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E-GEOD-4792 - Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1
Released on 10 June 2010, last updated on 27 March 2012
The segregation of maternal centromeres away from the paternal ones during the first division of meiosis depends on the attachment of sister kinetochores to microtubules emanating from the same spindle pole. In budding yeast monopolar attachment requires the recruitment to kinetochores of a protein complex called monopolin. The biochemical function of monopolin was unknown. Here, we have identified the casein kinase I Hrr25 as a hitherto unknown subunit of monopolin. Hrr25 differs from other monopolin components by its enzymatic activity and strong evolutionary conservation. We demonstrate that Hrr25’s kinase activity and its interaction with monopolin are both required for monopolar attachment. Accordingly, Hrr25 is associated with centromeres in meiosis I. Our results revealed a surprising new role for casein kinases and provide a hypothesis for the mechanism of monopolar attachment during meiosis I in sexually reproducing organisms: casein kinase I-dependent phosphorylation of kinetochore proteins. Keywords: ChIP-chip, Meiosis, Cell cycle, Saccharomyces cerevisiae, Chromosome VI tiling array, Hrr25, Mam1 • Experimental factors Distribution of the Hrr25 and Mam1 at Meiosis I. Distribution of Hrr25 in Mam1 deletion mutant (S.cerevisiae). All experiments were performed in cells with the same genetic background (Saccharomyces cerevisiae SK1). • Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Cerevisiae chromosome VI array was used. • Quality control steps taken Confirmation using different subunits of the same complex. Confirmation of protein distribution using deletion mutant strain. Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used. Swapping SUP fraction. Q-PCR. 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 VI were 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 (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 3’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). • 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-value≤0.025). Secondly, reliability of binding ratio was judged by change p-value (p-value≤0.025). 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
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Aliona Bogdanova, Joao Matos, Juraj Gregan, Karl Mechtler, Katsuhiko Shirahige, Kim Nasmyth, Mark Petronczki, Martin Schwickart, Saori Mori, Wolfgang Zachariae
Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1. Petronczki M, Matos J, Mori S, Gregan J, Bogdanova A, Schwickart M, Mechtler K, Shirahige K, Zachariae W, Nasmyth K.