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，对于忠实的染色体分离至关重要。我们从遗传学上确定了两个因子，Mis 6和Ams 2，每个因子都是正确定位Cnp 1（CENP-A的裂变酵母同源物）所需的。本研究对Mis 6、Ams 2和Cnp1.1的分子特征进行了研究，并鉴定了Mis 6 -302突变体的多拷贝抑制基因Sim 4/Mix 1，它与Mis 6形成稳定的复合物。我们发现，Mis 6-Sim 4亚复合物是必需的有丝分裂定位的Mad 2，但不是Bub 1，纺锤体检查点蛋白质在着丝粒。此外，我们证明了Nuf 2是必需的维持Mis 6-复合物在有丝分裂的动粒。Mis 6复合物在Mad 2依赖性检查点被激活的条件下与Mad 2物理相互作用。异位表达的Mis 6 ^<1-265>片段沿着有丝分裂纺锤体定位，突出Mis 6与纺锤体微管的潜在结合能力 ...更多信息 S.我们认为Mis 6复合物与Nuf 2复合物共同监控纺锤体-动粒的附着状态，并作为Mad 2在未附着的动粒上积累的平台。2.我们证明了在细胞周期中至少有两个不同的Cnp 1负载阶段。GATA型转录因子Ams 2在S期促进组蛋白基因的转录激活，并有助于将Cnp 1有效装载到重复的着丝粒中。在Ams 2-null细胞中，Cnp 1在S期通过后未能定位于着丝粒;然而，它通过后备重新加载途径再次逐渐积累，这发生在G2期，即有丝分裂中DNA复制和核分裂之间的差距期。在Ams 2-null细胞中G2期长度的缩短导致Cnp 1在着丝粒处积累的显著减少，从而导致有丝分裂细胞死亡和染色体错误分离。Cnp 1负载的灵活性可以解释当真实的着丝粒被损坏时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