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Chromosoma
2017 Mar 01;1262:279-286. doi: 10.1007/s00412-016-0592-7.
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Bub1 targeting to centromeres is sufficient for Sgo1 recruitment in the absence of kinetochores.
Williams SJ
,
Abrieu A
,
Losada A
.
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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 (câe). 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. câe 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 meanâ±âSD. 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 2â7). 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 (bâe) 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. bâe 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 (bâd). 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
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