MOE Laboratory of Biosystems Homeostasis and Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China.
MOE Laboratory of Biosystems Homeostasis and Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China.
J Biol Chem. 2019 Feb 1;294(5):1437-1450. doi: 10.1074/jbc.RA118.006046. Epub 2018 Nov 29.
The inner centromere region of a mitotic chromosome critically regulates sister chromatid cohesion and kinetochore-microtubule attachments. However, the molecular mechanism underlying inner centromere assembly remains elusive. Here, using CRISPR/Cas9-based gene editing in HeLa cells, we disrupted the interaction of Shugoshin 1 (Sgo1) with histone H2A phosphorylated on Thr-120 (H2ApT120) to selectively release Sgo1 from mitotic centromeres. Interestingly, cells expressing the H2ApT120-binding defective mutant of Sgo1 have an elevated rate of chromosome missegregation accompanied by weakened centromeric cohesion and decreased centromere accumulation of the chromosomal passenger complex (CPC), an integral part of the inner centromere and a key player in the correction of erroneous kinetochore-microtubule attachments. When artificially tethered to centromeres, a Sgo1 mutant defective in binding protein phosphatase 2A (PP2A) is not able to support proper centromeric cohesion and CPC accumulation, indicating that the Sgo1-PP2A interaction is essential for the integrity of mitotic centromeres. We further provide evidence indicating that Sgo1 protects centromeric cohesin to create a binding site for the histone H3-associated protein kinase Haspin, which not only inhibits the cohesin release factor Wapl and thereby strengthens centromeric cohesion but also phosphorylates histone H3 at Thr-3 to position CPC at inner centromeres. Taken together, our findings reveal a positive feedback-based mechanism that ensures proper assembly of the functional inner centromere during mitosis. They further suggest a causal link between centromeric cohesion defects and chromosomal instability in cancer cells.
有丝分裂染色体的着丝粒内部区域对姐妹染色单体的黏合以及动粒-微管连接有严格的调控作用。然而，着丝粒内部组装的分子机制仍不清楚。在这里，我们使用 CRISPR/Cas9 基因编辑技术在 HeLa 细胞中敲除 Shugoshin 1（Sgo1）与组蛋白 H2A 上 Thr-120 位磷酸化（H2ApT120）的相互作用，从而有选择地将 Sgo1 从有丝分裂着丝粒中释放出来。有趣的是，表达 Sgo1 的 H2ApT120 结合缺陷突变体的细胞，染色体错分离的比率增加，着丝粒黏合减弱，染色体乘客复合物（CPC）在着丝粒处的积累减少。CPC 是着丝粒内部的一个组成部分，也是纠正错误的动粒-微管连接的关键因子。当人为地将 Sgo1 突变体（其与蛋白磷酸酶 2A（PP2A）的结合缺陷）锚定在着丝粒上时，它不能支持适当的着丝粒黏合和 CPC 积累，这表明 Sgo1-PP2A 相互作用对于有丝分裂着丝粒的完整性是必不可少的。我们进一步提供的证据表明，Sgo1 保护着丝粒黏合蛋白以形成与组蛋白 H3 相关蛋白激酶 Haspin 的结合位点，Haspin 不仅抑制黏合释放因子 Wapl，从而增强着丝粒黏合，而且还磷酸化组蛋白 H3 的 Thr-3 位，将 CPC 定位在内着丝粒上。总之，我们的研究结果揭示了一个正反馈机制，该机制确保了有丝分裂过程中功能性着丝粒内部的正确组装。它们还表明，着丝粒黏合缺陷与癌细胞中的染色体不稳定性之间存在因果关系。
