argument for a role of Sgo1 in condensin loading is that centromeric condensin levels increase upon Sgo1 overexpression. The increase in condensin binding could, however, result from the delay in anaphase onset caused by Sgo1 overexpression. Would condensin levels remain unchanged if anaphase onset were delayed by other means</italic>?</p><p>To exclude the possibility that Brn1’s chromosomal enrichment in <italic>SGO1-</italic>overexpressing cells was a consequence of the metaphase arrest caused by high levels of Sgo1, we overexpressed <italic>SGO1</italic> in cells arrested in mitosis by treatment with nocodazole. Measurement of Brn1 association with two centromeric sites and a pericentromeric site by ChIP-qPCR showed that <italic>SGO1</italic> overexpression also increased Brn1 levels in cells arrested in mitosis, arguing against the idea that the centromeric enrichment of Brn1 is solely due to the cell cycle block caused by <italic>SGO1</italic> overexpression. This data is presented as <xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref> in the revised version.</p><p><italic>Ectopic recruitment of condensin by Sgo1 fused to TetR seems rather inefficient, with a two-fold enrichment (and considerable variation?). The authors should at least test whether ectopic recruitment can be measured at both sides of the TetO repeats. Yet a much better test whether condensin recruitment by Sgo1 is significant would be to employ the TetO-TetR approach to tether to centromeres an Sgo1 mutant that can otherwise not bind (e.g., Sgo1-100 or Sgo1-700) and assay whether this restores condensin levels and chromosome bi-orientation</italic>.</p><p>In the original manuscript, we used a primer set ∼800 bp to the left of the <italic>tetO</italic> repeats. Following sonication during the ChIP protocol, the majority of fragments are much shorter than this (in the range of ∼200 bp), so a primer set at this distance is expected to only inefficiently detect proteins recruited to the <italic>tetO</italic> repeats themselves. As suggested by the reviewers, we have now analyzed a site on the other side of the <italic>tetO</italic> repeats which is much closer (∼50bp) to the tethering site. As can be seen in <xref ref-type="fig" rid="fig6">Figure 6D, F</xref> in the revised manuscript, both Brn1 (condensin) and Rts1 (PP2A) are efficiently recruited to this site, and Ipl1 (CPC) less so. This result now provides compelling evidence for the direct recruitment of condensin by Sgo1.</p><p>As further suggested by the reviewers, we also tethered the Sgo1-100, Sgo1-700 and Sgo1-3A proteins to the centromere in a strain that otherwise lacks <italic>SGO1</italic>. As we showed in <xref ref-type="fig" rid="fig1">Figure 1A</xref>, normally, both Sgo1-100 and Sgo1-700 fail to be maintained on centromeres. However, Sgo1-100 can bind condensin, whereas Sgo1-700 cannot (<xref ref-type="fig" rid="fig9">Figure 9B</xref>). This finding is recapitulated in the tethering experiment. Only Sgo1-100 and not Sgo1-700 is capable of recruiting Brn1 (condensin) to the tethering site. As expected, tethered Sgo1-3A, which localizes to centromeres and associates with condensin is also able to recruit condensin to the tethering site. This data is shown in <xref ref-type="fig" rid="fig9">Figure 9D</xref>. Together with the finding that only the <italic>sgo1-700</italic> mutant loses the bias to sister kinetochore biorientation (<xref ref-type="fig" rid="fig9">Figure 9F</xref>), this provides strong evidence that Sgo1 biases sister kinetochores for biorientation through condensin recruitment.</p><p>We also tested the ability of the tethered Sgo1 proteins to rescue sister kinetochore biorientation after treating an unsynchronous population with nocodazole and examining segregation of the chromosome to which Sgo1-TetR-GFP is tethered in anaphase cells after nocodazole washout. Similar to the assays in <xref ref-type="fig" rid="fig1 fig7">Figures 1C and 7B</xref>, accurate segregation in this assay would require the activity of the error correction machinery. As expected, due to the impaired ability of all Sgo1 mutant proteins to maintain Ipl1 association with the centromere (<xref ref-type="fig" rid="fig9">Figure 9D</xref>) only tethered wild type Sgo1 was able to partially rescue the segregation of the chromosome to which it was tethered (chromosome IV; shown in <xref ref-type="fig" rid="fig9s1">Figure 9–figure supplement 1</xref>). For technical reasons (both <italic>SGO1-tetR-GFP</italic> and <italic>tetR-tdTomato</italic> are integrated into the same genomic locus), it was necessary to perform this experiment in diploid cells and therefore we cannot presently test the ability of these tethered constructs to retain sister kinetochore bias (we cannot easily arrest and release the cells from G1 as they are not sensitive to mating pheromone). Moreover, other unpublished findings in the lab have suggested that the dynamicity of Sgo1 on centromeres is key to its regulation and important for accurate segregation so it is likely that Sgo1 tethering affects its function. Indeed, even tethered wild type Sgo1-tetR-GFP is not fully able to rescue the mis-segregation of chromosome IV in our experiment (<xref ref-type="fig" rid="fig9s1">Figure 9–figure supplement 1</xref>). Nevertheless, our demonstration of a lack of sister kinetochore bias in the <italic>sgo1-700</italic> mutant, which cannot bind condensin (<xref ref-type="fig" rid="fig9">Figure 9B</xref>) or recruit it to centromeres (<xref ref-type="fig" rid="fig9">Figure 9B</xref>) provides compelling evidence that the Sgo1-condensin interact is critical in biasing sister kinetochores to biorient.</p><p><italic>An explanation for the altered binding profiles of condensin measured by ChIP-seq might be changes in cohesin accumulation at centromeres in the Sgo1 mutants. The authors need to check by ChIP-qPCR (and ideally also ChIP-seq) whether cohesin levels at centromeres are affected in the Sgo1 mutant</italic>.</p><p>We performed both these experiments and the data is shown in <xref ref-type="fig" rid="fig5">Figure 5</xref> and associated figure supplements. Cohesin association with chromosomes is indistinguishable in wild type and <italic>sgo1</italic>? cells.</p><p>Overall, we feel our study now argues strongly for a direct role of Sgo1 in condensin recruitment to the pericentromere.</p><p><italic>2) The authors monitor the splitting of a sister centromere pair as a measure for sister chromatid bi-orientation (</italic><xref ref-type="fig" rid="fig1 fig5"><italic>Figures 1A-C,F, 5A</italic></xref><italic>,</italic> <xref ref-type="fig" rid="fig3s2"><italic>Figure 3–figure supplement 2</italic></xref><italic>). The reduction of sister centromere splitting observed in the Sgo1 and condensin mutants could, however, equally well result from an increase in centromeric sister chromatid cohesion. An increase in centromeric cohesion would prevent splitting of bi-oriented sister chromatids</italic>.</p><p><italic>One possibility to distinguish whether chromatid pairs have attached to spindle microtubules from only one spindle pole or whether chromatids have bi-oriented but their centromeres cannot separate would be to score centromere-spindle pole body (SPB) distances in the live cell microscopy assay (</italic><xref ref-type="fig" rid="fig5"><italic>Figures 5B-F</italic></xref><italic>). If centromeres were bi-oriented but merely failed to split, they should be positioned at equal distances to both SPBs. If sister centromeres failed to bi-orient, they should frequently be found close to one SPB. Alternatively, the authors could release cells from the Cdc20 arrest and allow them to enter anaphase, then score whether sister centromeres segregate to one pole or to opposite poles (see Indjeian and Murray, CurBiol 2007)</italic>.</p><p>As mentioned above and below, we have obtained no evidence that Sgo1 affects cohesion in budding yeast mitosis. Importantly, the distance between separated sister centromeres in <italic>sgo1</italic>? and <italic>YCS5-aid</italic> cells is not significantly different to that of wild type cells (<xref ref-type="fig" rid="fig5">Figure 5</xref>). This is strong evidence that cohesion is not affected in these cells and that the decreased frequency of sister centromere is due to defective sister-kinetochore biorientation. Furthermore, as suggested by the reviewers, we measured the distance from <italic>CEN4-GFP</italic> foci to the nearest SPB, in <italic>sgo1</italic>?, <italic>YCS5-aid</italic> and <italic>ipl1-as</italic> cells with a single <italic>CEN4-GFP</italic> focus after nocodazole washout (shown in <xref ref-type="fig" rid="fig9s1">Figure 9–figure supplement 1</xref>). Taking the median <italic>CEN4-SPB</italic> distance for wild type as the typical distance, we scored the percentage of cells with shorter or longer distances in each case. In all three mutants, the fraction of cells with a <italic>CEN4-SPB</italic> shorter than the wild type median was increased, indicating that unpaired sister centromeres tend to reside closer to the SPB, supporting the idea that chromosomes fail to align in the centre of the spindle, as expected when biorientation fails. This effect was less pronounced in the condensin mutant, but this is expected as this has a milder biorientation defect overall (<xref ref-type="fig" rid="fig7s1">Figure 7–figure supplement 1</xref>). Indeed, Ipl1 is localized in <italic>YCS5-aid</italic> cells (<xref ref-type="fig" rid="fig7">Figure 7G</xref>) and the error correction process is partially functional (<xref ref-type="fig" rid="fig7">Figure 7F</xref>).</p><p>The mis-segregation of chromosomes following release from nocodazole treatment into anaphase is well established to occur in <italic>sgo1</italic> mutants (<xref ref-type="bibr" rid="bib11">Fernius and Hardwick, 2007</xref>; <xref ref-type="bibr" rid="bib22">Indjeian and Murray, 2007</xref>) and we have recapitulated these findings in <xref ref-type="fig" rid="fig9s1">Figure 9–figure supplement 1</xref>, using tethered <italic>sgo1</italic> alleles. However, similar experiments with condensin mutants are difficult to interpret as condensin is critical for chromosome arm segregation in anaphase (Renshaw et al., Dev Cell 2010). The situation is further complicated by the fact that due to the partial functionality of the error correction machinery in condensin mutants (<xref ref-type="fig" rid="fig7">Figure 7F</xref>), condensin is unlikely to be essential for sister kinetochore biorientation, but rather provide a bias that facilitates sister kinetochore capture and efficient error correction. Analysis of the <italic>sgo1-700</italic> mutant that fails to associate with condensin (<xref ref-type="fig" rid="fig7">Figure 7</xref>) provides strong evidence that Sgo1 promotes the bias to sister kinetochore biorientation through condensin recruitment.</p><p><italic>The data in</italic> <xref ref-type="fig" rid="fig6 fig7"><italic>Figures 6 and 7</italic></xref> <italic>show a mild, real defect in biorientation but don't specifically address the of kineteochore geometry. The authors should either perform further experiments to support their hypothesis or confine their “geometric bias” to speculation in the Discussion</italic>.</p><p>We agree with the reviewers that condensin mutants show a biorientation defect. We also agree that direct evidence for a role of condensin in kinetochore geometry is lacking. We have therefore restricted our references to a possible role in geometry to speculation.</p><p><italic>3) Most of the graphs that represent the results from ChIP-qPCR experiments (</italic><xref ref-type="fig" rid="fig1 fig2 fig5 fig7"><italic>Figures 1A, E, 2C, D, 5G, 7C</italic></xref><italic>,</italic> <xref ref-type="fig" rid="fig5s3"><italic>Figure 5–figure supplement 3</italic></xref><italic>) lack information that is essential to evaluate the data. What do the column plots represent? Do they show mean values, and if yes, from how many PCR runs and how many independent immunoprecipitation experiments? What do the error bars represent? The legend for</italic> <xref ref-type="fig" rid="fig4"><italic>Figures 4D-F</italic></xref> <italic>is missing completely</italic>.</p><p>We apologize for this oversight. We have now included a more detailed description of the procedures used in the Materials and methods and detailed figure legends throughout, explaining how each experiment was carried out, the procedures used to process the data and the meaning of the figures presented. As detailed in the figure legends, the vast majority of ChIP-qPCR experiments show mean values of three independent repeats. qPCR was always performed in triplicate (this information is now included in the Materials and methods). The number of independent immunoprecipitation experiments is given in the figure legends (typically 3). Error bars represent standard error. The exception to this are time course experiments where the day-to-day variability of cell cycle progression means data from different days cannot be combined usefully. Here, experiments are repeated, typically 3 times in total, and a representative experiment is shown. Overall, the pattern does not vary, though the absolute values at each timepoint can.</p><p><italic>Taking into account the possible variability between individual ChIP-qPCR experiments, careful statistical analysis will be required to evaluate the significance of the observed differences. For example, the values measured at CEN4 for the same wild-type strain (AM3513) in two independent Ipl1 HA-ChIP experiments vary 4-fold (compare</italic> <xref ref-type="fig" rid="fig2"><italic>Figures 2C and D</italic></xref><italic>), calling into question whether the 2-fold reduction in ChIP signals in the Sgo1 mutants (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2D</italic></xref><italic>) is significant. Similarly, the ChIP signal at CEN4 measured for the Sgo1-100 mutant in G1 phase is more than 2-fold higher than the signal for wildtype Sgo1 (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1B</italic></xref><italic>). Is the 4-fold increase in Sgo1 ChIP signals during mitosis (e.g., 2-fold when compared to the Sgo1-100 mutant) then still significant</italic>?</p><p>Again, we apologize: these discrepancies are due to our failure to provide adequate figure legends describing the procedures used to obtain the data and can all be accounted for by different growth conditions. In particular, the ChIP-qPCR signals obtained in experiments where nocodazole was directly added to cycling cells are typically much higher than experiments where cells were first arrested in G1 before being released into nocodazole. The fraction of cells that were approaching, or in, mitosis at the time of nocodazole will spend a much greater time in mitosis, where our proteins of interest are presumably continually recruited, thereby accounting for these differences. Of course, repeats are carried out using exactly the same conditions, with all strains within one repeat grown and processed on the same day, allowing direct comparison for those conditions. Specifically, for the experiment shown in <xref ref-type="fig" rid="fig2">Figure 2C</xref> in the old version (now <xref ref-type="fig" rid="fig3">Figure 3D</xref>), cells were first arrested in G1 before release into nocodazole (this was to allow Sgo1-aid degradation in a single cell cycle), whereas cells for the experiment shown in <xref ref-type="fig" rid="fig2">Figure 2D</xref> in the old version (now 3E) were arrested directly in nocodazole. The difference between the levels of Sgo1 and <italic>Sgo1-100</italic> mutant proteins is likely due to slight changes in the timing of alpha factor release in the handling of these cultures (see above about the interpretation of time course experiments). The main point of these experiments was to show that residual association to centromeres occurs even though it is not maintained in nocodazole-arrested cells. We do not think it is appropriate to take any further interpretation based on the absolute values from these experiments as due to the inherent variability of time course experiments; to do so would require exquisitely controlled conditions and much more closely spaced sampling.</p></body></sub-article></article> |