Nucleosome eviction in mitosis assists condensin loading and chromosome condensation
2016; Springer Nature; Volume: 35; Issue: 14 Linguagem: Inglês
10.15252/embj.201592849
ISSN1460-2075
AutoresEsther Toselli‐Mollereau, Xavier Robellet, Lydia Fauque, Sébastien Lemaire, Christoph Schiklenk, Carlo Klein, Clémence Hocquet, Pénélope Legros, Lia N’Guyen, Léo Mouillard, Émilie Chautard, Didier Auboeuf, Christian H. Haering, Pascal Bernard,
Tópico(s)Chromosomal and Genetic Variations
ResumoArticle6 June 2016free access Transparent process Nucleosome eviction in mitosis assists condensin loading and chromosome condensation Esther Toselli-Mollereau Esther Toselli-Mollereau Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Xavier Robellet Xavier Robellet Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Lydia Fauque Lydia Fauque Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Sébastien Lemaire Sébastien Lemaire Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Christoph Schiklenk Christoph Schiklenk Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Carlo Klein Carlo Klein Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Clémence Hocquet Clémence Hocquet Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Pénélope Legros Pénélope Legros Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Lia N'Guyen Lia N'Guyen Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Léo Mouillard Léo Mouillard Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Emilie Chautard Emilie Chautard Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Didier Auboeuf Didier Auboeuf Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Christian H Haering Christian H Haering Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Pascal Bernard Corresponding Author Pascal Bernard orcid.org/0000-0003-2732-9685 Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Esther Toselli-Mollereau Esther Toselli-Mollereau Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Xavier Robellet Xavier Robellet Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Lydia Fauque Lydia Fauque Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Sébastien Lemaire Sébastien Lemaire Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Christoph Schiklenk Christoph Schiklenk Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Carlo Klein Carlo Klein Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Clémence Hocquet Clémence Hocquet Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Pénélope Legros Pénélope Legros Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Lia N'Guyen Lia N'Guyen Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Léo Mouillard Léo Mouillard Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Emilie Chautard Emilie Chautard Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Didier Auboeuf Didier Auboeuf Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Christian H Haering Christian H Haering Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Pascal Bernard Corresponding Author Pascal Bernard orcid.org/0000-0003-2732-9685 Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France Search for more papers by this author Author Information Esther Toselli-Mollereau1,‡, Xavier Robellet1,‡, Lydia Fauque1,‡, Sébastien Lemaire1, Christoph Schiklenk2, Carlo Klein2, Clémence Hocquet1, Pénélope Legros1, Lia N'Guyen1, Léo Mouillard1, Emilie Chautard1, Didier Auboeuf1, Christian H Haering2 and Pascal Bernard 1 1Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Lyon, France 2Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +33 672 728 197; E-mail: [email protected] The EMBO Journal (2016)35:1565-1581https://doi.org/10.15252/embj.201592849 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA. Synopsis Fission yeast condensin accumulates at nucleosome-depleted regions in the vicinity of highly expressed genes. Nucleosomes hinder condensin localization, and their eviction coupled with transcription seems a key feature of condensin-binding sites. Condensin accumulates at nucleosome-depleted regions during mitosis in fission yeast. Nucleosome eviction by transcriptional co-activators such as Gcn5, Mst2 and RSC is necessary for condensin binding and proper chromosome condensation. Nucleosome constitutes an obstacle for condensin localization. The presence of free, non-nucleosomal DNA might be an important feature of condensin-binding sites in eukaryotes. Introduction In most eukaryotes, chromatin fibres metamorphose into compact and individualized rod-shaped chromosomes during mitosis and meiosis. This profound reorganization, called chromosome condensation, is a strict prerequisite for the accurate segregation of chromosomes. From yeasts to human, chromosome condensation relies upon condensin complexes and topoisomerase II-α (Topo II). It is widely accepted that Topo II ensures decatenation of sister chromatids and chromosomes (Baxter et al, 2011; Charbin et al, 2014). In contrast, how condensins reconfigure chromosome structure in a cell cycle-regulated manner remains poorly understood. Condensins are ring-shaped ATPases that belong to the family of SMC (Structural Maintenance of Chromosomes) protein complexes, which also include cohesin, responsible for sister chromatid cohesion, and the Smc5/Smc6 complex, which is implicated in DNA damage repair (Thadani et al, 2012; Aragon et al, 2013; Hirano, 2016). Eukaryotic condensins are composed of the Smc2 and Smc4 ATPase subunits, called Cut14 and Cut3 in fission yeast, and three non-SMC subunits. Smc2 and Smc4 form a V-shaped heterodimer in which two ATPase heads face each other at the apices of two 50-nm-long coiled-coil arms. A kleisin subunit (called Cnd2 in fission yeast) associates with the ATPase heads, thereby creating a tripartite ring-like structure, and recruits two HEAT-repeat containing subunits. Most eukaryotes possess two condensins, called condensin I and II, which are made of the same Smc2/Smc4 heterodimer but contain different sets of the three non-SMC subunits. Budding and fission yeasts possess a single condensin complex, which is similar to condensin I by protein sequence. Condensin II is nuclear throughout the cell cycle and accumulates on chromosomes at the beginning of prophase. In contrast, condensin I is cytoplasmic during interphase and gains access to chromosomes only from prometaphase to telophase (Ono et al, 2003; Hirota et al, 2004). Fission yeast condensin shows a similar localization pattern as condensin I, with the bulk of the complexes binding chromosomes from prophase to telophase (Sutani et al, 1999). However, a fraction seems to persist on chromosomes during interphase (Aono et al, 2002; Nakazawa et al, 2015). Studies performed in a wide range of systems have substantiated the idea that condensins modify the topology of chromosomal DNA by introducing positive supercoils (Kimura & Hirano, 1997), by topological entrapment of DNA molecules within their ring-like structure (Cuylen et al, 2011) and/or by promoting the re-annealing of unwound genomic DNA segments (Sutani et al, 2015). It remains unclear, however, to what extent each of these molecular activities contribute to the shaping of chromosomes. Equally unclear is how condensins associate with and manipulate chromosomal DNA in the intricate context of the chromatin fibre. From yeasts to mammals, condensins are enriched at centromeres, telomeres and, along chromosome arms, nearby genes that are highly transcribed by either of the three RNA polymerases (D'Ambrosio et al, 2008; Schmidt et al, 2009; Dowen et al, 2013; Kim et al, 2013; Kranz et al, 2013; Nakazawa et al, 2015; Sutani et al, 2015). Several factors have been implicated in the recruitment of condensins along chromosomes. In budding yeast, the cohesin loading factor Scc2/4 has been reported to promote the full level of association of condensin at tRNA genes and at genes encoding ribosomal proteins (D'Ambrosio et al, 2008). The replication fork blocking protein Fob1, in a complex with the monopolin subunits Csm1 and Lrs4, recruits condensin at rDNA repeats (Johzuka & Horiuchi, 2009). Fission yeast monopolin associates with condensin and contributes to its localization at the rDNA repeats and also at the kinetochore, but plays no role along chromosome arms (Tada et al, 2011). In chicken DT40 cells, the chromokinesin Kif4 has been implicated in the localization of condensins (mostly condensin I) along the longitudinal axes of mitotic chromosomes (Samejima et al, 2012). In human cells, the zinc finger protein AKAP95 directly interacts with the kleisin CAP-H and takes part in the recruitment of condensin I onto chromatin (Steen et al, 2000; Eide et al, 2002). More recently, it has been reported that condensins I and II bind the N-terminal tail of histones H2A and H4, respectively (Liu et al, 2010; Tada et al, 2011). However, contacts with histones do not explain the pattern of condensins along chromosomes, and contrasting results have been obtained regarding the role played by histone tails, if any, in the chromosomal association of condensin I (Tada et al, 2011; Shintomi et al, 2015). Thus, the mechanisms through which condensins associate with chromatin remain unclear. The enrichment of condensin nearby highly expressed genes suggests a crucial link between the localization of condensins and a feature associated with high transcription levels. In line with this, chemical inhibition of RNA Pol II reduces condensin association with mitotic chromosomes during early mitosis in fission yeast (Sutani et al, 2015), and transcription factors have been implicated in the loading of condensin (D'Ambrosio et al, 2008; Iwasaki et al, 2015; Nakazawa et al, 2015). Paradoxically, transcription of DNA repeats by RNA Pol I or Pol II obstructs the stable association of condensin in budding yeast (Johzuka & Horiuchi, 2007; Clemente-Blanco et al, 2009, 2011). Fission yeast condensin appears excluded from gene bodies and accumulates towards the 3′ ends of highly expressed genes (Nakazawa et al, 2015; Sutani et al, 2015). Moreover, transcription by all three RNA Pols is usually repressed during mitosis in most eukaryotes (Gottesfeld & Forbes, 1997), when the association of condensin with chromosomes reaches its maximum. In budding yeast, the inhibition of RNA Pol I and Pol II by the Cdc14 phosphatase during anaphase is necessary for condensin binding (Clemente-Blanco et al, 2009, 2011). Thus, the association of condensin with chromatin appears both positively and negatively linked with transcription. What remains unclear, however, is the molecular determinant(s) that defines condensin association sites and what feature(s) associated with transcription takes part in condensin binding. Active gene promoters are associated with histone H3 acetylated at lysines 9 and 14 (H3K9ac and H3K14ac) (Pokholok et al, 2005; Roh et al, 2005; Wiren et al, 2005). Although the bulk of chromatin is deacetylated during mitosis in mammals (Kruhlak et al, 2001; Patzlaff et al, 2010), traces of H3K9ac and H3K14ac persist at some gene promoters (Wang & Higgins, 2013). Gcn5 is the histone acetyltransferase (HAT) subunit of the SAGA complex, an evolutionarily conserved modular transcription coactivator that acetylates nucleosomes, notably H3K9, H3K14 and H3K18 (Koutelou et al, 2010; Weake & Workman, 2012). Gcn5-containing SAGA occupies the promoters and coding regions of active genes. At promoters, Gcn5 occupancy increases with transcription rate (Robert et al, 2004; Govind et al, 2007; Johnsson et al, 2009; Xue-Franzen et al, 2013; Bonnet et al, 2014). By acetylating nucleosomes, SAGA promotes the local formation of an “open” chromatin structure where the transcription pre-initiation complex assembles. Using fission yeast as a model system, we show that condensin binding to chromosomes and mitotic chromosome condensation rely upon Gcn5 HAT activity. Although the majority of Gcn5 is transiently displaced from chromosomes in early mitosis and the bulk of chromatin is deacetylated, Gcn5 and acetylated H3 persist at promoters adjoining a number of highly expressed genes. There, Gcn5 and the ATP-dependent chromatin-remodelling complex RSC (remodels the structure of chromatin) evict nucleosomes and promote the efficient binding of condensin at these nucleosome-depleted regions. Our results suggest that nucleosomes constitute a barrier for the localization of condensin, which is overcome by Gcn5-mediated histone acetylation and chromatin remodelling. Besides providing unanticipated insights into the mechanism of condensin binding to chromatin, our study suggests that the presence of exposed, non-nucleosomal DNA may be an important feature that attracts condensins to highly expressed genes in eukaryotes. Results Gcn5 takes part in mitotic chromosome condensation Fission yeast cells carrying the thermosensitive cut3-477 mutation in the Smc4 condensin subunit cease to divide at 36°C, but continue to proliferate at the semi permissive temperature of 32°C, even though condensin binding to chromosomes is reduced and mitotic chromosome condensation is partly impaired (Saka et al, 1994; Tada et al, 2011; Robellet et al, 2014). To identify the factors that collaborate with condensin, we screened for mutations synthetically lethal with cut3-477 at 32°C (Robellet et al, 2014). We isolated gcn5-47, a nonsense G765A mutation in the gcn5 open reading frame that is predicted to eliminate the C-terminal bromodomain of the protein (Fig 1A and B). Deletion of the complete gcn5 open reading frame was also colethal with cut3-477 at 32°C (Fig 1A) and with the top2-250 allele of Topo II (Appendix Fig S1A). In sharp contrast, neither gcn5-47 nor gcn5Δ lowered the restrictive temperature of mutations eso1-H17 and rad21-K1, which affect sister-chromatid cohesion (Appendix Fig S1B). This indicates that lack of Gcn5 function does not confer a blind hypersensitivity to any perturbation in the structure of chromosomes. Thus, Gcn5 interacts positively and specifically with key chromosome condensation factors. Figure 1. Gcn5 is required for mitotic chromosome condensation Genetic interaction between condensin and Gcn5. Fivefold serial dilutions of fission yeast strains were spotted onto complete medium. Scheme of Gcn5-47. KAT: lysine acetyltransferase. Bromo: bromodomain. Condensation assay. 3D distances between two fluorescently marked loci on chromosome I were measured by live cell microscopy in fission yeast cells progressing from late G2 through mitosis. Time lapse recording for n > 32 cells for one (wild type) or two (gcn5-47 and cut14-208) biological replicates was aligned to anaphase onset (t = 0) and average distances (± s.d.) plotted. Values are listed in Appendix Table S1. Chromosome segregation in gcn5-47 or cut3-477 mutant cells. Cells growing at 32°C were fixed and processed for immunofluorescence against Mis6-HA and Cdc11-GFP to reveal centromeres (Cen) and spindle pole bodies (SPB), respectively. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). Left panel: single mutant cells cut3-477 or gcn5-47 exhibiting chromatin bridges (b) or trailing chromatin (t). Bar: 5 μm. Right panel: frequencies of chromatin bridges and trailing chromatin as a function of spindle length (SPB-SPB distance, n > 40 for each category). Spindle lengths in cells showing chromatin bridges. Boxes indicate 25th, median and 75th percentile. Whiskers are the minimum and maximum (n > 50). ***P < 0.001. Download figure Download PowerPoint To assess whether Gcn5 plays a role in mitotic chromosome condensation, we used a quantitative condensation assay that measures the three dimensional distances between two fluorescently labelled loci located on the left arm of chromosome I as cells pass through mitosis (Petrova et al, 2013). In wild-type cells, the distance between two loci separated by 0.5 or 1.0 Mb of DNA decreased about twofold from G2 phase until late anaphase (Fig 1C). In gcn5-47 mutant cells, the distances between the two loci combinations remained larger throughout the mitotic time course (Fig 1C), even though condensation was not as severely affected as in the cut14-208 condensin mutant. The distances between the fluorescently labelled loci were also slightly enlarged during interphase in gcn5-47 cells (Fig 1C). Reduced acetylation of nucleosomes might relax chromatin fibres during interphase. Alternatively, or additionally, lack of Gcn5 might impair condensin-mediated chromosome shaping throughout the cell cycle. Consistent with impaired condensation, gcn5-47 mutant cells exhibited frequent chromatin bridges or chromatin trailing in anaphase (Fig 1D) and failed to efficiently disentangle the rDNA repeats located on the arms of chromosome III (Fig EV1). These phenotypes are frequently observed as a consequence of defects in mitotic chromosome condensation (Tada et al, 2011), for example in the cut3-477 condensin mutant (Figs 1D and EV1). However, most chromatin bridges disappeared during late anaphase B in gcn5-47 mutant cells, unlike in the more severe cut3-477 mutant. Plotting the number of chromatin bridges as a function of spindle length suggested that chromosome arms eventually achieved complete separation in gcn5-47 cells, at a time when the mitotic spindle was 25% longer compared to wild-type cells (Fig 1E). Taken together, these data indicate that Gcn5 is required for the efficient condensation of chromosome arms during mitosis. Because condensation is partly impaired in the absence of Gcn5 function, the complete separation of chromosome arms necessitates a longer mitotic spindle. Click here to expand this figure. Figure EV1. Chromosomes III remain untangled during anaphase in gcn5 mutant cellsScheme of chromosome III in which ~100 copies of 10 kb rDNA repeats, each consisting of 5.8S, 18S and 28S rDNA genes, are located adjacent to the telomeres. Fib1, which binds the rDNA repeats, was used to monitor the segregation of the arms of chromosome III in late anaphase cells. Fission yeast cells exponentially growing at 32°C and expressing Fib1-RFP (rDNA) and Cdc11-GFP (SPB) were fixed, stained with DAPI and examined for chromosome segregation (DAPI) and rDNA segregation (n > 70). Bar: 5 microns. Download figure Download PowerPoint The role of Gcn5 in chromosome condensation relies on its acetyltransferase activity The function of Gcn5 as a transcriptional coactivator raised the possibility that its effect on chromosome condensation might be indirect, that is that Gcn5 controls the transcription of a bona fide condensation factor. However, our data suggest that this is unlikely. The mRNA levels of Topo II and all five condensin subunits were not notably reduced in gcn5-47 or gcn5Δ cells (Fig EV2A), nor were Cut3, Cnd2 or Topo II protein levels (Fig EV2B and C). Moreover, efficient co-immunoprecipitation of Cut3-HA with Cnd2-GFP suggested that the integrity of the condensin complex was not affected in the absence of Gcn5 (Fig EV2C). We also re-analysed available transcriptome data for wild-type and gcn5Δ mutant cells (Helmlinger et al, 2008) by comparing the mRNA levels of 236 genes that have been reported in pombase (www.pombase.org) to be required for chromosome segregation and/or condensation, or to genetically or physically interact with condensin. Using a threshold of at least 1.5-fold up- or downregulation, we found a single hit: the cnp3 gene, which encodes a centromeric protein (Fig EV2D). However, restoring the level of cnp3 mRNA by ectopic expression did not suppress the colethality between gcn5-47 and cut3-477 at 32°C (Fig EV2E and F), indicating that the functional interaction between Gcn5 and condensin was independent of cnp3. We conclude that Gcn5 plays a genuine role in mitotic chromosome condensation. Click here to expand this figure. Figure EV2. The link between Gcn5 and Cut3 is direct A. Steady-state level of condensin and Topo II mRNA in cells lacking functional Gcn5. About 500 ng of total RNA extracted from fission yeast cells exponentially growing at 32°C was reverse-transcribed in the presence (+) or absence (−) of reverse transcriptase (RT) and cDNAs were quantified by real-time qPCR. Indicated values correspond to the average and mean deviation from two independent experiments. B. Steady-state levels of Cut3-GFP and Top2-HA detected by Western blotting. α-Tubulin (Tub) was used a loading control. C. Integrity of the condensin complex as judged by co-immunoprecipitation. Cnd2-GFP was immunoprecipitated from indicated strains arrested in mitosis (septation indexes <4%). Levels of Cnd2-GFP and Cut3-HA in total and immunoprecipitated fractions were assessed by Western blotting. D, E. Steady-state level of cnp3 mRNA measured by RT-qPCR. About 500 ng of total RNA was reverse-transcribed in the presence (+) or absence (−) of RT. pCNP3 indicates that the eponym plasmid bearing the cnd3 gene was inserted into the genome. Averages and s.d. calculated from 2 biological replicates are shown. F. Restoring cnp3 mRNA level does not suppress the negative genetic interaction between gcn5-47 and cut3-477. Cells of indicated genotype were serially diluted fivefold and spotted onto complete media supplemented with phloxin B. Download figure Download PowerPoint The Gcn5-containing SAGA complex consists of three independent functional modules (Weake & Workman, 2012; see Fig 2A): the HAT module, which is composed of Gcn5, Ada2 and Ada3, the Spt module, which includes the Spt8 subunit implicated in the recruitment of the TATA-binding protein to certain promoters, and the ubiquitin protease module, which contains Sgf11 and Sgf73. To test whether SAGA components other than Gcn5 genetically interact with condensin, we combined deletions of representative subunits of each of the three modules with cut3-477 (Fig 2A). Lack of the ada2 or ada3 subunits of the HAT module was colethal with cut3-477 at 32°C, but deletion of subunits of the two other modules had no effect. This finding strongly suggests that condensin is closely linked to the Gcn5 HAT activity. We confirmed this conclusion by testing the catalytically inactive gcn5-E191Q mutant (Helmlinger et al, 2008) (Fig 2B). Figure 2. The link between condensin and Gcn5 relies on its ability to acetylate nucleosomes A–D. Genetic interactions between condensin and (A) subunits of the SAGA complex, (B) catalytically inactive Gcn5, (C) multiples HATs, or (D) Gcn5 and Mst2. Fivefold serial dilutions of fission yeast strains were spotted onto complete medium. Download figure Download PowerPoint Different HATs can have overlapping functions and Mst2 and Elp3 are partially redundant with Gcn5 (Nugent et al, 2010). Of five additional HATs—Hat1, Naa40, Rtt109, Mst2 and Elp3—tested, none showed an obvious negative genetic interaction with cut3-477 (Fig 2C). However, simultaneous deletion of Mst2 and Gcn5 reduced viability of the cut3-477 mutant even further than depletion of Gcn5 alone, which suggests that the two HATs might be partially redundant (Fig 2D). Thus, Gcn5 functionally interacts with condensin through its acetyltransferase activity, possibly in the context of the SAGA complex, and Mst2 is partly redundant with Gcn5 for this function. Gcn5 and Mst2 facilitate condensin binding to mitotic chromosomes To test whether Gcn5 and Mst2 might affect the association of condensin with chromosomes, we assessed chromosomal condensin levels using chromatin immunoprecipitation (ChIP) against the kleisin subunit Cnd2 tagged with GFP. Previous genomewide mapping experiments have shown that the condensin-binding profile along chromosome arms in mitosis consists of low-occupancy binding sites and hot spots of association, which correlate with highly expressed genes (D'Ambrosio et al, 2008; Schmidt et al, 2009; Nakazawa et al, 2015; Sutani et al, 2015). We quantified Cnd2-GFP binding using qPCR at the kinetochore (cnt1), pericentric heterochromatin (dh1) and 14 loci along chromosome arms that represent 9 high-occupancy and 5 low-occupancy binding sites. Since highly expressed genes can be vulnerable to misleading ChIP enrichments (Teytelman et al, 2013), we first verified that the association of Cnd2-GFP with these genes was reduced in the condensin mutant cut3-477 (Fig EV3A–C), as we would expect for bona fide condensin-binding sites. We then arrested wild-type or gcn5 mutant cells in pro/metaphase by the nda3-KM311 tubulin mutation and determined the chromosomal association of Cnd2-GFP by ChIP. In cells lacking Gcn5, binding of Cnd2-GFP was significantly reduced at the kinetochore domain and at all high-occupancy condensin-binding sites (Fig 3A). ChIP signals were even further decreased in cells lacking both Gcn5 and Mst2 (Fig 3A). The reduced ChIP signals were not due to a decrease in Cnd2-GFP protein levels in gcn5Δ mst2Δ cells (Fig 3B). In sharp contrast, Cnd2-GFP occupancy at pericentric heterochromatin (dh1) and at all low-occupancy condensin-binding sites remained unchanged. Click here to expand this figure. Figure EV3. Condensin association sites in fission yeast and their transcription A. Condensin binding assessed by ChIP. Fission yeast cells were arrested in pro/metaphase at 19°C by the nda3-KM311 mutation and processed for ChIP against Cnd2-GFP. % IP correspond to the averages, and s.d. calculated from 6 ChIPs performed on 3 biological replicates. B. Mitotic indexes. Averages and s.d. calculated from the 3 biological replicates used in (A) are shown. C. Steady-state level of Cnd2-GFP. Exponentially growing cells were shifted at 36°C for 2.5 h to inactivate cut3-477, and whole cell extracts assessed for Cnd2-GFP levels by Western blotting using anti-GFP (A11122) antibody. Tubulin (Tub) served as loading control. D, E. RNA Pol II occupancy assessed by ChIP in mitotically arrested cells. Cells expressing Cnd2-GFP were arrested in mitosis and pr
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