Centromeres contain specialized chromatin that includes the centromere-specific histone H3 variant, spCENP-A/Cnp1. Here we report identification of five fission yeast centromere proteins, Mis14-18. Mis14 is recruited to kinetochores independently of CENP-A, and, conversely, CENP-A does not require Mis14 to associate with centromeres. In contrast, Mis15, Mis16 (strong similarity with human RbAp48 and RbAp46), Mis17, and Mis18 are all part of the CENP-A recruitment pathway. Mis15 and Mis17 form an evolutionarily conserved complex that also includes Mis6. Mis16 and Mis18 form a complex and maintain the deacetylated state of histones specifically in the central core of centromeres. Mis16 and Mis18 are the most upstream factors in kinetochore assembly as they can associate with kinetochores in all kinetochore mutants except for mis18 and mis16, respectively. RNAi knockdown in human cells shows that Mis16 function is conserved as RbAp48 and RbAp46 are both required for localization of human CENP-A.

Defects in kinetochore proteins often lead to aneuploidy and cancer. Mis12-Mtw1 is a conserved, essential kinetochore protein family. Here, we show that a Mis12 core complex exists in Schizosaccharomyces pombe and human cells. Nine polypeptides bind to human hMis12; two of these, HEC1 and Zwint-1, are authentic kinetochore proteins. Four other human proteins of unknown function (c20orf172, DC8, PMF1 and KIAA1570) correspond to yeast Mis12-Mtw1 complex components and are shown to be required for chromosome segregation in HeLa cells using RNA interference (RNAi). Surprisingly, hMis12 also forms a stable complex with the centromeric heterochromatin components HP1alpha and HP1gamma. Double HP1 RNAi abolishes kinetochore localization of hMis12 and DC8. Therefore, centromeric HP1 may be the base to anchor the hMis12 core complex that is enriched with coiled coils and extends to outer Zwint-1 during mitosis.

Heterochromatin protein 1 (HP1) has an essential role in heterochromatin formation and mitotic progression through its interaction with various proteins. We have identified a unique HP1alpha-binding protein, POGZ (pogo transposable element-derived protein with zinc finger domain), using an advanced proteomics approach. Proteins generally interact with HP1 through a PxVxL (where x is any amino-acid residue) motif; however, POGZ was found to bind to HP1alpha through a zinc-finger-like motif. Binding by POGZ, mediated through its zinc-finger-like motif, competed with PxVxL proteins and destabilized the HP1alpha-chromatin interaction. Depletion experiments confirmed that the POGZ HP1-binding domain is essential for normal mitotic progression and dissociation of HP1alpha from mitotic chromosome arms. Furthermore, POGZ is required for the correct activation and dissociation of Aurora B kinase from chromosome arms during M phase. These results reveal POGZ as an essential protein that links HP1alpha dissociation with Aurora B kinase activation during mitosis.

We have comprehensively mapped long-range associations between chromosomal regions throughout the fission yeast genome using the latest genomics approach that combines next generation sequencing and chromosome conformation capture (3C). Our relatively simple approach, referred to as enrichment of ligation products (ELP), involves digestion of the 3C sample with a 4 bp cutter and self-ligation, achieving a resolution of 20 kb. It recaptures previously characterized genome organizations and also identifies new and important interactions. We have modeled the 3D structure of the entire fission yeast genome and have explored the functional relationships between the global genome organization and transcriptional regulation. We find significant associations among highly transcribed genes. Moreover, we demonstrate that genes co-regulated during the cell cycle tend to associate with one another when activated. Remarkably, functionally defined genes derived from particular gene ontology groups tend to associate in a statistically significant manner. Those significantly associating genes frequently contain the same DNA motifs at their promoter regions, suggesting that potential transcription factors binding to these motifs are involved in defining the associations among those genes. Our study suggests the presence of a global genome organization in fission yeast that is functionally similar to the recently proposed mammalian transcription factory.