First person – Masashi Nambu

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Masashi Nambu is first author on ‘ Direct evaluation of cohesin-mediated sister kinetochore associations at meiosis I in fission yeast’, published in JCS. Masashi works in the lab of Ayumu Yamamoto at Faculty of Science, and Graduate School of Integrated Science and Technology, Shizuoka University, Japan, investigating the development of ‘direct’ evaluation of kinetochore association and the contribution of cohesion and its regulators.

Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a “gene drive,” to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an “insertion/deletion” (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of “super-mendelian” inheritance from both male and female mice.

Direct evaluation of cohesin-mediated sister kinetochore associations at meiosis I in fission yeast

Kinetochores drive chromosome segregation by mediating chromosome interactions with the spindle. In higher eukaryotes, sister kinetochores are separately positioned on opposite sides of sister centromeres during mitosis, but associate with each other during meiosis I. Kinetochore association facilitates the attachment of sister chromatids to the same pole, enabling the segregation of homologous chromosomes toward opposite poles. In the fission yeast, Schizosaccharomyces pombe, Rec8-containing meiotic cohesin is suggested to establish kinetochore associations by mediating cohesion of the centromere cores. However, cohesin-mediated kinetochore associations on intact chromosomes have never been demonstrated directly. Here, we describe a novel method for the direct evaluation of kinetochore associations on intact chromosomes in live S. pombe cells, and demonstrate that sister kinetochores and the centromere cores are positioned separately on mitotic chromosomes but associate with each other on meiosis I chromosomes. Furthermore, we demonstrate that kinetochore association depends on meiotic cohesin and the cohesin regulators, Moa1 and Mrc1, and requires mating-pheromone signaling for its establishment. These results confirm cohesin-mediated kinetochore association and its regulatory mechanisms, along with the usefulness of the developed method for its analysis.

Aurora B/C-dependent phosphorylation promotes Rec8 cleavage in mammalian oocytes

To generate haploid gametes, cohesin is removed in a stepwise manner from chromosome arms in meiosis I and the centromere region in meiosis II, to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease Separase[1, 2]. In yeast, Rec8 is kept in a non-phosphorylated state by the action of PP2A-B56, which is localised to the centromere region, thereby preventing cohesin removal from this region in meiosis I[3-5]. However, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and whether centromeric cohesin protection is indeed brought about by dephosphorylation of Rec8 preventing cleavage, in mammalian meiosis. The identity of one or several potential Rec8-specific kinase(s) is also unknown. This is due to technical challenges, as Rec8 is poorly conserved preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals, and additionally, there is no turn-over of Rec8 after cohesion establishment, preventing phospho mutant analysis of functional Rec8. To address how Rec8 cleavage is brought about in mammals, we adapted a biosensor for Separase to study Rec8 cleavage in single mouse oocytes by live imaging, and identified phosphorylation sites promoting cleavage. We found that Rec8 cleavage by Separase depends on Aurora B/C kinase activity, and identified a residue promoting cleavage and being phosphorylated in an Aurora B/C kinase-dependent manner. Accordingly, inhibition of Aurora B/C kinase during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.

A Novel Meiosis-Related lncRNA, Rbakdn, Contributes to Spermatogenesis by Stabilizing Ptbp2

Spermatocyte meiosis is the cornerstone of mammalian production. Thousands of long noncoding RNAs (lncRNAs) have been reported to be functional in various cellular processes, but the function of lncRNAs in meiosis remains largely unknown. Here, we profiled lncRNAs in spermatocytes at stage I of meiosis and identified a testis-specific lncRNA, Rbakdn, as a vital regulator of meiosis. Rbakdn is dynamically expressed during meiosis I, and Rbakdn knockdown inhibits meiosis in vitro. Furthermore, Rbakdn knockdown in testes in mice by intratesticular injection disturbs meiosis, reduces testicular volume, and increases apoptosis of spermatocytes, resulting in vacuolation of the seminiferous tubules. Rbakdn can bind to Ptbp2, an RNA-binding protein that is important in the regulation of the alternative splicing of many genes in spermatogenesis. Rbakdn knockdown leads to a decrease in Ptbp2 through the ubiquitination degradation pathway, indicating that Rbakdn maintains the stability of Ptbp2. In conclusion, our study identified an lncRNA, Rbakdn, with a crucial role in meiosis.

The Cdc14 Phosphatase Controls Resolution of Recombination Intermediates and Crossover Formation during Meiosis

Meiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. The coordinated resolution of meiotic recombination intermediates is required for crossover formation, ultimately necessary for the accurate completion of both rounds of chromosome segregation. Numerous master kinases orchestrate the correct assembly and activity of the repair machinery. Although much less is known, the reversal of phosphorylation events in meiosis must also be key to coordinate the timing and functionality of repair enzymes. Cdc14 is a crucial phosphatase required for the dephosphorylation of multiple CDK1 targets in many eukaryotes. Mutations that inactivate this phosphatase lead to meiotic failure, but until now it was unknown if Cdc14 plays a direct role in meiotic recombination. Here, we show that the elimination of Cdc14 leads to severe defects in the processing and resolution of recombination intermediates, causing a drastic depletion in crossovers when other repair pathways are compromised. We also show that Cdc14 is required for the correct activity and localization of the Holliday Junction resolvase Yen1/GEN1. We reveal that Cdc14 regulates Yen1 activity from meiosis I onwards, and this function is essential for crossover resolution in the absence of other repair pathways. We also demonstrate that Cdc14 and Yen1 are required to safeguard sister chromatid segregation during the second meiotic division, a late action that is independent of the earlier role in crossover formation. Thus, this work uncovers previously undescribed functions of the evolutionary conserved Cdc14 phosphatase in the regulation of meiotic recombination.

Loss of centromeric RNA activates the spindle assembly checkpoint in mammalian female meiosis I

The repetitive sequences of DNA centromeric regions form the structural basis for kinetochore assembly. Recently they were found to be transcriptionally active in mitosis, with their RNAs providing noncoding functions. Here we explore the role, in mouse oocytes, of transcripts generated from within the minor satellite repeats. Depletion of minor satellite transcripts delayed progression through meiosis I by activation of the spindle assembly checkpoint. Arrested oocytes had poorly congressed chromosomes, and centromeres were frequently split by microtubules. Thus, we have demonstrated that the centromeric RNA plays a specific role in female meiosis I compared with mitosis and is required for maintaining the structural integrity of centromeres. This may contribute to the high aneuploidy rates observed in female meiosis.

Selective dephosphorylation by PP2A-B55 directs the meiosis I - meiosis II transition in oocytes

Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we find that protein levels are broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We find that the phosphatase PP2A-B55 is reactivated at the meiosis I/II transition resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI / MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after meiosis I. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.

Accumulation of Securin on Spindle During Female Meiosis I

Chromosome segregation during female meiosis is frequently incorrect with severe consequences including termination of further development or severe disorders, such as Down syndrome. Accurate chromosome segregation requires tight control of a protease called separase, which facilitates the separation of sister chromatids by cohesin cleavage. There are several control mechanisms in place, including the binding of specific protein inhibitor securin, phosphorylation by cyclin-dependent kinase 1 (CDK1), and complex with SGO2 and MAD2 proteins. All these mechanisms restrict the activation of separase for the time when all chromosomes are properly attached to the spindle. In our study, we focused on securin and compared the expression profile of endogenous protein with exogenous securin, which is widely used to study chromosome segregation. We also compared the dynamics of securin proteolysis in meiosis I and meiosis II. Our study revealed that the expression of both endogenous and exogenous securin in oocytes is compartmentalized and that this protein accumulates on the spindle during meiosis I. We believe that this might have a direct impact on the regulation of separase activity in the vicinity of the chromosomes.