POLO kinase regulates the Drosophila centromere cohesion protein MEI-S332

Accurate segregation of chromosomes is critical to ensure that each daughter cell receives the full genetic complement. Maintenance of cohesion between sister chromatids, especially at centromeres, is required to segregate chromosomes precisely during mitosis and meiosis. The Drosophila protein MEI-S332, the founding member of a conserved protein family, is essential in meiosis for maintaining cohesion at centromeres until sister chromatids separate at the metaphase II/anaphase II transition. MEI-S332 localizes onto centromeres in prometaphase of mitosis or meiosis I, remaining until sister chromatids segregate. We elucidated a mechanism for controlling release of MEI-S332 from centromeres via phosphorylation by POLO kinase. We demonstrate that POLO antagonizes MEI-S332 cohesive function and that full POLO activity is needed to remove MEI-S332 from centromeres, yet this delocalization is not required for sister chromatid separation. POLO phosphorylates MEI-S332 in vitro, POLO and MEI-S332 bind each other, and mutation of POLO binding sites prevents MEI-S332 dissociation from centromeres.     

SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana

In meiosis, chromosome cohesion is maintained by the cohesin complex, which is released in a two‐step manner. At meiosis I, the meiosis‐specific cohesin subunit Rec8 is cleaved by the protease Separase along chromosome arms, allowing homologous chromosome segregation. Next, in meiosis II, cleavage of the remaining centromere cohesin results in separation of the sister chromatids. In eukaryotes, protection of centromeric cohesion in meiosis I is mediated by SHUGOSHINs (SGOs). The Arabidopsis genome contains two SGO homologs. Here we demonstrate that Atsgo1 mutants show a premature loss of cohesion of sister chromatid centromeres at anaphase I and that AtSGO2 partially rescues this loss of cohesion. In addition to SGOs, we characterize PATRONUS which is specifically required for the maintenance of cohesion of sister chromatid centromeres in meiosis II. In contrast to the Atsgo1 Atsgo2 double mutant, patronus T‐DNA insertion mutants only display loss of sister chromatid cohesion after meiosis I, and additionally show disorganized spindles, resulting in defects in chromosome segregation in meiosis. This leads to reduced fertility and aneuploid offspring. Furthermore, we detect aneuploidy in sporophytic tissue, indicating a role for PATRONUS in chromosome segregation in somatic cells. Thus, ploidy stability is preserved in Arabidopsis by PATRONUS during both meiosis and mitosis.     

SOLO: a meiotic protein required for centromere cohesion,coorientation, and SMC1 localization in Drosophila melanogaster

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.     

A proposed role for the Polycomb group protein dRING in meiotic sister-chromatid cohesion

ORD protein is required for accurate chromosome segregation during male and female meiosis in Drosophila melanogaster. Null ord mutations result in random segregation of sister chromatids during both meiotic divisions because cohesion is completely abolished prior to kinetochore capture of microtubules during meiosis I. Previous analyses of mutant ord alleles have led us to propose that the C-terminal half of the ORD protein mediates protein-protein interactions that are essential for sister-chromatid cohesion. To identify proteins that interact with ORD, we conducted a yeast two-hybrid screen using an ORD bait and isolated dRING, a core subunit of the Drosophila Polycomb repressive complex 1. We show that a missense mutation in ORD completely ablates the two-hybrid interaction with dRING and prevents nuclear retention of the mutant ORD protein in male meiotic cells. Using affinity-purified antibodies generated against full-length recombinant dRING, we demonstrate that dRING protein is expressed in the male and female gonads and colocalizes extensively with ORD on the chromatin of primary spermatocytes during G2 of meiosis. Our results suggest a novel role for the Polycomb group protein dRING and are consistent with the model that interaction of dRING and ORD is required to promote the proper segregation of meiotic chromosomes.Communicated by R. Paro     

TH2A is phosphorylated at meiotic centromere by Haspin

Histone phosphorylation is sometimes associated with mitosis and meiosis. We have recently identified a phosphorylation of the 127th threonine on TH2A (pTH2A), a germ cell-specific H2A variant, in condensed spermatids and mitotic early preimplantation embryos of mice. Here, we further report the existence of pTH2A at the centromeres in metaphase I spermatocytes and oocytes. Moreover, we identified Haspin, a known kinase for the 3rd threonine on H3, is responsible for pTH2A in vivo. In contrast to the severe meiotic defect in oocytes treated with a Haspin inhibitor, pTH2A-deficient mice, in which the 127th threonine was replaced by alanine, maintained the fertility and exhibited no obvious defect in both oocytes and spermatogenesis. Interestingly, pTH2A was significantly decreased in aged oocytes, suggesting that its accumulation is regulated by centromeric cohesins. Collectively, our study proposes a new set of kinase-histone pair at meiotic centromere, which is highly coordinated during meiosis.     

HP1 links centromeric heterochromatin to centromere cohesion in mammals

Heterochromatin protein‐1 (HP1) is a key component of heterochromatin. Reminiscent of the cohesin complex which mediates sister‐chromatid cohesion, most HP1 proteins in mammalian cells are displaced from chromosome arms during mitotic entry, whereas a pool remains at the heterochromatic centromere region. The function of HP1 at mitotic centromeres remains largely elusive. Here, we show that double knockout (DKO) of HP1α and HP1γ causes defective mitosis progression and weakened centromeric cohesion. While mutating the chromoshadow domain (CSD) prevents HP1α from protecting sister‐chromatid cohesion, centromeric targeting of HP1α CSD alone is sufficient to rescue the cohesion defects in HP1 DKO cells. Interestingly, HP1‐dependent cohesion protection requires Haspin, an antagonist of the cohesin‐releasing factor Wapl. Moreover, HP1α CSD directly binds the N‐terminal region of Haspin and facilitates its centromeric localization. The need for HP1 in cohesion protection can be bypassed by centromeric targeting of Haspin or inhibiting Wapl activity. Taken together, these results reveal a redundant role for HP1α and HP1γ in the protection of centromeric cohesion through promoting Haspin localization at mitotic centromeres in mammalian cells.     

A new role for the mitotic RAD21/SCC1 cohesin in meiotic chromosome cohesion and segregation in the mouse

The evolutionarily conserved cohesin complex is required for the establishment and maintenance of sister chromatid cohesion, in turn essential for proper chromosome segregation. RAD21/SCC1 is a regulatory subunit of the mitotic cohesin complex, as it links together all other subunits of the complex. The destruction of RAD21/SCC1 along chromosomal arms and later at centromeres results in the dissociation of the cohesin complex, facilitating chromosome segregation. Here, we report for the first time that mammalian RAD21/SCC1 associates with the axial/lateral elements of the synaptonemal complex along chromosome arms and on centromeres of mouse spermatocytes. Importantly, RAD21/SCC1 is lost from chromosome arms in late prophase I but persists on centromeres. The loss of centromeric RAD21/SCC1 coincides with the separation of sister chromatids at anaphase II. These findings support a role for mammalian RAD21/SCC1 in maintaining sister chromatid cohesion in meiosis.     

A kinase‐dependent role for Haspin in antagonizing Wapl and protecting mitotic centromere cohesion

Sister‐chromatid cohesion mediated by the cohesin complex is fundamental for precise chromosome segregation in mitosis. Through binding the cohesin subunit Pds5, Wapl releases the bulk of cohesin from chromosome arms in prophase, whereas centromeric cohesin is protected from Wapl until anaphase onset. Strong centromere cohesion requires centromeric localization of the mitotic histone kinase Haspin, which is dependent on the interaction of its non‐catalytic N‐terminus with Pds5B. It remains unclear how Haspin fully blocks the Wapl–Pds5B interaction at centromeres. Here, we show that the C‐terminal kinase domain of Haspin (Haspin‐KD) binds and phosphorylates the YSR motif of Wapl (Wapl‐YSR), thereby directly inhibiting the YSR motif‐dependent interaction of Wapl with Pds5B. Cells expressing a Wapl‐binding‐deficient mutant of Haspin or treated with Haspin inhibitors show centromeric cohesion defects. Phospho‐mimetic mutation in Wapl‐YSR prevents Wapl from binding Pds5B and releasing cohesin. Forced targeting Haspin‐KD to centromeres partly bypasses the need for Haspin–Pds5B interaction in cohesion protection. Taken together, these results indicate a kinase‐dependent role for Haspin in antagonizing Wapl and protecting centromeric cohesion in mitosis.     

A yeast centromere acts in cis to inhibit meiotic gene conversion of adjacent sequences

The centromere of chromosome III (CEN3) of yeast has been examined for its ability to inhibit meiotic recombination in adjacent sequences. The effect of the centromere was investigated when it was adjacent to both of the recombining sequences (homozygous) or adjacent to only one of the two recombining DNA segments (hemizygous). When homozygous, CEN3 exerts a bidirectional repression of crossing over and a strong inhibition of gene conversion. This suggests that CEN3 reduces the frequency of crossing over by interfering with the initiation of proximal recombination events. When hemizygous, CEN3 impairs the ability of adjacent sequences to act as the recipient of genetic information during gene conversion. These results support the idea that the initiating event in yeast meiotic recombination involves the recipient molecule.     

Vertebrate shugoshin links sister centromere cohesion and kinetochore microtubule stability in mitosis

Drosophila MEI-S332 and fungal Sgo1 genes are essential for sister centromere cohesion in meiosis I. We demonstrate that the related vertebrate Sgo localizes to kinetochores and is required to prevent premature sister centromere separation in mitosis, thus providing an explanation for the differential cohesion observed between the arms and the centromeres of mitotic sister chromatids. Sgo is degraded by the anaphase-promoting complex, allowing the separation of sister centromeres in anaphase. Intriguingly, we show that Sgo interacts strongly with microtubules in vitro and that it regulates kinetochore microtubule stability in vivo, consistent with a direct microtubule interaction. Sgo is thus critical for mitotic progression and chromosome segregation and provides an unexpected link between sister centromere cohesion and microtubule interactions at kinetochores.     

The cohesion protein ORD is required for homologue bias during meiotic recombination

During meiosis, sister chromatid cohesion is required for normal levels of homologous recombination, although how cohesion regulates exchange is not understood. Null mutations in orientation disruptor (ord) ablate arm and centromeric cohesion during Drosophila meiosis and severely reduce homologous crossovers in mutant oocytes. We show that ORD protein localizes along oocyte chromosomes during the stages in which recombination occurs. Although synaptonemal complex (SC) components initially associate with synapsed homologues in ord mutants, their localization is severely disrupted during pachytene progression, and normal tripartite SC is not visible by electron microscopy. In ord germaria, meiotic double strand breaks appear and disappear with frequency and timing indistinguishable from wild type. However, Ring chromosome recovery is dramatically reduced in ord oocytes compared with wild type, which is consistent with the model that defects in meiotic cohesion remove the constraints that normally limit recombination between sisters. We conclude that ORD activity suppresses sister chromatid exchange and stimulates inter-homologue crossovers, thereby promoting homologue bias during meiotic recombination in Drosophila.     

Control of centromere localization of the MEI-S332 cohesion protection protein

In mitosis and meiosis, cohesion is maintained at the centromere until sister-chromatid separation. Drosophila MEI-S332 is essential for centromeric cohesion in meiosis and contributes to, though is not absolutely required for, cohesion in mitosis. It localizes specifically to centromeres in prometaphase and delocalizes at the metaphase-anaphase transition. In mei-S332 mutants, centromeric sister-chromatid cohesion is lost at anaphase I, giving meiosis II missegregation. MEI-S332 is the founding member of a family of proteins important for chromosome segregation. One likely activity of these proteins is to protect the cohesin subunit Rec8 from cleavage at the metaphase I-anaphase I transition. Although the family members do not show high sequence identity, there are two short stretches of homology, and mutations in conserved residues affect protein function. Here we analyze the cis- and trans-acting factors required for MEI-S332 localization. We find a striking correlation between domains necessary for MEI-S332 centromere localization and conserved regions within the protein family. Drosophila MEI-S332 expressed in human cells localizes to mitotic centromeres, further highlighting this functional conservation. MEI-S332 can localize independently of cohesin, assembling even onto unreplicated chromatids. However, the separase pathway that regulates cohesin dissociation is needed for MEI-S332 delocalization at anaphase.     

A new seed-based assay for meiotic recombination in Arabidopsis thaliana

Meiotic recombination is a fundamental biological process that plays a central role in the evolution and breeding of plants. We have developed a new seed-based assay for meiotic recombination in Arabidopsis. The assay is based on the transformation of green and red fluorescent markers expressed under a seed-specific promoter. A total of 74 T-DNA markers were isolated, sequenced and mapped both physically and genetically. Lines containing red and green markers that map 1-20 cM apart were crossed to produce tester lines with the two markers linked in cis yielding seeds that fluoresced both in red and green. We show that these lines can be used for efficient scoring of recombinant types (red only or green only fluorescing seeds) in a seed population derived from a test cross (backcross) or self-pollination. Two tester lines that were characterized during several generations of backcross and self-pollination, one in the background of ecotype Landsberg and one in the ecotype Columbia, are described. We discuss the number of plants and seeds to be scored in order to obtain reliable and reproducible crossing over rate values. This assay offers a relatively high-throughput method, with the benefit of seed markers (similar to the maize classical genetic markers) combined with the advantages of Arabidopsis. It advances the prospect to better understand the factors that affect the rate of meiotic crossover in plants and to stimulate this process for more efficient breeding and mapping.     

Shugoshins: from protectors of cohesion to versatile adaptors at the centromere

Sister chromatids are held together by a protein complex named cohesin. Shugoshin proteins protect cohesin from cleavage by separase during meiosis I in eukaryotes and from phosphorylation-mediated removal during mitosis in vertebrates. This protection is crucial for chromosome segregation during mitosis and meiosis. Mechanistically, shugoshins shield cohesin by forming a complex with the phosphatase PP2A, which dephosphorylates cohesin, leading to its retention at centromeres during the onset of meiotic anaphase and vertebrate mitotic prophase I. In addition to this canonical function, shugoshins have evolved novel, species-specific cellular functions, the mechanisms of which remain a subject of intense debate, but are likely to involve spatio-temporally coordinated interactions with the chromosome passenger complex, the spindle checkpoint and the anaphase promoting complex. Here, we compare and contrast these remarkable features of shugoshins in model organisms and humans.     

A Mec1- and PP4-dependent checkpoint couples centromere pairing to meiotic recombination

The faithful alignment of homologous chromosomes during meiotic prophase requires the coordination of DNA double-strand break (DSB) repair with large-scale chromosome reorganization. Here we identify the phosphatase PP4 (Pph3/Psy2) as a mediator of this process in Saccharomyces cerevisiae. In pp4 mutants, early stages of crossover repair and homology-independent pairing of centromeres are coordinately blocked. We traced the loss of centromere pairing to the persistent phosphorylation of the chromosomal protein Zip1 on serine 75. Zip1-S75 is a consensus site for the ATR-like checkpoint kinase Mec1, and centromere pairing is restored in mec1 mutants. Importantly, Zip1-S75 phosphorylation does not alter chromosome synapsis or DSB repair, indicating that Mec1 separates centromere pairing from the other functions of Zip1. The centromeric localization and persistent activity of PP4 during meiotic prophase suggest a model whereby Zip1-S75 phosphorylation dynamically destabilizes homology-independent centromere pairing in response to recombination initiation, thereby coupling meiotic chromosome dynamics to DSB repair.     

Roles and regulation of the Drosophila centromere cohesion protein MEI-S332 family

In meiosis, a physical attachment, or cohesion, between the centromeres of the sister chromatids is retained until their separation at anaphase II. This cohesion is essential for ensuring accurate segregation of the sister chromatids in meiosis II and avoiding aneuploidy, a condition that can lead to prenatal lethality or birth defects. The Drosophila MEI-S332 protein localizes to centromeres when sister chromatids are attached in mitosis and meiosis, and it is required to maintain cohesion at the centromeres after cohesion along the sister chromatid arms is lost at the metaphase I/anaphase I transition. MEI-S332 is the founding member of a family of proteins that protect centromeric cohesion but whose members also affect kinetochore behaviour and spindle microtubule dynamics. We compare the Drosophila MEI-S332 family members, evaluate the role of MEI-S332 in mitosis and meiosis I, and discuss the regulation of localization of MEI-S332 to the centromere and its dissociation at anaphase. We analyse the relationship between MEI-S332 and cohesin, a protein complex that is also necessary for sister-chromatid cohesion in mitosis and meiosis. In mitosis, centromere localization of 相似文献