Acetylation of Rec8 cohesin complexes regulates reductional chromosome segregation in meiosis.

For establishing sister chromatid cohesion and proper chromosome segregation in mitosis in fission yeast, the acetyltransferase Eso1 plays a key role. Eso1 acetylates cohesin complexes, at two conserved lysine residues K105 and K106 of the cohesin subunit Psm3. Although Eso1 also contributes to reductional chromosome segregation in meiosis, the underlying molecular mechanisms have remained elusive. Here, we purified meiosis-specific Rec8 cohesin complexes localized at centromeres and identified a new acetylation at Psm3-K1013, which largely depends on the meiotic kinetochore factor meikin (Moa1). Our molecular genetic analyses indicate that Psm3-K1013 acetylation cooperates with canonical acetylation at Psm3-K105 and K106, and plays a crucial role in establishing reductional chromosome segregation in meiosis.

Recent advances in mechanisms ensuring the pairing, synapsis and segregation of XY chromosomes in mice and humans.

Sex chromosome aneuploidies are among the most common variations in human whole chromosome copy numbers, with an estimated prevalence in the general population of 1:400 to 1:1400 live births. Unlike whole-chromosome aneuploidies of autosomes, those of sex chromosomes, such as the 47, XXY aneuploidy that causes Klinefelter Syndrome (KS), often originate from the paternal side, caused by a lack of crossover (CO) formation between the X and Y chromosomes. COs must form between all chromosome pairs to pass meiotic checkpoints and are the product of meiotic recombination that occurs between homologous sequences of parental chromosomes. Recombination between male sex chromosomes is more challenging compared to both autosomes and sex chromosomes in females, as it is restricted within a short region of homology between X and Y, called the pseudo-autosomal region (PAR). However, in normal individuals, CO formation occurs in PAR with a higher frequency than in any other region, indicating the presence of mechanisms that promote the initiation and processing of recombination in each meiotic division. In recent years, research has made great strides in identifying genes and mechanisms that facilitate CO formation in the PAR. Here, we outline the most recent and relevant findings in this field. XY chromosome aneuploidy in humans has broad-reaching effects, contributing significantly also to Turner syndrome, spontaneous abortions, oligospermia, and even infertility. Thus, in the years to come, the identification of genes and mechanisms beyond XY aneuploidy is expected to have an impact on the genetic counseling of a wide number of families and adults affected by these disorders.

Direct observation of a crescent-shape chromosome in expanded Bacillus subtilis cells.

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins - the key drivers of chromosomal organization - along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

AurkA/TPX2 co-overexpression in nontransformed cells promotes genome instability through induction of chromosome mis-segregation and attenuation of the p53 signalling pathway.

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells.

Uncharted territories: Solving the mysteries of male meiosis in flies.

The segregation of homologous chromosomes during meiosis typically requires tight end-to-end chromosome pairing. However, in Drosophila spermatogenesis, male flies segregate their chromosomes without classic synaptonemal complex formation and without recombination, instead compartmentalizing homologs into subnuclear domains known as chromosome territories (CTs). How homologs find each other in the nucleus and are separated into CTs has been one of the biggest riddles in chromosome biology. Here, we discuss our current understanding of pairing and CT formation in flies and review recent data on how homologs are linked and partitioned during meiosis in male flies.

Gene regulation during meiosis.

Meiosis is essential for gamete production in all sexually reproducing organisms. It entails two successive cell divisions without DNA replication, producing haploid cells from diploid ones. This process involves complex morphological and molecular differentiation that varies across species and between sexes. Specialized genomic events like meiotic recombination and chromosome segregation are tightly regulated, including preparation for post-meiotic development. Research in model organisms, notably yeast, has shed light on the genetic and molecular aspects of meiosis and its regulation. Although mammalian meiosis research faces challenges, particularly in replicating gametogenesis in vitro, advances in genetic and genomic technologies are providing mechanistic insights. Here we review the genetics and molecular biology of meiotic gene expression control, focusing on mammals.

Homologous chromosome pairing: The linchpin of accurate segregation in meiosis.

Meiosis is a specialized cell division that occurs in sexually reproducing organisms, generating haploid gametes containing half the chromosome number through two rounds of cell division. Homologous chromosomes pair and prepare for their proper segregation in subsequent divisions. How homologous chromosomes recognize each other and achieve pairing is an important question. Early studies showed that in most organisms, homologous pairing relies on homologous recombination. However, pairing mechanisms differ across species. Evidence indicates that chromosomes are dynamic and move during early meiotic stages, facilitating pairing. Recent studies in various model organisms suggest conserved mechanisms and key regulators of homologous chromosome pairing. This review summarizes these findings and compare similarities and differences in homologous chromosome pairing mechanisms across species.

A dynamic population of prophase CENP-C is required for meiotic chromosome segregation.

The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. These two functions, however, happen at different times in the cell cycle. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. We have investigated the dynamics of function of CENP-C during the extended meiotic prophase of Drosophila oocytes and found that maintaining high levels of CENP-C for metaphase I requires expression during prophase. In contrast, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.

Centromere structure and function: lessons from Drosophila.

The fruit fly Drosophila melanogaster serves as a powerful model organism for advancing our understanding of biological processes, not just by studying its similarities with other organisms including ourselves but also by investigating its differences to unravel the underlying strategies that evolved to achieve a common goal. This is particularly true for centromeres, specialized genomic regions present on all eukaryotic chromosomes that function as the platform for the assembly of kinetochores. These multiprotein structures play an essential role during cell division by connecting chromosomes to spindle microtubules in mitosis and meiosis to mediate accurate chromosome segregation. Here, we will take a historical perspective on the study of fly centromeres, aiming to highlight not only the important similarities but also the differences identified that contributed to advancing centromere biology. We will discuss the current knowledge on the sequence and chromatin organization of fly centromeres together with advances for identification of centromeric proteins. Then, we will describe both the factors and processes involved in centromere organization and how they work together to provide an epigenetic identity to the centromeric locus. Lastly, we will take an evolutionary point of view of centromeres and briefly discuss current views on centromere drive.

Meiosis: Dances Between Homologs.

The raison d'être of meiosis is shuffling of genetic information via Mendelian segregation and, within individual chromosomes, by DNA crossing-over. These outcomes are enabled by a complex cellular program in which interactions between homologous chromosomes play a central role. We first provide a background regarding the basic principles of this program. We then summarize the current understanding of the DNA events of recombination and of three processes that involve whole chromosomes: homolog pairing, crossover interference, and chiasma maturation. All of these processes are implemented by direct physical interaction of recombination complexes with underlying chromosome structures. Finally, we present convergent lines of evidence that the meiotic program may have evolved by coupling of this interaction to late-stage mitotic chromosome morphogenesis.

Cell cycle-dependent organization of a bacterial centromere through multi-layered regulation of the ParABS system.

The accurate distribution of genetic material is crucial for all organisms. In most bacteria, chromosome segregation is achieved by the ParABS system, in which the ParB-bound parS sequence is actively partitioned by ParA. While this system is highly conserved, its adaptation in organisms with unique lifestyles and its regulation between developmental stages remain largely unexplored. Bdellovibrio bacteriovorus is a predatory bacterium proliferating through polyploid replication and non-binary division inside other bacteria. Our study reveals the subcellular dynamics and multi-layered regulation of the ParABS system, coupled to the cell cycle of B. bacteriovorus. We found that ParA:ParB ratios fluctuate between predation stages, their balance being critical for cell cycle progression. Moreover, the parS chromosomal context in non-replicative cells, combined with ParB depletion at cell division, critically contribute to the unique cell cycle-dependent organization of the centromere in this bacterium, highlighting new levels of complexity in chromosome segregation and cell cycle control.

Female meiotic drive in plants: mechanisms and dynamics.

Female meiosis is fundamentally asymmetric, creating an arena for genetic elements to compete for inclusion in the egg to maximize their transmission. Centromeres, as mediators of chromosomal segregation, are prime candidates to evolve via 'female meiotic drive'. According to the centromere-drive model, the asymmetry of female meiosis ignites a coevolutionary arms race between selfish centromeres and kinetochore proteins, the by-product of which is accelerated sequence divergence. Here, I describe and compare plant models that have been instrumental in uncovering the mechanistic basis of female meiotic drive (maize) and the dynamics of active selfish centromeres in nature (monkeyflowers). Then, I speculate on the mechanistic basis of drive in monkeyflowers, discuss how centromere strength influences chromosomal segregation in plants, and describe new insights into the evolution of plant centromeres.

Genetic dissection of crossover mutants defines discrete intermediates in mouse meiosis.

Crossovers (COs), the exchange of homolog arms, are required for accurate chromosome segregation during meiosis. Studies in yeast have described the single-end invasion (SEI) intermediate: a stabilized 3' end annealed with the homolog as the first detectible CO precursor. SEIs are thought to differentiate into double Holliday junctions (dHJs) that are resolved by MutLgamma (MLH1/MLH3) into COs. Currently, we lack knowledge of early steps of mammalian CO recombination or how intermediates are differentiated in any organism. Using comprehensive analysis of recombination in thirteen different genetic conditions with varying levels of compromised CO resolution, we infer CO precursors include asymmetric SEI-like intermediates and dHJs in mouse. In contrast to yeast, MLH3 is structurally required to differentiate CO precursors into dHJs. We verify conservation of aspects of meiotic recombination and show unique features in mouse, providing mechanistic insight into CO formation.

Mad2 is dispensable for accurate chromosome segregation but becomes essential when oocytes are subjected to environmental stress.

Accurate chromosome segregation, monitored by the spindle assembly checkpoint (SAC), is crucial for the production of euploid cells. Previous in vitro studies by us and others showed that Mad2, a core member of the SAC, performs a checkpoint function in oocyte meiosis. Here, through an oocyte-specific knockout approach in mouse, we reconfirmed that Mad2-deficient oocytes exhibit an accelerated metaphase-to-anaphase transition caused by premature degradation of securin and cyclin B1 and subsequent activation of separase in meiosis I. However, it was surprising that the knockout mice were completely fertile and the resulting oocytes were euploid. In the absence of Mad2, other SAC proteins, including BubR1, Bub3 and Mad1, were normally recruited to the kinetochores, which likely explains the balanced chromosome separation. Further studies showed that the chromosome separation in Mad2-null oocytes was particularly sensitive to environmental changes and, when matured in vitro, showed chromosome misalignment, lagging chromosomes, and aneuploidy with premature separation of sister chromatids, which was exacerbated at a lower temperature. We reveal for the first time that Mad2 is dispensable for proper chromosome segregation but acts to mitigate environmental stress in meiotic oocytes.

Confinement plus myosin-II suppression maximizes heritable loss of chromosomes, as revealed by live-cell ChReporters.

The mechanical environment of a cell can have many effects, but whether it impacts the DNA sequence of a cell has remained unexamined. To investigate this, we developed a live-cell method to measure changes in chromosome numbers. We edited constitutive genes with GFP or RFP tags on single alleles and discovered that cells that lose Chromosome reporters (ChReporters) become non-fluorescent. We applied our new tools to confined mitosis and to inhibition of the putative tumor suppressor myosin-II. We quantified compression of mitotic chromatin in vivo and demonstrated that similar compression in vitro resulted in cell death, but also rare and heritable ChReptorter loss. Myosin-II suppression rescued lethal multipolar divisions and maximized ChReporter loss during three-dimensional (3D) compression and two-dimensional (2D) lateral confinement, but not in standard 2D culture. ChReporter loss was associated with chromosome mis-segregation, rather than just the number of divisions, and loss in vitro and in mice was selected against in subsequent 2D cultures. Inhibition of the spindle assembly checkpoint (SAC) caused ChReporter loss in 2D culture, as expected, but not during 3D compression, suggesting a SAC perturbation. Thus, ChReporters enable diverse studies of viable genetic changes, and show that confinement and myosin-II affect DNA sequence and mechano-evolution.

Tfs1, transcription elongation factor TFIIS, has an impact on chromosome segregation affected by pka1 deletion in Schizosaccharomyces pombe.

The cAMP-dependent protein kinase (PKA) pathway in Schizosaccharomyces pombe plays an important role in microtubule organization and chromosome segregation. Typically, loss of functional Pka1 induces sensitivity to the microtubule-destabilizing drug thiabendazole (TBZ) and chromosome mis-segregation. To determine the mechanism via which Pka1 is involved in these events, we explored the relevance of transcription factors by creating a double-deletion strain of pka1 and 102 individual genes encoding transcription factors. We found that rst2∆, tfs1∆, mca1∆, and moc3∆ suppressed the TBZ-sensitive phenotype of the pka1∆ strain, among which tfs1∆ was the strongest suppressor. All single mutants (rst2∆, tfs1∆, mca1∆, and moc3∆) showed a TBZ-tolerant phenotype. Tfs1 has two transcriptional domains (TFIIS and Zn finger domains), both of which contributed to the suppression of the pka1∆-induced TBZ-sensitive phenotype. pka1∆-induced chromosome mis-segregation was rescued by tfs1∆ in the presence of TBZ. tfs1 overexpression induced the TBZ-sensitive phenotype and a high frequency of chromosome mis-segregation, suggesting that the amount of Tfs1 must be strictly controlled. However, Tfs1-expression levels did not differ between the wild-type and pka1∆ strains, and the Tfs1-GFP protein was localized to the nucleus and cytoplasm in both strains, which excludes the direct regulation of expression and localization of Tfs1 by Pka1. Growth inhibition by TBZ in pka1∆ strains was notably rescued by double deletion of rst2 and tfs1 rather than single deletion of rst2 or tfs1, indicating that Rst2 and Tfs1 contribute independently to counteract TBZ toxicity. Our findings highlight Tfs1 as a key transcription factor for proper chromosome segregation.

Whole-genome doubling drives oncogenic loss of chromatin segregation.

Whole-genome doubling (WGD) is a recurrent event in human cancers and it promotes chromosomal instability and acquisition of aneuploidies1-8. However, the three-dimensional organization of chromatin in WGD cells and its contribution to oncogenic phenotypes are currently unknown. Here we show that in p53-deficient cells, WGD induces loss of chromatin segregation (LCS). This event is characterized by reduced segregation between short and long chromosomes, A and B subcompartments and adjacent chromatin domains. LCS is driven by the downregulation of CTCF and H3K9me3 in cells that bypassed activation of the tetraploid checkpoint. Longitudinal analyses revealed that LCS primes genomic regions for subcompartment repositioning in WGD cells. This results in chromatin and epigenetic changes associated with oncogene activation in tumours ensuing from WGD cells. Notably, subcompartment repositioning events were largely independent of chromosomal alterations, which indicates that these were complementary mechanisms contributing to tumour development and progression. Overall, LCS initiates chromatin conformation changes that ultimately result in oncogenic epigenetic and transcriptional modifications, which suggests that chromatin evolution is a hallmark of WGD-driven cancer.

Caenorhabditis elegans spermatocytes can segregate achiasmate homologous chromosomes apart at higher than random frequency during meiosis I.

Chromosome segregation errors during meiosis are the leading cause of aneuploidy. Faithful chromosome segregation during meiosis in most eukaryotes requires a crossover which provides a physical attachment holding homologs together in a "bivalent." Crossovers are critical for homologs to be properly aligned and partitioned in the first meiotic division. Without a crossover, individual homologs (univalents) might segregate randomly, resulting in aneuploid progeny. However, Caenorhabditis elegans zim-2 mutants, which have crossover defects on chromosome V, have fewer dead embryos than that expected from random segregation. This deviation from random segregation is more pronounced in zim-2 males than that in females. We found three phenomena that can explain this apparent discrepancy. First, we detected crossovers on chromosome V in both zim-2(tm574) oocytes and spermatocytes, suggesting a redundant mechanism to make up for the ZIM-2 loss. Second, after accounting for the background crossover frequency, spermatocytes produced significantly more euploid gametes than what would be expected from random segregation. Lastly, trisomy of chromosome V is viable and fertile. Together, these three phenomena allow zim-2(tm574) mutants with reduced crossovers on chromosome V to have more viable progeny. Furthermore, live imaging of meiosis in spo-11(me44) oocytes and spermatocytes, which exhibit crossover failure on all 6 chromosomes, showed 12 univalents segregating apart in roughly equal masses in a homology-independent manner, supporting the existence of a mechanism that segregates any 2 chromosomes apart.

DASH/Dam1 complex mutants stabilize ploidy in histone-humanized yeast by weakening kinetochore-microtubule attachments.

Forcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. Here, we show that aneuploidy in histone-humanized yeasts is specific to a subset of chromosomes that are defined by their centromeric evolutionary origins but that these aneuploidies are not adaptive. Instead, we find that a set of missense mutations in outer kinetochore proteins drives adaptation to human histones. Furthermore, we characterize the molecular mechanism underlying adaptation in two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. We propose a model through which weakened microtubule attachments promote increased kinetochore-microtubule turnover and thus suppress chromosome instability. In sum, our data show how a set of point mutations evolved in histone-humanized yeasts to counterbalance human histone-induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.

Chromosome-specific behaviors during early meiosis.

Inheriting the wrong number of chromosomes is one of the leading causes of infertility and birth defects in humans. However, in many organisms, individual chromosomes vary dramatically in both organization, sequence, and size. Chromosome segregation systems must be capable of accounting for these differences to reliably segregate chromosomes. During gametogenesis, meiosis ensures that all chromosomes segregate properly into gametes (i.e., egg or sperm). Interestingly, not all chromosomes exhibit the same dynamics during meiosis, which can lead to chromosome-specific behaviors and defects. This review will summarize some of the chromosome-specific meiotic events that are currently known and discuss their impact on meiotic outcomes.