Centromeric chromatin in mitosis reorganizes into a functional structure stabilized by cohesin [NG-Capture-C]

Three-dimensional organization of mitotic chromosomes is established by cohesin and condensin complexes. In the centromere, cohesin maintains sister chromatid pairing until anaphase onset, while condensin provides elastic resistance to spindle forces. However, how condensin and cohesin structure vertebrate centromeres remains unclear. By super-resolution imaging, Capture-C analysis and polymer modeling we show that vertebrate centromeres are partitioned into two condensin-dependent subdomains during mitosis. This bipartite sub-structure is found in human, mouse and chicken centromeres, and is therefore a fundamental feature of vertebrate centromere. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores with each subdomain capable of binding a distinct microtubule bundle. Cohesin helps to link the centromere subdomains, limiting their separation in response to spindle forces and preventing merotelic attachments. The two-domain structure described here may have implications for avoiding chromosomal instability as uncoupling of centromere subdomains is a common feature of lagging chromosomes in cancer cells. Overall design: NG-Capture-C analysis to inveistigate chromatin organisation of non-repetitive centromres such as Z and 5 in chicken DT40 CDK1as cells at G2 and prometaphase.

黏连蛋白（cohesin）与凝缩蛋白（condensin）复合物可构建有丝分裂染色体的三维组织结构。在着丝粒（centromere）区域内，黏连蛋白可维持姐妹染色单体配对直至分裂后期起始，而凝缩蛋白则能对纺锤体作用力提供弹性抗性。然而，目前尚不清楚凝缩蛋白与黏连蛋白如何塑造脊椎动物的着丝粒结构。

本研究通过超分辨率成像、Capture-C分析与聚合物建模，证实有丝分裂过程中脊椎动物着丝粒可被划分为两个依赖于凝缩蛋白的亚结构域。该二分亚结构广泛存在于人类、小鼠与鸡的着丝粒中，因此是脊椎动物着丝粒的核心特征之一。

超分辨率成像与电子断层扫描结果显示，具有二分结构的着丝粒可组装形成二分动粒（kinetochore），每个亚结构域均能结合特定的微管束。黏连蛋白可介导着丝粒亚结构域之间的连接，限制其在纺锤体作用力下发生分离，并阻止错端附着（merotelic attachments）的形成。

本研究揭示的双结构域结构，或可为规避染色体不稳定性提供新思路——着丝粒亚结构域的解耦，是癌细胞中滞后染色体的常见特征。

实验设计：本研究采用NG-Capture-C分析，对鸡DT40 CDK1as细胞在G2期与前中期的非重复性着丝粒（如Z型与5型着丝粒）的染色质组织情况进行探究。