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Chromatin encapsulates not only genomic sequence information but also its structural organization. Chromosome folding patterns may juxtapose distal functional elements of DNA in space and are thought to regulate gene expression. Directly visualizing chromosome architecture to study this grammar of chromosome folding has been but a longstanding ambition in the field of epigenetics. An emergent imaging technology called multiplexed DNA-FISH has made it possible to localize the positions of thousands of genomic loci with nanometer precision inside single cells. Given a cell’s ploidy, deriving chromatin conformations from this data initially assumed detecting a fixed number of bright signals and connecting them in sequential genomic order. But during cell cycle chromosomes copies are subject to change; it is also subject to copy number variations such as amplifications or deletions; it is subjected further still to technical noise such as false positives appearing as true signal, or signal dropout. In my thesis, I sought to develop a different framework that accounts for various sources of noise that have gone unappreciated in multiplexed DNA imaging, to show that by embracing the complexity of noise we can in fact uncover new biology previously unseen. First, I describe spatial genome alignment and its derivative polymer fiber karyotyping, methods for accurately resolving chromatin structures in a copy number agnostic fashion. We apply this method to multiplexed DNA-FISH data of mouse embryonic stem cells (mESC) and the mouse cortex. In dividing mESCs, we provide the first reconstructions of tightly intertwined sister chromatids decondensed in interphase – convoluted structures which previously could not be parsed by computer vision or by human eye. We go on to uncover unusual structures such as replicated homologs interacting in one single chromosome territory, suggestive of mitotic crossover. In the mouse cortex, we uncover tightly paired chromosomes in non-dividing neurons suggestive of sister chromatids in replication. Second, I summarize the field of spatial multi-omics, describing where spatial DNA imaging fits within the technological landscape, as well as describing other emerging technologies. I illustrate the principles of two main methods for achieving spatial resolution: imaging in situ and sequencing-based “release-and-capture” techniques, and further provide a broad survey of different multiplexing strategies for indexing cellular contents. I compare and contrast the two main families of spatial technologies, delineating how the distinct advantages of each lends them to different experimental applications. Given the new capabilities we and others uncovered using spatial multi-omics, such as detecting copy number variations and visualizing histone modifications, that extend beyond transcriptomic profiling, I provide a future perspective on how spatial multi-mics and the many channels of information it captures could inform future clinical applications.

Saccharomyces cerevisiae is at the forefront of defining the major recombination mechanisms/models that repair targeted double-strand breaks during mitosis. Each of these models predicts specific molecular intermediates as well as genetic outcomes. Recent use of single-nucleotide polymorphisms to track the exchange of sequences in recombination products has provided an unprecedented level of detail about the corresponding intermediates and the extents to which different mechanisms are utilized. This approach also has revealed complexities that are not predicted by canonical models, suggesting that modifications to these models are needed. Current data are consistent with the initiation of most inter-homolog spontaneous mitotic recombination events by a double-strand break. In addition, the sister chromatid is preferred over the homolog as a repair template.

Following DNA replication, equal amounts of chromatin proteins are distributed over sister chromatids by re-deposition of parental chromatin proteins and deposition of newly synthesized chromatin proteins. Molecular mechanisms balancing the allocation of new and old chromatin proteins remain largely unknown. Here, we studied the genome-wide distribution of new chromatin proteins relative to parental DNA template strands and replication initiation zones using the double-click-seq. Under control conditions, new chromatin proteins were preferentially found on DNA replicated by the lagging strand machinery. Strikingly, replication stress induced by hydroxyurea or curaxin treatment and inhibition of ataxia telangiectasia and Rad3-related protein (ATR) or p53 inactivation inverted the observed chromatin protein deposition bias to the strand replicated by the leading strand polymerase in line with previously reported effects on replication protein A occupancy. We propose that asymmetric deposition of replication protein occupancy. propose asymmetric deposition newly synthesized chromatin proteins onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis.

To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal segregation at anaphase. Emerging evidence has shown that persistent DNA entanglements connecting the sister chromatids lead to the formation of ultrafine anaphase bridges (UFBs). If UFBs are not resolved soon after anaphase, they can induce chromosome missegregation. PICH (PLK1-interacting checkpoint helicase) is a DNA translocase that localizes on chromosome arms, centromeres and UFBs. It plays multiple essential roles in mitotic chromosome organization and segregation. PICH also recruits other associated proteins to UFBs, and together they mediate UFB resolution. Here, the proposed mechanism behind PICH’s functions in chromosome organization and UFB resolution will be discussed. We summarize the regulation of PICH action at chromosome arms and centromeres, how PICH recognizes UFBs and recruits other UFB-associated factors, and finally how PICH promotes UFB resolution together with other DNA processing enzymes.

Meiosis generates genetic diversity in natural populations and during breeding. We review meiosis research in major crop species, including an in-depth resource of cloned crop meiotic mutants and an overview of genome-wide recombination maps., Meiosis is a key feature of sexual reproduction. During meiosis homologous chromosomes replicate, recombine, and randomly segregate, followed by the segregation of sister chromatids to produce haploid cells. The unique genotypes of recombinant gametes are an essential substrate for the selection of superior genotypes in natural populations and in plant breeding. In this review we summarize current knowledge on meiosis in diverse monocot and dicot crop species and provide a comprehensive resource of cloned meiotic mutants in six crop species (rice, maize, wheat, barley, tomato, and Brassica species). Generally, the functional roles of meiotic proteins are conserved between plant species, but we highlight notable differences in mutant phenotypes. The physical lengths of plant chromosomes vary greatly; for instance, wheat chromosomes are roughly one order of magnitude longer than those of rice. We explore how chromosomal distribution for crossover recombination can vary between species. We conclude that research on meiosis in crops will continue to complement that in Arabidopsis, and alongside possible applications in plant breeding will facilitate a better understanding of how the different stages of meiosis are controlled in plant species.

The B chromosome of maize undergoes nondisjunction at the second pollen mitosis as part of its accumulation mechanism. Previous work identified 9-Bic-1 (9-B inactivated centromere-1), which comprises an epigenetically silenced B chromosome centromere that was translocated to the short arm of chromosome 9(9S). This chromosome is stable in isolation, but when normal B chromosomes are added to the genotype, it will attempt to undergo nondisjunction during the second pollen mitosis and usually fractures the chromosome in 9S. These broken chromosomes allow a test of whether the inactive centromere is reactivated or whether a de novo centromere is formed elsewhere on the chromosome to allow recovery of fragments. Breakpoint determination on the B chromosome and chromosome 9 showed that mini chromosome B1104 has the same breakpoint as 9-Bic-1 in the B centromere region and includes a portion of 9S. CENH3 binding was found on the B centromere region and on 9S, suggesting both centromere reactivation and de novo centromere formation. Another mini chromosome, B496, showed evidence of rearrangement, but it also only showed evidence for a de novo centromere. Other mini chromosome fragments recovered were directly derived from the B chromosome with breakpoints concentrated near the centromeric knob region, which suggests that the B chromosome is broken at a low frequency due to the failure of the sister chromatids to separate at the second pollen mitosis. Our results indicate that both reactivation and de novo centromere formation could occur on fragments derived from the progenitor possessing an inactive centromere. Supplementary Information The online version contains supplementary material available at 10.1007/s10577-021-09670-5.

Mutations in diploid budding yeast occur in meiosis at higher frequencies than in cells grown vegetatively. Such meiotic mutations are thought to result from the repair of double-strand breaks (DSBs) in meiosis, during the process of recombination. Here, we report studies of mutagenicity in haploid strains that may undergo meiosis due to the expression of both mating-type alleles, MATa and MATα. We measure the rate of mutagenicity in the reporter gene CAN1, and find it to be fivefold higher than in mitotic cells, as determined by fluctuation analysis. This enhanced meiotic mutagenicity is shown to depend on the presence of SPO11, the gene responsible for meiotic DSBs. Mutations in haploid meiosis must result from repair of the DSBs through interaction with the sister chromatid, rather than with non-sister chromatids as in diploids. Thus, mutations in diploid meiosis that are not ostensibly associated with recombination events can be explained by sister-chromatid repair. The spectrum of meiotic mutations revealed by Sanger sequencing is similar in haploid and in diploid meiosis. Compared to mitotic mutations in CAN1, long Indels are more frequent among meiotic mutations. Both, meiotic and mitotic mutations are more common at G/C sites than at A/T, in spite of an opposite bias in the target reporter gene. We conclude that sister-chromatid repair of DSBs is a major source of mutagenicity in meiosis.

The spermatogenesis of fish remained unclear about the cytological process, especially on the meiotic process of spermatocytes after meiosis I. In the present study, microstructure and ultrastructure of spermatogenesis in Cyprinus carpio (abbreviated CC) were studied, the meiosis process was traced by chromosomal observation and the corresponding immunohistochemical staining of testis where spermatocytes at different stages were distinguished. Results firstly described the telomere-led bouquet configuration of prophase I-stage cells in fish, which ensure the normal chromosomal behavior just like in other eukaryotes. Compared to the canonical meiotic process, the spermatocytes at leptotene, zygotene, pachytene, diplotene and diakinesis stages in CC were normal and also proceeded metaphase I interacting with the meiotic spindle as a unit, while the subsequently meiosis II in CC demonstrated contradicted findings in most organisms, in which no obvious spindle-dependent sister chromatids aligning and segregating could be observed, but some circular condensed nucleus structures. Furthermore, protein antibody of RAD51, DMC1, RNA-Pol II CTD P–S5, H3K36me3 were used respectively as probes, marked initially between the primary spermatocytes at prophase I and metaphase I stage, and between the spermatids and spermatozoa. The systematical study in cytological characteristics of the whole meiotic process in fish spermatogenesis in this paper, provide a standard reference for fish meiosis, which can be of great significance in the practice of fish genetics and breeding.

DNA Topoisomerase II (TopoII) uses ATP hydrolysis to decatenate chromosomes so that sister chromatids can faithfully segregate in mitosis. When the TopoII enzyme cycle stalls due to failed ATP hydrolysis, the onset of anaphase is delayed, presumably to allow extra time for decatenation to be completed. Recent evidence revealed that, unlike the spindle assembly checkpoint, this TopoII checkpoint response requires Aurora B and Haspin kinases and is triggered by SUMOylation of the C-terminal domain of TopoII.

High hyperdiploid acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. The main driver event of this disease is a nonrandom aneuploidy consisting of gains of whole chromosomes but without overt evidence of chromosomal instability (CIN). Here, we investigated the frequency and severity of defective sister chromatid cohesion—a phenomenon related to CIN—in primary pediatric ALL. We found that a large proportion (86%) of hyperdiploid cases displayed aberrant cohesion, frequently severe, to compare with 49% of ETV6/RUNX1‐positive ALL, which mostly displayed mild defects. In hyperdiploid ALL, cohesion defects were associated with increased chromosomal copy number heterogeneity, which could indicate increased CIN. Furthermore, cohesion defects correlated with RAD21 and NCAPG mRNA expression, suggesting a link to reduced cohesin and condensin levels in hyperdiploid ALL. Knockdown of RAD21 in an ALL cell line led to sister chromatid cohesion defects, aberrant mitoses, and increased heterogeneity in chromosomal copy numbers, similar to what was seen in primary hyperdiploid ALL. In summary, our study shows that aberrant sister chromatid cohesion is frequent but heterogeneous in pediatric high hyperdiploid ALL, ranging from mild to very severe defects, and possibly due to low cohesin or condensin levels. Cases with high levels of aberrant chromosome cohesion displayed increased chromosomal copy number heterogeneity, possibly indicative of increased CIN. These abnormalities may play a role in the clonal evolution of hyperdiploid pediatric ALL.

Human eggs frequently contain an incorrect number of chromosomes, a condition termed aneuploidy. Aneuploidy affects ∼10–25% of eggs in women in their early 30s, and more than 50% of eggs from women over 40. Most aneuploid eggs cannot develop to term upon fertilization, making aneuploidy in eggs a leading cause of miscarriages and infertility. The cellular origins of aneuploidy in human eggs are incompletely understood. Aneuploidy arises from chromosome segregation errors during the two meiotic divisions of the oocyte, the progenitor cell of the egg. Chromosome segregation is driven by a microtubule spindle, which captures and separates the paired chromosomes during meiosis I, and sister chromatids during meiosis II. Recent studies reveal that defects in the organization of the acentrosomal meiotic spindle contribute to human egg aneuploidy. The microtubules of the human oocyte spindle are very frequently incorrectly attached to meiotic kinetochores, the multi-protein complexes on chromosomes to which microtubules bind. Multiple features of human oocyte spindles favour incorrect attachments. These include spindle instability and many age-related changes in chromosome and kinetochore architecture. Here, we review how the unusual spindle assembly mechanism in human oocytes contributes to the remarkably high levels of aneuploidy in young human eggs, and how age-related changes in chromosome and kinetochore architecture cause aneuploidy levels to rise even higher as women approach their forties.

Cohesin is essential for sister chromatid cohesion, which ensures equal segregation of the chromatids to daughter cells. However, the molecular mechanism by which cohesin mediates this function is elusive. Scc3, one of the four core subunits of cohesin, is vital to cohesin activity. However, the mechanism by which Scc3 contributes to the activity and identity of its functional domains is not fully understood. Here, we describe an in-frame five-amino acid insertion mutation after glutamic acid 704 (scc3-E704ins) in yeast Scc3, located in the middle of the second armadillo repeat. Mutated cohesin-scc3-E704ins complexes are unable to establish cohesion. Detailed molecular and genetic analyses revealed that the mutated cohesin has reduced affinity to the Scc2 loader. This inhibits its enrichment at centromeres and chromosomal arms. Mutant complexes show a slow diffusion rate in live cells suggesting that they induce a major conformational change in the complex. The analysis of systematic mutations in the insertion region of Scc3 revealed two conserved aspartic acid residues that are essential for the activity. The study offers a better understanding of the contribution of Scc3 to cohesin activity and the mechanism by which cohesin tethers the sister chromatids during the cell cycle.

Among the inheritance of cellular components during cell division, deoxyribonucleic acid (DNA) and its condensate (chromosome) are conventionally visualized using chemical tag-labeled nucleotide analogs. However, associated mutagenesis with nucleotide analogs in the visualization of chromosomes is cause for concern. This study investigated the efficiency of using stable isotope labels in visualizing the replicating cultured human cell-chromosomes, in the absence of analog labels, at a high spatial resolution of 100 nm. The distinct carbon isotope ratio between sister chromatids reflected the semi-conservative replication of individual DNA strands through cell cycles and suggested the renewal of histone molecules in daughter chromosomes. Thus, this study provides a new, powerful approach to trace and visualize cellular components with stable isotope labeling.Key words: stable isotope, chromosome replication, semi-conservative replication, imaging, mass spectrometry.

Mutations that compromise mismatch repair (MMR) or DNA polymerase ε or δ exonuclease domains produce mutator phenotypes capable of fueling cancer evolution. Here, we investigate how combined defects in these pathways expands genetic heterogeneity in cells of the budding yeast, Saccharomyces cerevisiae, using a single-cell resolution approach that tallies all mutations arising from individual divisions. The distribution of replication errors present in mother cells after the initial S-phase was broader than expected for a single uniform mutation rate across all cell divisions, consistent with volatility of the mutator phenotype. The number of mismatches that then segregated to the mother and daughter cells co-varied, suggesting that each division is governed by a different underlying genome-wide mutation rate. The distribution of mutations that individual cells inherit after the second S-phase is further broadened by the sequential actions of semiconservative replication and mitotic segregation of chromosomes. Modeling suggests that this asymmetric segregation may diversify mutation burden in mutator-driven tumors., Dowsett et al use a single-cell resolution approach to analyse the distribution of mutations across several divisions in yeast diploid strains mutated in mismatch repair and polymerase delta proofreading. They find that the underlying mutation rate varies from one division to another, and that new mutations segregate unequally between sister chromatids at each division, expanding genetic heterogeneity in the population.

DNA breaks may arise accidentally in vegetative cells or in a programmed manner in meiosis. The usage of a DNA template makes homologous recombination potentially error-free, however, recombination is not always accurate. Cells possess a remarkable capacity to tailor processing of recombination intermediates to fulfill a particular need. Vegetatively growing cells aim to maintain genome stability and therefore repair accidental breaks largely accurately, using sister chromatids as templates, into mostly non-crossovers products. Recombination in meiotic cells is instead more likely to employ homologous chromosomes as templates and result in crossovers to allow proper chromosome segregation and promote genetic diversity. Here we review models explaining the processing of recombination intermediates in vegetative and meiotic cells and its regulation, with a focus on MLH1-MLH3-dependent crossing-over during meiotic recombination.

Aneuploidy of meiotic origin is a major contributor to age-related subfertility and an increased risk of miscarriage in women. Although age-related aneuploidy has been studied in rodents, the mare may be a more appropriate animal model to study reproductive aging. Similar to women, aged mares show reduced fertility and an increased incidence of early pregnancy loss; however, it is not known whether aging predisposes to aneuploidy in equine oocytes. We evaluated the effect of advanced mare age on (1) gene expression for cohesin components, (2) incidence of aneuploidy and (3) chromosome centromere cohesion (measured as the distance between sister kinetochores) in oocytes matured in vitro. Oocytes from aged mares showed reduced gene expression for the centromere cohesion stabilizing protein, Shugoshin 1. Moreover, in vitro matured oocytes from aged mares showed a higher incidence of aneuploidy and premature sister chromatid separation, and weakened centromeric cohesion. We therefore propose the mare as a valid model for studying effects of aging on centromeric cohesion; cohesion loss predisposes to disintegration of bivalents and premature separation of sister chromatids during the first meiotic division, leading to embryonic aneuploidy; this probably contributes to the reduced fertility and increased incidence of pregnancy loss observed in aged mares.

During mitosis microtubules self-organize to form a bipolar mitotic spindle structure, which positions the sister chromatids on the spindle mid-plane and separates them afterwards. Previous studies have identified many spindle associated proteins. Yet, we do not fully understand how these nanoscopic proteins lead to force generation through interactions of individual microtubules, motor proteins and chromosomes, and how a large number of these local interactions ultimately determine the structure and mechanics of the spindle in micron scale. Here we review the current understanding and open questions related to the structure and mechanics of the mitotic spindle. We then discuss how a combination of electron microscopy and computational modeling can be used to tackle some of these open questions.

Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance., Bulky DNA adducts are important replication-blocking lesions. Here the authors reveal that homologous recombination at bulky adducts in mammalian cells involves post-replicative gap repair in a PrimPol dependent manner.

Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we define the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.

All physical connections between sister chromatids must be broken before cells can divide, and eukaryotic cells have evolved multiple ways in which to process branchpoints connecting DNA molecules separated both spatially and temporally. A single DNA link between chromatids has the potential to disrupt cell cycle progression and genome integrity, so it is highly likely that cells require a nuclease that can process remaining unresolved and hemi-resolved DNA junctions and other branched species at the very late stages of mitosis. We argue that ANKLE1 probably serves this function in human cells (LEM-3 in Caenorhabditis elegans). LEM-3 has previously been shown to be located at the cell mid-body, and we show here that human ANKLE1 is a nuclease that cleaves a range of branched DNA species. It thus has the substrate selectivity consistent with an enzyme required to process a variety of unresolved and hemi-resolved branchpoints in DNA. Our results suggest that ANKLE1 acts as a catch-all enzyme of last resort that allows faithful chromosome segregation and cell division to occur., Graphical abstract Unlabelled Image, Highlights • Human ANKLE1 is a nuclease that cleaves branched DNA. • ANKLE1 cleaves a variety of branched DNA species, including four-way junctions, flaps and Y-structures. • The activity of ANKLE1 is consistent with a catch-all nuclease to sever links between chromosomes prior to cell division. • ANKLE1 is probably the enzyme of last resort processing links between chromosomes.

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1,2 analysis has revealed a complex genomic landscape of internal chromosomal structures in vertebrate cells3-7, but the identical sequence of sister chromatids has made it difficult to determine how they topologically interact in replicated chromosomes. Here we describe sister-chromatid-sensitive Hi-C (scsHi-C), which is based on labelling of nascent DNA with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes reveal that sister-chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister-chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired and are characterized by facultative heterochromatin and insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister-chromatid topologies and our scsHi-C technology will make it possible to investigate how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

Smc5/6, an atypical SMC complex with two RING-type subunits Roger Solé-Soler ; Jordi Torres-Rosell Crossmark: Check for Updates Biochem Soc Trans (2020) 48 (5): 2159-2171. https://doi.org/10.1042/BST20200389 Article history Share Icon Share Cite Icon Cite Get Permissions The Smc5/6 complex plays essential roles in chromosome segregation and repair, by promoting disjunction of sister chromatids. The core of the complex is constituted by an heterodimer of Structural Maintenance of Chromosomes (SMC) proteins that use ATP hydrolysis to dynamically associate with and organize chromosomes. In addition, the Smc5/6 complex contains six non-SMC subunits. Remarkably, and differently to other SMC complexes, the Nse1 and Nse2 subunits contain RING-type domains typically found in E3 ligases, pointing to the capacity to regulate other proteins and complexes through ubiquitin-like modifiers. Nse2 codes for a C-terminal SP-RING domain with SUMO ligase activity, assisting Smc5/6 functions in chromosome segregation through sumoylation of several chromosome-associated proteins. Nse1 codes for a C-terminal NH-RING domain and, although it has been proposed to have ubiquitin ligase activity, no Smc5/6-dependent ubiquitylation target has been described to date. Here, we review the function of the two RING domains of the Smc5/6 complex in the broader context of SMC complexes as global chromosome organizers of the genome. Work in the J.T.-R. lab is supported by grant and PGC2018-097796-B-I00 from Ministerio de Ciencia, Innovación y Universidades and grant 2017-SGR-569 from AGAUR-Generalitat de Catalunya. R.S.-S. is a recipient of an FPU16/07021 fellowship from Ministerio de Ciencia, Innovación y Universidades. The IRBLLEIDA Institute is part of CERCA Programme/Generalitat de Catalunya

Chromosome segregation requires both compaction and disentanglement of sister chromatids. We describe SisterC, a chromosome conformation capture assay that distinguishes interactions between and along identical sister chromatids. SisterC employs 5-bromo-2′-deoxyuridine (BrdU) incorporation during S-phase to label newly replicated strands, followed by Hi-C and then the destruction of 5-bromodeoxyuridine-containing strands via Hoechst/ultraviolet treatment. After sequencing of the remaining intact strands, this allows assignment of Hi-C products as inter- and intra-sister interactions based on the strands that reads are mapped to. We performed SisterC on mitotic Saccharomyces cerevisiae cells. We find precise alignment of sister chromatids at centromeres. Along arms, sister chromatids are less precisely aligned, with inter-sister connections every ~35 kilobase (kb). Inter-sister interactions occur between cohesin binding sites that are often offset by 5 to 25 kb. Along sister chromatids, cohesin results in the formation of loops of up to 50 kb. SisterC allows study of the complex interplay between sister chromatid compaction and their segregation during mitosis. It remains impossible using conventional Hi-C to differentiate interactions between and along sister chromatids. SisterC relies on selective destruction of nascent DNA and in combination with Hi-C offers a means to study intra- and inter-sister interactions independently.

The faithful segregation of duplicated sister chromatids rely on the remarkable ability of kinetochores to sustain stable load bearing attachments with the dynamic plus ends of kinetochore–microtubules (kMTs). The outer layer of the kinetochore recruits several motor and non-motor microtubule-associated proteins (MAPs) that help the kinetochores establish and maintain a load bearing dynamic attachment with kMTs. The primary kMT-binding protein, the Ndc80 complex (Ndc80c), which is highly conserved among diverse organisms from yeast to humans, performs this essential function with assistance from other MAPs. These MAPs are not an integral part of the kinetochore, but they localize to the kinetochore periodically throughout mitosis and regulate the strength of the kinetochore microtubule attachments. Here, we attempt to summarize the recent advances that have been made toward furthering our understanding of this co-operation between the Ndc80c and these MAPs, focusing on the spindle and kinetochore-associated 1 (Ska1) complex (Ska1c) and Cdc10-dependent transcript 1 (Cdt1) in humans.

Homologous recombination (HR) repairs double-strand breaks (DSBs) occurring in sister chromatids using the intact sisters as the repair template. HR is initiated by DSB resection, which generates 3′ single-strand DNA (ssDNA). RAD51 recombinase polymerizes on the ssDNA and undergoes strand exchange with intact sister chromatids, generating junction molecules (JMs). The separation of JMs completes HR-dependent DSB repair. Defective resolution of JMs not only leaves DSBs unrepaired but also has the broken sisters remain entangled with the intact sisters, leading to the formation of isochromatid-type breaks, where both sister chromatids are broken at the same sites, in mitotic chromosome spreads. The MRE11 nuclease plays a key role in HR, and it is generally believed that MRE11 does so by initiating DSB resection. We here showed that the loss of MRE11 reduced the efficiency of HR in human TK6 cells without affecting DSB resection, indicating a role for MRE11 in HR also at a post-resection step. MRE11-deficient TK6 cells showed proficient induction of RAD51 foci by ionizing-radiation (IR) and olaparib but significantly delayed their resolution. Although exposure of G2-phase cells to IR cleaves only one of two sister chromatids, the loss of the MRE11-nuclease activity increased the number of isochromosome-type breaks in subsequent M phase. The overexpression of GEN1 resolvase suppressed the formation of IR-induced isochromatid-type breaks in MRE11-nuclease-deficient TK6 cells. These data indicate that MRE11 plays an important role in HR by processing JMs. We propose the dual roles of MRE11 in HR at DSB resection and post-resection steps.

Asymmetric cell division (ACD) produces daughter cells with separate distinct cell fates and is critical for the development and regulation of multicellular organisms. Epigenetic mechanisms are key players in cell fate determination. Centromeres, epigenetically specified loci defined by the presence of the histone H3-variant, centromere protein A (CENP-A), are essential for chromosome segregation at cell division. ACDs in stem cells and in oocyte meiosis have been proposed to be reliant on centromere integrity for the regulation of the non-random segregation of chromosomes. It has recently been shown that CENP-A is asymmetrically distributed between the centromeres of sister chromatids in male and female Drosophila germline stem cells (GSCs), with more CENP-A on sister chromatids to be segregated to the GSC. This imbalance in centromere strength correlates with the temporal and asymmetric assembly of the mitotic spindle and potentially orientates the cell to allow for biased sister chromatid retention in stem cells. In this essay, we discuss the recent evidence for asymmetric sister centromeres in stem cells. Thereafter, we discuss mechanistic avenues to establish this sister centromere asymmetry and how it ultimately might influence cell fate.

As an important factor of chromosome abnormality in offspring, elderly pregnant women have attracted more and more attention. Studies have confirmed that the offspring of older pregnant women, whether conceived naturally or through assisted reproductive technology, are more likely to develop chromosome abnormalities than younger pregnant women, and the reasons have not been fully explained. The spindle assembly checkpoint monitors the correct connection between all mobile axes and the spindle. The bipolar spindle is the key to cell division to guide the correct separation of chromosomes and is the main gatekeeper to prevent the development of oocytes carrying DNA damage. Older pregnant women lead to the error of sister chromatid separation and the decrease of chromosome cohesion. The integrity of telomeres also affects the folding of DNA and the ability of cells to replicate. In addition, more oxidative stress makes the ovaries of older pregnant women unable to support fertilization and embryonic development. With the oxygen free radicals increase, the pathway of ovarian cell apoptosis and mitochondrial repair were changed. Therefore, the analysis of the causes of fetal chromosome abnormalities in elderly pregnant women is helpful to improve the understanding of the particularity of pregnancy in elderly pregnant women, so as to reduce the birth of defective babies and improve the birth quality.

In mice, the pseudoautosomal region of the sex chromosomes undergoes a dynamic structural rearrangement to promote a high rate of DNA double-strand breaks and to ensure X-Y recombination. Sex chromosomes in males of most eutherian mammals share only a small homologous segment, the pseudoautosomal region (PAR), in which the formation of double-strand breaks (DSBs), pairing and crossing over must occur for correct meiotic segregation(1,2). How cells ensure that recombination occurs in the PAR is unknown. Here we present a dynamic ultrastructure of the PAR and identify controlling cis- and trans-acting factors that make the PAR the hottest segment for DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyperaccumulate in the PAR, its chromosome axes elongate and the sister chromatids separate. These processes are linked to heterochromatic mo-2 minisatellite arrays, and require MEI4 and ANKRD31 proteins but not the axis components REC8 or HORMAD1. We propose that the repetitive DNA sequence of the PAR confers unique chromatin and higher-order structures that are crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, notably, oocytes can be reprogrammed to exhibit spermatocyte-like levels of DSBs in the PAR simply by delaying or preventing synapsis. Thus, the sexually dimorphic behaviour of the PAR is in part a result of kinetic differences between the sexes in a race between the maturation of the PAR structure, formation of DSBs and completion of pairing and synapsis. Our findings establish a mechanistic paradigm for the recombination of sex chromosomes during meiosis.

Heterosis, a phenomenon in which hybrid offspring shows better performance in many aspects than either of their homozygous parents, has been widely applied for improving crop productivity and adaptability. However, progenies generated via sexual reproduction are diverse owing to the genetic recombination during meiosis, which is not conducive to the application of heterosis. Apomixis is a type of asexual reproduction through which plants can produce clonal seeds without undergoing normal meiosis and fertilization. Therefore, apomixis has been a research hotspot for a long time because of its potential in fixing heterosis. Over 400 plant species exhibit apomictic phenomena in nature, and many genes linked with apomictic loci have been discovered in various species. These genes are involved in distinct regulatory mechanisms of apomixis. Even after decades of effort, these genes have not been successfully applied for crop apomixis because of complexity of the underlying mechanisms and lack of apomictic crops in nature. Due to the apomixis of plants also can be induced artificially by allowing plants to undergo unusual meiosis and fertilization, synthetic apomixis based on reproductive mechanisms has been proposed to fix heterosis. In MiMe ( mitosis instead of meiosis ), meiotic division is completely replaced by a mitotic-like division and diploid gametes that are genetically identical to their mother are produced. MiMe is induced by the absence of genes required for the pairing and recombination of homologous chromosomes, maintenance of sister chromatid cohesionas well as for the transition of germ cells from meiosis I to II. Following normal fertilization of clonal diploid gametes, the ploidy of offspring is doubled in each generation. To achieve synthetic apomixis and obtain clonal seeds, chromosome doubling following normal fertilization must be prevented. An effective way for this prevention is the elimination of one set of chromosomes from either parent. When a plant with altered CENH3 (centromere-specific histone H3) protein is crossed with a wild-type plant, haploid progenies are induced through the elimination of chromosomes from the cenh3 mutant. Furthermore, combining MiMe plants with a genome elimination line via crossing is an effective strategy to produce clonal seeds. In addition, upon expression of BBM1 (BABY BOOM 1), which plays an important role in the initiation of embryogenesis, in the egg cell, haploid progenies are generated that produce seeds with haploid embryos following self-fertilization. Moreover, MTL (matrilineal) disruption in plants using CRISPR/Cas9 gene editing technology also can generate haploid progenies. Therefore, the combination of MiMe with BBM1 - or MTL -induced production of haploid progenies can achieve synthetic apomixis in plants and produce clonal seeds. In this review, we summarize advances in synthetic apomixis at the molecular level and discuss the improvement of synthetic apomixis as well as its potential application in crop breeding.

Separation of eukaryotic sister chromatids during the cell cycle is timed by the spindle assembly checkpoint (SAC) and ultimately triggered when separase cleaves cohesion-mediating cohesin1,2,3. Silencing of the SAC during metaphase activates the ubiquitin ligase APC/C (anaphase-promoting complex, also known as the cyclosome) and results in the proteasomal destruction of the separase inhibitor securin1. In the absence of securin, mammalian chromosomes still segregate on schedule, but it is unclear how separase is regulated under these conditions4,5. Here we show that human shugoshin 2 (SGO2), an essential protector of meiotic cohesin with unknown functions in the soma6,7, is turned into a separase inhibitor upon association with SAC-activated MAD2. SGO2–MAD2 can functionally replace securin and sequesters most separase in securin-knockout cells. Acute loss of securin and SGO2, but not of either protein individually, resulted in separase deregulation associated with premature cohesin cleavage and cytotoxicity. Similar to securin8,9, SGO2 is a competitive inhibitor that uses a pseudo-substrate sequence to block the active site of separase. APC/C-dependent ubiquitylation and action of the AAA-ATPase TRIP13 in conjunction with the MAD2-specific adaptor p31comet liberate separase from SGO2–MAD2 in vitro. The latter mechanism facilitates a considerable degree of sister chromatid separation in securin-knockout cells that lack APC/C activity. Thus, our results identify an unexpected function of SGO2 in mitotically dividing cells and a mechanism of separase regulation that is independent of securin but still supervised by the SAC., This work was supported by a grant (STE997/4-2) from the Deutsche Forschungsgemeinschaft (DFG) to O.S. and by MINECO (BFU2017-89408-R) and Junta de Castilla y Leon (CSI239P18). CIC-IBMCC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia cofunded by the Castilla–León autonomous government and the European Regional Development Fund (CLC–2017–01).

Plant fertility is highly sensitive to elevated temperature. Here, we report that hot spells induce the formation of dyads and triads by disrupting the biogenesis or stability of the radial microtubule arrays (RMAs) at telophase II. Heat-induced meiotic restitution in Arabidopsis is predominantly SDR-type (Second Division Restitution) indicating specific interference with RMAs formed between separated sister chromatids. In addition, elevated temperatures caused distinct deviations in cross-over formation in male meiosis. Synapsis at pachytene was impaired and the obligate cross-over per chromosome was discarded, resulting in partial univalency in meiosis I (MI). At diakinesis, interconnections between non-homologous chromosomes tied separate bivalents together, suggesting heat induces ectopic events of non-homologous recombination. Summarized, heat interferes with male meiotic cross-over designation and cell wall formation, providing a mechanistic basis for plant karyotype change and genome evolution under high temperature conditions., de Storme and Geelen show that heat stress has pleiotropic effects on male meiosis in Arabidopsis, causing deviations in cross-over formations, reproduction, and fertility. They show that heat also affects cell wall formation, providing mechanistic insights into karyotype change under high temperatures.

We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG. Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines. The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase. CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.

Structural Maintenance of Chromosomes 2 (SMC2) and Structural Maintenance of Chromosomes 4 (SMC4) are the core components of the condensin complexes, which are required for chromosome assembly and faithful segregation during cell division. Because of the crucial functions of both proteins in cell division, much work has been done in various vertebrates, but little information is known about their roles in plants. Here, we identified ZmSMC2 and ZmSMC4 in maize (Zea mays) and confirmed that ZmSMC2 associates with ZmSMC4 via their hinge domains. Immunostaining revealed that both proteins showed dynamic localization during mitosis. ZmSMC2 and ZmSMC4 are essential for proper chromosome segregation and for H3 phosphorylation at Serine 10 (H3S10ph) at pericentromeres during mitotic division. The loss of function of ZmSMC2 and ZmSMC4 enlarges mitotic chromosome volume and impairs sister chromatid separation to the opposite poles. Taken together, these findings confirm and extend the coordinated role of ZmSMC2 and ZmSMC4 in maintenance of normal chromosome architecture and accurate segregation during mitosis.

Before fertilization, ovulated mammalian oocytes are arrested at the metaphase of second meiosis (MII), which is maintained by the so-called cytostatic factor (CSF). It is well known that the continuous synthesis and accumulation of cyclin B is critical for maintaining the CSF-mediated MII arrest. Recent studies by us and others have shown that Ccnb3 is required for the metaphase-to-anaphase transition during the first meiosis of mouse oocytes, but whether Ccnb3 plays a role in MII arrest and exit remains unknown. Here, we showed that the protein level of Ccnb3 gradually decreased during oocyte meiotic maturation, and exogenous expression of Ccnb3 led to release of MII arrest, degradation of securin, separation of sister chromatids, extrusion of the second polar body (PB2), and finally entry into interphase. These phenotypes could be rescued by inhibition of Wee1B or CDK2. Our results indicate that Ccnb3 plays a critical regulatory role in MII arrest and exit in mouse oocytes.

Nowadays, life is highly influenced by intense growth of various industries, high levels of pollution, and other environmental factors with harmful effects on human health. Therefore, cytogenetic monitoring is essential for detection of changes in the structure of chromosomes, which occur because of the effects of various genotoxic agents. In this review, we shall apprize the theoretical and experimental aspects of several tests for assessment of genotoxicity in humans such as Micronucleus assay, Comet assay, Chromosomal aberrations assessment and Sister chromatid exchanges analysis. These methods are accepted by the World Health Organization as standard tests for genotoxicological screening in humans. The methods are sensitive and confirm the cellular genotoxic effects of various genotoxicants.

Epigenetic mechanisms play essential roles in determining distinct cell fates during the development of multicellular organisms. Histone proteins represent crucial epigenetic components that help specify cell identities. Previous work has demonstrated that during the asymmetric cell division of the Drosophila male germline stem cells (GSCs), histones H3 and H4 are asymmetrically inherited, such that preexisting (old) histones are segregated towards the self-renewing GSC whereas newly synthesized (new) histones are enriched towards the differentiating daughter cell. In order to further understand the molecular mechanisms underlying this striking phenomenon, two key questions must be answered: when and how old and new histones are differentially incorporated by sister chromatids, and how epigenetically distinct sister chromatids are specifically recognized and segregated. Here we discuss recent advances in our understanding of the molecular mechanisms and cellular bases underlying these fundamental and important biological processes responsible for generating two distinct cells through one cell division.

Cohesin organizes the genome by forming intra-chromosomal loops and inter-sister chromatid linkages. During gamete formation by meiosis, chromosomes are reshaped to support crossover recombination and two consecutive rounds of chromosome segregation. Here we show that Eco1 acetyltransferase positions both chromatin loops and sister chromatid cohesion to organize meiotic chromosomes into functional domains in budding yeast. Eco1 acetylates the Smc3 cohesin subunit in meiotic S phase to establish chromatin boundaries, independently of DNA replication. Boundary formation by Eco1 is critical for prophase exit and for the maintenance of cohesion until meiosis II, but is independent of the ability of Eco1 to antagonize the cohesin-release factor, Wpl1. Conversely, prevention of cohesin release by Wpl1 is essential for centromeric cohesion, kinetochore monoorientation and co-segregation of sister chromatids in meiosis I. Our findings establish Eco1 as a key determinant of chromatin boundaries and cohesion positioning, revealing how local chromosome structuring directs genome transmission into gametes.

Homology-directed repair of DNA double-strand breaks (DSBs) can be a highly faithful pathway. Non-crossover repair dominates in mitotically growing cells, likely through a preference for synthesis-dependent strand annealing (SDSA). While genetic studies highlight a key role for the RecQ helicase BLM/Rqh1 (in human and S. pombe cells, respectively) in promoting noncrossover repair, how homology-directed repair mechanism choice is orchestrated in time and space is not well understood. Here, we develop a microscopy-based assay in living fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. We observe highly efficient homology search and homology-directed repair in this system. Surprisingly, we find that the initial distance between the DSB and the donor sequence does not correlate with the duration of repair. Instead, we observe that repair is likely to involve multiple site-specific and Rad51-dependent co-localization events between the DSB and donor sequence, suggesting that efficient interhomologue repair in fission yeast often involves multiple strand invasion events. By contrast, we find that loss of Rqh1 leads to successful repair through a single strand invasion event, suggesting that multiple strand invasion cycles reflect ongoing SDSA. However, failure to repair is also more likely in rqh1Δ cells, which could reflect increased strand invasion at non-homologous sites. This work has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM and characterized by aberrant sister chromatid crossovers and inefficient repair.

Kinetochores are macromolecular protein assemblies that attach sister chromatids to spindle microtubules and mediate accurate chromosome segregation during mitosis. The outer kinetochore consists of the KMN network, a protein super-complex comprising Knl1 (yeast Spc105), Mis12 (yeast Mtw1), and Ndc80 (yeast Ndc80), which harbors sites for microtubule binding. Within the KMN network, Spc105 acts as an interaction hub of components involved in spindle assembly checkpoint (SAC) signaling. It is known that Spc105 forms a complex with kinetochore component Kre28. However, where Kre28 physically localizes in the budding yeast kinetochore is not clear. The exact function of Kre28 at the kinetochore is also unknown. Here, we investigate how Spc105 and Kre28 interact and how they are organized within bioriented yeast kinetochores using genetics and cell biological experiments. Our microscopy data show that Spc105 and Kre28 localize at the kinetochore with a 1:1 stoichiometry. We also show that the Kre28-Spc105 interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the kinetochores. We created several truncation mutants of kre28 that affect Spc105 loading at the kinetochores. When over-expressed, these mutants sustain the cell viability, but SAC signaling and kinetochore biorientation are impaired. Therefore, we conclude that Kre28 contributes to chromosome biorientation and high-fidelity segregation at least indirectly by regulating Spc105 localization at the kinetochores.

Warsaw breakage syndrome (WABS) is a very rare recessive hereditary disease caused by mutations in the gene coding for the DNA helicase DDX11, involved in genome stability maintenance and sister cohesion establishment. Typical clinical features observed in WABS patients include growth retardation, facial dysmorphia, microcephaly, hearing loss due to cochlear malformations and, at cytological level, sister chromatid cohesion defects. Molecular bases of WABS have not yet been elucidated, due to lack of disease animal model systems and limited knowledge of the DDX11 physiological functions. However, WABS is considered to belong to the group of cohesinopathies, genetic disorders due to mutations of subunits or regulators of cohesin, the protein complex responsible for tethering sister chromatids from the time of their synthesis till they separate in mitosis. Recent evidences suggest that cohesin and its regulators have additional key roles in chromatin organization by promoting the formation of chromatin loops. This "non-canonical" function of cohesin is expected to impact gene transcription during cell differentiation and embryonic development and its dis-regulation, caused by mutation/loss of genes encoding cohesin subunits or regulators, could originate the developmental defects observed in cohesinopathies. Ethiopathogenesis of WABS is discussed in line with these recent findings and evidence of a possible role of DDX11 as a cohesin regulator.