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Guse A
,
Carroll CW
,
Moree B
,
Fuller CJ
,
Straight AF
.
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During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain--required for new CENP-A histone assembly--is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.
Figure 2. CENP-A chromatin specifically recruits kinetochore proteins as a response to a mimic of kinetochore detachment from microtubules(a) A schematic showing the experimental procedure. (b) Quantification of immunofluorescence analysis of CENP-C, Ndc80, CENP-E, Mad2, Rod or ZW10 recruitment to chromatin arrays with (+) and without (â) nocodazole (NOC). The levels are rescaled so that CENP-A arrays with (+) nocodazole are set at 1. Error bars represent SEM, n = 3 (p < 0.05 between â and + nocodazole for CENP-E, Mad2, Rod and ZW10 binding to CENP-A chromatin arrays). (c) Western blot analysis of CENP-C, Ndc80, Rod and ZW10 recruitment to CENP-A and H3 chromatin arrays with and without (+/â) nocodazole (NOC) in CSF and cycled egg extracts. H4 levels are shown as a loading control.
Figure 3. Kinetochores assembled on reconstituted CENP-A chromatin bind microtubules and generate a mitotic checkpoint signal(a) Representative images of microtubule polymerization induced by sperm or reconstituted CENP-A and H3 chromatin. Microtubules (green) and Mad2 (magenta) levels are shown. Scale bar, 10μm (b) Quantification of tubulin and DNA associated with CENP-A and H3 chromatin beads. Error bars represent SEM, n = 5 (c) Quantification of tubulin and Mad2 levels associated with CENP-A and H3 chromatin beads after cold shock (4°C) and nocodazole (NOC) treatment. Error bars represent SEM, n = 5 (d) Western blot showing phospho-Wee1 (P-Wee1) levels as an indicator of the cell cycle stage and tubulin levels as a loading control. Samples from different time points after release from mitotic arrest (t 0â², t 10â², t 20â², t 30â², t 40â²) are shown for CENP-A and H3 chromatin arrays, each incubated with nocodazole (+) or with DMSO (â) as a control. (e) Quantification of four independent experiments showing the phospho-Wee1 signal intensity (P-Wee1 signal) over time (min). Error bars represent SEM, n = 4.
Figure 4. The CENP-A C-terminus is required for centromere and kinetochore assembly in Xenopusegg extract(a) A schematic showing the different CENP-A/H3 chimeras used in this study. The numbers on top represent the amino acid (AA) within HsCENP-A. (b) Quantification of immunofluorescence analysis of CENP-C, CENP-K and CENP-N recruitment to wild type and chimeric arrays. The relative amounts of each centromere protein bound to the arrays are shown relative to CENP-A arrays set to 1. Error bars represent SEM, n = 3 (p ⤠0.05 for all proteins binding to CENP-A arrays compared to chimeric arrays except for the H3 arrays containing the CENP-A C-terminus). (c) Quantification of immunofluorescence analysis of Ndc80, CENP-E, Mad2 recruitment to chimeric chromatin arrays with (+) and without (â) nocodazole (NOC). Values are displayed relative to CENP-A arrays in the presence of nocodazole set to 1. Error bars represent SEM, n = 4. The efficiencies of recruitment of kinetochore proteins to CENP-A and H3+CAC arrays in nocodazole were not statistically distinguishable (p ⥠0.26 for Ndc80, CENP-E and Mad2). (d) Quantification of microtubule binding to CENP-A, H3, H3+HsCAC and H3+XlCAC chromatin arrays represented as percentage of beads associated with tubulin levels above threshold (dark grey bars, left y-axis). Average DNA levels on chromatin beads are shown representing the levels of chromatin arrays bound to beads (light grey bars, right y-axis). Error bars represent SEM, n = 4 for CENP-A and H3 arrays, n = 5 for H3+HsCAC arrays and n = 2 for H3+XlCAC arrays. (e) Western blot analysis shows phospho-Wee1 (P-Wee1) levels as an indicator of the cell cycle stage at t 0â² (0 min) and t 40â² (40 min) after mitotic exit. Tubulin levels are shown as a loading control. (f) Quantification of the phospho-Wee1 signal intensity over time (P-Wee1). Error bars represent SEM, n = 5.
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