Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Chromosoma
2017 Mar 01;1262:279-286. doi: 10.1007/s00412-016-0592-7.
Show Gene links
Show Anatomy links
Bub1 targeting to centromeres is sufficient for Sgo1 recruitment in the absence of kinetochores.
Williams SJ
,
Abrieu A
,
Losada A
.
???displayArticle.abstract???
Centromeric chromatin containing the histone H3 variant centromere protein A (CENP-A) directs kinetochore assembly through a hierarchical binding of CENPs, starting with CENP-C and CENP-T. Centromeres are also the chromosomal regions where cohesion, mediated by cohesin, is most prominently maintained in mitosis. While most cohesin dissociates from chromosome arms in prophase, Shugoshin 1 (Sgo1) prevents this process at centromeres. Centromeric localization of Sgo1 depends on histone H2A phosphorylation by the kinase Bub1, but whether additional interactions with kinetochore components are required for Sgo1 recruitment is unclear. Using the Xenopus egg cell-free system, we here show that both CENP-C and CENP-T can independently drive centromeric accumulation of Sgo1 through recruitment of Bub1 to the KNL1, MIS12, NDC80 (KMN) network. The spindle assembly checkpoint (SAC) kinase Mps1 is also required for this pathway even in the absence of checkpoint signaling. Sgo1 recruitment is abolished in chromosomes lacking kinetochore components other than CENP-A. However, forced targeting of Bub1 to centromeres is sufficient to restore Sgo1 localization under this condition.
Fig. 1. Both CENP-C and CENP-T promote Sgo1 recruitment to centromeres. a Representative examples of replicated chromosomes assembled in the egg extracts and stained with the indicated antibodies. Insets highlight an individual centromere within each chromosome mass. Scale bar, 10 μm. b Immunoblot analysis of the extracts used to assemble the chromosomes shown in (c–e). Increasing amounts of mock-depleted CSF extract, expressed as percentage, and aliquots of extracts depleted with specific antibodies, as indicated, were analyzed side by side to estimate the extent of each depletion. H1 served as a loading control. c–e Representative examples of chromosomes assembled in the indicated extracts and stained with antibodies against Sgo1 (c), Bub1 (d), and phosphoH2A (pH2A) (e). For validation of the pH2A antibody see Online Resource 1. Scale bar, 10 μm. f Quantification of average fluorescence in centromere pairs per nucleus (chromosome mass), expressed as a percentage of the average obtained in mock depleted extracts. Bars represent mean ± SD. More than ten nuclei were measured per condition in each of the three independent experiments
Fig. 2. Reduced Sgo1 recruitment to chromatin in the absence of CENP-C, CENP-T, Bub1, or Mps1. Immunoblot analysis of chromatin fractions from replicated chromosomes assembled in the indicated extracts and purified by centrifugation through a sucrose cushion (lanes 2–7). Chromatin purified in the same way from a mock assembly reaction without sperm served as control (no sp, lane 1). Histone H1 was used as loading control. Quantification of the Sgo1 signals, normalized to the H1 signals, and expressed relative to the Sgo1 signal in the chromatin obtained in the mock-depleted extract
Fig. 3. Mps1 is required for Bub1 and Sgo1 targeting to the centromere. a Immunoblot analysis of the extracts used in (b–e) to estimate the efficiency of the depletion and to confirm that these depletions did not alter Sgo1 levels in the soluble extracts. RbAp48 was used as loading control. b–e Representative examples of chromosomes assembled in extracts lacking Mps1 or Bub1, as well as in mock-depleted extracts, and stained with antibodies against Bub1 (b), Sgo1 (c), pH2A (d), or a Bub1 antibody (Bub1*) different from the one used in (b) and Fig. 1. CENP-C or CENP-A was used to label centromeres. Insets highlight an individual centromere pair within each chromosome mass. Scale bar, 10 μm
Fig. 4. Complete absence of Bub1 and Sgo1 in CSF chromosomes lacking CENP-C. a, b Depletion of CENP-C is sufficient to fully prevent Sgo1 (a) and Bub1 (b) recruitment to centromeres of chromosomes assembled in CSF extracts. Note that these chromosomes are not replicated and therefore contain each a single kinetochore labeled by CENP-A. Scale bar, 10 μm
Fig. 5. Forced targeting of Bub1 to the centromere rescues Sgo1 targeting in the absence of kinetochores. a Schematic representation of the constructs used in (b–d). b Representative examples of chromosomes assembled in mock-depleted extracts and in CENP-C-depleted extracts supplemented with buffer, cenC, or cenBub1 and stained with the indicated antibodies. Insets highlight an individual centromere within each chromosome mass. Scale bar, 10 μm. c Quantification of the number of chromosome masses showing Sgo1 staining at centromeres in two different experiments (expressed as percentage). More than 120 centromeres were scored per condition in each experiment. d Chromosomes assembled as in (b) stained with pH2A and DAPI. Scale bar, 10 μm
Abrieu,
Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint.
2001, Pubmed,
Xenbase
Abrieu,
Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint.
2001,
Pubmed
,
Xenbase
Basilico,
The pseudo GTPase CENP-M drives human kinetochore assembly.
2014,
Pubmed
Bernad,
Xenopus HJURP and condensin II are required for CENP-A assembly.
2011,
Pubmed
,
Xenbase
Boyarchuk,
Bub1 is essential for assembly of the functional inner centromere.
2007,
Pubmed
,
Xenbase
Carroll,
Dual recognition of CENP-A nucleosomes is required for centromere assembly.
2010,
Pubmed
Drinnenberg,
Recurrent loss of CenH3 is associated with independent transitions to holocentricity in insects.
2014,
Pubmed
Emanuele,
Measuring the stoichiometry and physical interactions between components elucidates the architecture of the vertebrate kinetochore.
2005,
Pubmed
,
Xenbase
Fachinetti,
A two-step mechanism for epigenetic specification of centromere identity and function.
2013,
Pubmed
Fukagawa,
The centromere: chromatin foundation for the kinetochore machinery.
2014,
Pubmed
Gascoigne,
Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes.
2011,
Pubmed
Hinshaw,
An Iml3-Chl4 heterodimer links the core centromere to factors required for accurate chromosome segregation.
2013,
Pubmed
Hiruma,
CELL DIVISION CYCLE. Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling.
2015,
Pubmed
Hori,
CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore.
2008,
Pubmed
Ji,
CELL DIVISION CYCLE. Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80C.
2015,
Pubmed
Kang,
Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells.
2011,
Pubmed
Kawashima,
Shugoshin enables tension-generating attachment of kinetochores by loading Aurora to centromeres.
2007,
Pubmed
Kawashima,
Phosphorylation of H2A by Bub1 prevents chromosomal instability through localizing shugoshin.
2010,
Pubmed
Kim,
Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores.
2015,
Pubmed
Kitajima,
Shugoshin collaborates with protein phosphatase 2A to protect cohesin.
2006,
Pubmed
Kitajima,
Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization.
2005,
Pubmed
Krenn,
Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction.
2012,
Pubmed
Krizaic,
The distinct functions of CENP-C and CENP-T/W in centromere propagation and function in Xenopus egg extracts.
2015,
Pubmed
,
Xenbase
Liu,
Phosphorylation-enabled binding of SGO1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis.
2013,
Pubmed
Liu,
Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates.
2009,
Pubmed
Liu,
Mitotic Transcription Installs Sgo1 at Centromeres to Coordinate Chromosome Segregation.
2015,
Pubmed
Logsdon,
Both tails and the centromere targeting domain of CENP-A are required for centromere establishment.
2015,
Pubmed
Losada,
Cohesin in cancer: chromosome segregation and beyond.
2014,
Pubmed
Losada,
Cohesin release is required for sister chromatid resolution, but not for condensin-mediated compaction, at the onset of mitosis.
2002,
Pubmed
,
Xenbase
Losada,
Identification of Xenopus SMC protein complexes required for sister chromatid cohesion.
1998,
Pubmed
,
Xenbase
McCleland,
The highly conserved Ndc80 complex is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity.
2003,
Pubmed
,
Xenbase
McGuinness,
Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells.
2005,
Pubmed
McKinley,
The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface.
2015,
Pubmed
Milks,
Dissection of CENP-C-directed centromere and kinetochore assembly.
2009,
Pubmed
,
Xenbase
Morin,
CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint.
2012,
Pubmed
,
Xenbase
Nerusheva,
Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation.
2014,
Pubmed
Nishiyama,
Aurora B and Cdk1 mediate Wapl activation and release of acetylated cohesin from chromosomes by phosphorylating Sororin.
2013,
Pubmed
,
Xenbase
Przewloka,
CENP-C is a structural platform for kinetochore assembly.
2011,
Pubmed
Rivera,
Shugoshin regulates cohesion by driving relocalization of PP2A in Xenopus extracts.
2009,
Pubmed
,
Xenbase
Rivera,
Xenopus Shugoshin 2 regulates the spindle assembly pathway mediated by the chromosomal passenger complex.
2012,
Pubmed
,
Xenbase
Sumara,
The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase.
2002,
Pubmed
,
Xenbase
Tang,
Human Bub1 protects centromeric sister-chromatid cohesion through Shugoshin during mitosis.
2004,
Pubmed
Vleugel,
Arrayed BUB recruitment modules in the kinetochore scaffold KNL1 promote accurate chromosome segregation.
2013,
Pubmed
Vleugel,
Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores.
2015,
Pubmed
Yamagishi,
Two histone marks establish the inner centromere and chromosome bi-orientation.
2010,
Pubmed
Yamagishi,
Heterochromatin links to centromeric protection by recruiting shugoshin.
2008,
Pubmed
Yamagishi,
MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components.
2012,
Pubmed
