Spo13 facilitates monopolin recruitment to kinetochores and regulates maintenance of centromeric cohesion
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE4813 | GEO | 2006/05/16
SECONDARY ACCESSION(S): PRJNA95617
REPOSITORIES: GEO
Similar Datasets
Project description:Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres. Keywords: ChIP-chip, Mitosis, Meiosis, Cell cycle, Saccharomyces cerevisiae, Chromosome VI tiling array, Sgo1, Pp2A, Cse4, Ndc10, Rts1, Rec8
2006-05-10 | GSE4791 | GEO
Project description:BACKGROUND: Cells undergoing meiosis perform two consecutive divisions after a single round of DNA replication. During the first meiotic division, homologous chromosomes segregate to opposite poles. This is achieved by (1) the pairing of maternal and paternal chromosomes via recombination producing chiasmata, (2) coorientation of homologous chromosomes such that sister chromatids attach to the same spindle pole, and (3) resolution of chiasmata by proteolytic cleavage by separase of the meiotic-specific cohesin Rec8 along chromosome arms. Crucially, cohesin at centromeres is retained to allow sister centromeres to biorient at the second division. Little is known about how these meiosis I-specific events are regulated. RESULTS: Here, we show that Spo13, a centromere-associated protein produced exclusively during meiosis I, is required to prevent sister kinetochore biorientation by facilitating the recruitment of the monopolin complex to kinetochores. Spo13 is also required for the reaccumulation of securin, the persistence of centromeric cohesin during meiosis II, and the maintenance of a metaphase I arrest induced by downregulation of the APC/C activator CDC20. CONCLUSION: Spo13 is a key regulator of several meiosis I events. The presence of Spo13 at centromere-surrounding regions is consistent with the notion that it plays a direct role in both monopolin recruitment to centromeres during meiosis I and maintenance of centromeric cohesion between the meiotic divisions. Spo13 may also limit separase activity after the first division by ensuring securin reaccumulation and, in doing so, preventing precocious removal from chromatin of centromeric cohesin. Keywords: Meiosis, Cell cycle, Saccharomyces cerevisiae, Chromosome VI tiling array, Spo13, ChIP-chip ⢠Experimental factors Distribution of the Spo13 at Meiosis I. Experiment was performed in Saccharomyces cerevisiae SK1 strain. ⢠Experimental design ChIP analysis: Hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Cerevisiae chromosome VI array was used. ⢠Quality control steps taken Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used. Sup swapping. Samples used, extract preparation and labelling: ⢠The origin of each biological sample Saccharomyces cerevisiae (SK1). ⢠Manipulation of biological samples and protocols used Chromatin immunoprecipitation (ChIP) and hybridization to Affimetrix high-density oligonucleotide arrays of S. cerevisiae chromosome VIwere performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). ⢠Technical protocols for preparing the hybridization extract The chromatin-immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. ⢠Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (Lengronne et al. Nature 2004). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3âbiotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). ⢠Measurement data and specifications: Arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the cel files data and processed data files can be downloaded from GEO database. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site. The analysis is available on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-valueâ¤0.025). Secondly, reliability of binding ratio was judged by change p-value (p-valueâ¤0.0025). Thirdly, clusters consisting of at least 900bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. ⢠Array Design: General array design: in situ synthesized arrays by Affymetrix Availability of arrays: commercially available from Affymetrix Location and ID of each spot on arrays: available from Affymetrix on request Probe type: oligonucleotide The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACFC6, P/N# 510636 GSM108198 was used for normalization of GSM108511.
2010-06-11 | E-GEOD-4813 | biostudies-arrayexpress
Project description:The biorientation of sister chromatids on the mitotic spindle, essential for accurate sister chromatid segregation, relies on critical centromere components including cohesin, the centromere-specific H3 variant CENP-A, and centromeric DNA. Centromeric DNA is highly variable between chromosomes yet must accomplish a similar function. Moreover, how the 50 nm cohesin ring, proposed to encircle sister chromatids, accommodates inter-sister centromeric distances of hundreds of nanometers on the metaphase spindle is a conundrum. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We used ChIP-seq and super-resolution microscopy to examine the geometry of essential centromeric components on human chromosomes. ChIP-seq of SA1, SA2, and Rad21 in human cells demonstrates that cohesin subunits are depleted in -satellite arrays where CENP-A nucleosomes and kinetochores assemble. Cohesin is instead enriched at pericentromeric DNA. Structured illumination microscopy of sister centromeres is consistent, revealing a non-overlapping pattern of CENP-A and cohesin.
2023-10-11 | GSE240957 | GEO
Project description:During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Diminished Smc5/6 function causes severe defects in nuclear division, but the underlying causes of these defects remain unclear. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of joint-molecule intermediates. Furthermore, we find that Smc5/6 specifically promotes resolution of joint molecules via the XPF-family endonuclease, Mus81-Mms4Eme1. We propose that Smc5/6 acts as a chaperone for M-bM-^@M-^XmitoticM-bM-^@M-^Y-like recombination processes during meiosis. ChIP-chip was used to compare Smc5 localization in wild-type and spo11 strains
2013-12-27 | E-GEOD-44852 | biostudies-arrayexpress
Project description:During meiosis, a single round of DNA replication is followed by two consecutive rounds of nuclear divisions called meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate, while sister chromatids remain together. Determining how this unusual chromosome segregation behavior is established is central to understanding germ cell development. Here we show that preventing microtubule-kinetochore interactions during premeiotic S phase and prophase I is essential for establishing the meiosis I chromosome segregation pattern. Premature interactions of kinetochores with microtubules transform meiosis I into a mitosis-like division by disrupting two key meiosis I events: coorientation of sister kinetochores and protection of centromeric cohesin removal from chromosomes. Furthermore we find that restricting outer kinetochore assembly contributes to preventing premature engagement of microtubules with kinetochores. We propose that inhibition of microtubule-kinetochore interactions during premeiotic S phase and prophase I is central to establishing the unique meiosis I chromosome segregation pattern. The association of the cohesion factors Rec8, Pds5, and Sgo1 were measured by ChIP-chip analysis in wild-type and CUP-CLB3 strains.
2012-10-05 | E-GEOD-41339 | biostudies-arrayexpress
Project description:During meiosis, a single round of DNA replication is followed by two consecutive rounds of nuclear divisions called meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate, while sister chromatids remain together. Determining how this unusual chromosome segregation behavior is established is central to understanding germ cell development. Here we show that preventing microtubule-kinetochore interactions during premeiotic S phase and prophase I is essential for establishing the meiosis I chromosome segregation pattern. Premature interactions of kinetochores with microtubules transform meiosis I into a mitosis-like division by disrupting two key meiosis I events: coorientation of sister kinetochores and protection of centromeric cohesin removal from chromosomes. Furthermore we find that restricting outer kinetochore assembly contributes to preventing premature engagement of microtubules with kinetochores. We propose that inhibition of microtubule-kinetochore interactions during premeiotic S phase and prophase I is central to establishing the unique meiosis I chromosome segregation pattern.
2012-10-05 | GSE41339 | GEO
Project description:Meiosis produces gametes through a specialised, two-step cell division, which is highly error-prone in humans. Reductional meiosis I, where maternal and paternal chromosomes (homologs) segregate, is followed by equational meiosis II, where sister chromatids separate. Uniquely during meiosis I, sister kinetochores are monooriented and pericentromeric cohesin is protected. Here, we demonstrate that these key adaptations for reductional chromosome segregation are achieved through separable control of multiple kinases by the meiosis I-specific budding yeast Spo13 protein. Recruitment of Polo kinase to kinetochores directs monoorientation, while, independently, cohesin protection is achieved by controlling the effects of cohesin kinases. Therefore, reductional chromosome segregation, the defining feature of meiosis, is established by multifaceted kinase control by a master regulator. The recent identification of Spo13 orthologs, fission yeast Moa1 and mouse MEIKIN, suggests that kinase coordination by a master meiosis I regulator may be a general feature in the establishment of reductional chromosome segregation.
2019-11-13 | PXD012627 | Pride
Project description:Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres. Keywords: ChIP-chip, Mitosis, Meiosis, Cell cycle, Saccharomyces cerevisiae, Chromosome VI tiling array, Sgo1, Pp2A, Cse4, Ndc10, Rts1, Rec8 â?¢ Experimental factors Distribution of Cse4, Ndc10, Rts1 in mitotic G2 phase and meiosis I (S. cerevisiae). Distribution of Rec8 in meiosis I (S.cerevisiae). All experiments were performed in cells with the same genetic background (Saccharomyces cerevisiae SK1). â?¢ Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with SUP (supernatant) fraction. SUP fraction is same as WCE fraction. â?¢ Quality control steps taken Duplication, confirmation using different tags, and different subunits of the same complex. Confirmation of protein distribution using deletion mutant strain. Confirmation of several loci by q-PCR. Checking of the ChIP fraction by Western blotting. Checking of the ChIP fraction by swapping SUP fraction. Samples used, extract preparation and labelling: â?¢ The origin of each biological sample Saccharomyces cerevisiae (SK1) . â?¢ Manipulation of biological samples and protocols used Chromatin immunoprecipitation (ChIP) and hybridization to Affimetrix high-density oligonucleotide arrays of S. cerevisiae chromosome VI was performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). â?¢ Technical protocols for preparing the hybridization extract The chromatin-immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. â?¢ Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://everythingchromosomevi.gsc.riken.go.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3â??biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). â?¢ Measurement data and specifications: Arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the raw data files can be downloaded from GEO database. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site at http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf. The analysis is available on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-valueâ?¤0.025 for cerevisiae). Secondly, reliability of binding ratio was judged by change p-value (p-valueâ?¤0.025 for cerevisiae). Thirdly, clusters consisting of at least 900bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. â?¢ Array Design: General array design: in situ synthesized arrays by Affymetrix Availability of arrays: commercially available from Affymetrix Location and ID of each spot on arrays: available from Affymetrix on request Probe type: oligonucleotide The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACFC6, P/N# 510636
2010-06-11 | E-GEOD-4791 | biostudies-arrayexpress
Project description:During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Diminished Smc5/6 function causes severe defects in nuclear division, but the underlying causes of these defects remain unclear. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of joint-molecule intermediates. Furthermore, we find that Smc5/6 specifically promotes resolution of joint molecules via the XPF-family endonuclease, Mus81-Mms4Eme1. We propose that Smc5/6 acts as a chaperone for ‘mitotic’-like recombination processes during meiosis.
2013-12-27 | GSE44852 | GEO
Project description:The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres, where it provides the cohesive counter-force to bi-polar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometeres. This ‘centromere breathing’ presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast S. cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, that has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is re-loaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Keywords: ChIP-chip
2007-09-30 | GSE8661 | GEO