For proper partitioning of chromosomes in mitosis, the chromosomal passenger complex (CPC) including Aurora B and survivin must be localized at the center of paired kinetochores, at the site called the inner centromere. It is largely unknown what defines the inner centromere and how the CPC is targeted to this site. Here, we show that the phosphorylation of histone H3-threonine 3 (H3-pT3) mediated by Haspin cooperates with Bub1-mediated histone 2A-serine 121 (H2A-S121) phosphorylation in targeting the CPC to the inner centromere in fission yeast and human cells. H3-pT3 promotes nucleosome binding of survivin, whereas phosphorylated H2A-S121 facilitates the binding of shugoshin, the centromeric CPC adaptor. Haspin colocalizes with cohesin by associating with Pds5, whereas Bub1 localizes at kinetochores. Thus, the inner centromere is defined by intersection of two histone kinases.

The genomic stability of all organisms depends on the precise partition of chromosomes to daughter cells. The spindle assembly checkpoint (SAC) senses unattached kinetochores and prevents premature entry to anaphase, thus ensuring that all chromosomes attach to opposite spindle poles (bi-orientation) during mitosis. MPS1 is an evolutionarily conserved protein kinase required for the SAC and chromosome bi-orientation. Yet, its primary cellular substrate has remained elusive. We show that fission yeast Mph1 (MPS1 homologue) phosphorylates the kinetochore protein Spc7 (KNL1/Blinkin homologue) at the MELT repeat sequences. This phosphorylation promotes the in vitro binding to the Bub1-Bub3 complex, which is required for kinetochore-based SAC activation (Mad1-Mad2-Mad3 localization) and chromosome alignment. Accordingly, a non-phosphorylatable spc7-12A mutation abolishes kinetochore targeting of Bub1-Bub3, whereas a phospho-mimetic spc7-12E mutation forces them to localize at kinetochores throughout the entire cell cycle, even in the absence of Mph1. Thus, MPS1/Mph1 kinase locating at the unattached kinetochores initially creates a mark, which is crucial for SAC activation and chromosome bi-orientation. This mechanism seems to be conserved in human cells.

Reductional chromosome segregation in germ cells, where sister chromatids are pulled to the same pole, accompanies the protection of cohesin at centromeres from separase cleavage. Here, we show that mammalian shugoshin Sgo2 is expressed in germ cells and is solely responsible for the centromeric localization of PP2A and the protection of cohesin Rec8 in oocytes, proving conservation of the mechanism from yeast to mammals. However, this role of Sgo2 contrasts with its mitotic role in protecting centromeric cohesin only from prophase dissociation, but never from anaphase cleavage. We demonstrate that, in somatic cells, shugoshin colocalizes with cohesin in prophase or prometaphase, but their localizations become separate when centromeres are pulled oppositely at metaphase. Remarkably, if tension is artificially removed from the centromeres at the metaphase-anaphase transition, cohesin at the centromeres can be protected from separase cleavage even in somatic cells, as in germ cells. These results argue for a unified view of centromeric protection by shugoshin in mitosis and meiosis.

Successful partition of replicated genomes at cell division requires chromosome attachment to opposite poles of mitotic spindle (bi-orientation). Any defects in this regulation bring about chromosomal instability, which may accelerate tumour progression in humans. To achieve chromosome bi-orientation at prometaphase, the chromosomal passenger complex (CPC), composed of catalytic kinase Aurora B and regulatory components (INCENP, Survivin and Borealin), must be localized to centromeres to phosphorylate kinetochore substrates. Although the CPC dynamically changes the subcellular localization, the regulation of centromere targeting is largely unknown. Here we isolated a fission yeast cyclin B mutant defective specifically in chromosome bi-orientation. Accordingly, we identified Cdk1 (also known as Cdc2)-cyclin-B-dependent phosphorylation of Survivin. Preventing Survivin phosphorylation impairs centromere CPC targeting as well as chromosome bi-orientation, whereas phosphomimetic Survivin suppresses the bi-orientation defect in the cyclin B mutant. Survivin phosphorylation promotes direct binding with shugoshin, which we now define as a conserved centromeric adaptor of the CPC. In human cells, the phosphorylation of Borealin has a comparable role. Thus, our study resolves the conserved mechanisms of CPC targeting to centromeres, highlighting a key role of Cdk1-cyclin B in chromosome bi-orientation.

Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive.Here we have identified meiosis-specific kinetochore factor MEIKIN in mouse, which functions in meiosis I but neither in meiosis II nor in mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated largely by the activity of Polo-like kinase PLK1, which is enriched to kinetochores depending on MEIKIN. Our integrative analysis indicates that MEIKIN is the long awaited key regulator of meiotic kinetochore function, which is conserved from yeasts to humans. 2In mitosis, sister chromatid cohesion is established depending on cohesin in S phase and maintained until metaphase when the sister chromatids are captured by spindle microtubules from opposite poles and aligned on the spindle equator. For the onset of anaphase, the anaphase-promoting complex (APC) triggers the degradation of securin, an inhibitory chaperone for separase that cleaves cohesin RAD21 and removes cohesin along the entire chromosome. This removal of cohesin triggers the separation of sister chromatids and their movement to opposite poles, a process called equational division [1][2][3] . During meiosis, however, meiotic cohesin REC8 largely replaces RAD21 along the entire chromosomes; one round of DNA replication is followed by two rounds of nuclear division, which results in four haploid nuclei or gametes (Fig. 1a).In the first division of meiosis (meiosis I), homologous chromosomes connected by chiasmata are captured from the opposite poles, while sisters are captured from the same pole (mono-orientation). At the onset of anaphase I, REC8 cohesin is cleaved by separase along the arm regions, but protected at centromeres until metaphase II 4-6 . Thus, mono-orientation and centromeric cohesion protection are two hallmarks of meiotic kinetochore function, which are widely conserved among eukaryotic organisms 7-9 (Fig. 1a). There is accumulating evidence that cohesion protection is mediated by the centromeric protein shugoshin (SGO) and its partner protein phosphatase 2A (PP2A) [10][11][12][13][14][15] , which antagonizes REC8 phosphorylation, a prerequisite of cleavage 16, 17 . So far, meiosis-specific kinetochore proteins have been identified only in two yeasts (S. cerevisiae Spo13 and Mam1 (monopolin subunit), and S. pombe Moa1) [18][19][20][21][22][23] . Puzzlingly, however, because their structural and functional similarities remain to be identified, conservation of meiotic kinetochore regulation is questionable even between yeasts 8, 9 . Therefore, in this study, we address the long-standing question of whether meiotic kinetochore regulation is conserved from yeasts to mammals, and, if so, how. Mammalian meiotic kinetochore protein MEIKINFission yeast Moa1 interacts directly with the conserved kinetochore protein Cnp3 (CENP-C homolog), and localizes to the kinetochore in meiosis I 24 . To identify an equivalent meiosis...