Molecular basis of centromeric chromatin required for equal chromosome segregation

CENP-A是一种着丝粒特异性组蛋白H3变体，对忠实的染色体分离至关重要。我们从遗传学上鉴定了两个因子，Mis6和Ams2，它们都是Cnp1的正确着丝粒定位所必需的，Cnp1是CENP-A的裂变酵母同源物。在本项目中，我们阐明了Mis6、Ams2和Cnp1.1的分子特征。作为Mis6 -302突变体的多拷贝抑制因子，我们发现Sim4/Mix1与Mis6形成稳定的复合物。我们发现Mis6-Sim4亚复合物是Mad2的有丝分裂定位所必需的，而不是Bub1，即着丝粒上的纺锤体检查点蛋白。此外，我们证明在有丝分裂过程中，Nuf2对于维持着丝点上的mis6复合体是必需的。在Mad2依赖的检查点被激活的条件下，mis6复合物与Mad2进行物理相互作用。异位表达的Mis6^<1-265>片段定位于有丝分裂纺锤体，突出了Mis6与纺锤体微管的潜在结合能力。我们提出Mis6复合物与nuf2复合物合作，监测纺锤体-着丝点的附着状态，并作为Mad2在未附着的着丝点上积累的平台。我们证明在细胞周期中至少有两个不同的Cnp1装载阶段。gata型转录因子Ams2在S期促进组蛋白基因的转录激活，并帮助Cnp1有效地装载到复制的着丝粒中。在ams2缺失的细胞中，经过S期后，Cnp1不能定位到着丝粒；然而，在有丝分裂中DNA复制和核分裂之间的间隙期G2期间，它通过备份重新加载途径逐渐积累。在ams2缺失的细胞中，G2长度的缩短导致着丝粒中Cnp1积累的显著减少，导致有丝分裂细胞死亡并伴有染色体错分离。当原着丝粒受损时，Cnp1加载的灵活性可能解释了着丝粒形成的可塑性。少

CENP-A is a centromere-specific histone H3 variant, essential for faithful chromosome segregation. We genetically identified two factors, Mis6 and Ams2, each of which is required for the correct centromere localisation of Cnp1, the fission yeast homologue of CENP-A. In this project, we elucidated following molecular features of Mis6, Ams2 and Cnp1.1.As a multicopy suppressor for mis6-302 mutant, we identified Sim4/Mix1 which forms a stable complex with Mis6. We showed that the Mis6-Sim4 subcomplex is required for mitotic localization of Mad2, but not Bub1, spindle checkpoint protein at centromeres. Furthermore, we demonstrated that Nuf2 is required for maintaining the Mis6-complex on the kinetochore during mitosis. The Mis6-complex physically interacts with Mad2 under the condition that the Mad2-dependent checkpoint is activated. Ectopically expressed Mis6^<1-265> fragment localizes along the mitotic spindle, highlighting the potential binding ability of Mis6 to the spindle microtubule … More s. We propose that the Mis6-complex, in collaboration with the Nuf2-complex, monitors the spindle-kinetochore attachment state and act as a platform for Mad2 to accumulate at unattached kinetochores.2.We demonstrated that there are at least two distinct phases of Cnp1 loading during the cell cycle. A GATA-type transcription factor, Ams2, promotes transcriptional activation of histone genes during S phase and aids in efficient loading of Cnp1 into duplicated centromeres. In Ams2-null cells, Cnp1 fails to localise to centromeres following the passage of S phase ; however, it accumulates again gradually via a backup reloading pathway, which occurs during G2, the gap phase between DNA replication and nuclear division in mitosis. Shortening of G2 length in Ams2-null cells results in marked reduction of Cnp1 accumulation at centromeres, leading to mitotic cell death with chromosome missegregation. The flexibility of Cnp1 loading may account for the plasticity of centromere formation when the authentic centromere is damaged. Less