Molecular basis of centromeric chromatin required for equal chromosome segregation

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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