Synaptonemal complex formation produces a particular arrangement of the lateral element-associated DNA.

During meiosis, homologous chromosomes exchange genetic material. This exchange or meiotic recombination is mediated by a proteinaceous scaffold known as the Synaptonemal complex (SC). Any defects in its formation produce failures in meiotic recombination, chromosome segregation and meiosis completion. It has been proposed that DNA repair events that will be resolved by crossover between homologous chromosomes are predetermined by the SC. Hence, structural analysis of the organization of the DNA in the SC could shed light on the process of crossover interference. In this work, we employed an ultrastructural DNA staining technique on mouse testis and followed nuclei of pachytene cells. We observed structures organized similarly to the SCs stained with conventional techniques. These structures, presumably the DNA in the SCs, are delineating the edges of both lateral elements and no staining was observed between them. DNA in the LEs resembles two parallel tracks. However, a bubble-like staining pattern in certain regions of the SC was observed. Furthermore, this staining pattern is found in SCs formed between non-homologous chromosomes, in SCs formed between sister chromatids and in SCs without lateral elements, suggesting that this particular organization of the DNA is determined by the synapsis of the chromosomes despite their lack of homology or the presence of partially formed SCs.

Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences.

Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, which cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired and accumulation of toxic DNA double strand breaks occurs. To repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, which may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA, and their segregation during cell division are the origin of subsequent aberrations such as translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, which results in tissue attrition, selection of malignant clones and cancer onset. Moreover, chromosomal instability and cell death are not exclusive of the somatic compartment, they also affect germinal cells, as evidenced by the infertility observed in patients with FA.

Quantitative cytogenetics reveals molecular stoichiometry and longitudinal organization of meiotic chromosome axes and loops.

During meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative, and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using Caenorhabditis elegans. Analyses of wild-type (WT) chromosomes and de novo circular minichromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with 2 functionally distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister-chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging 3 per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister-chromatid loops. Indeed, immunofluorescence/fluorescence in situ hybridization (immuno-FISH) assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring.

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

Holocentric chromosomes.

Holocentric chromosomes possess multiple kinetochores along their length rather than the single centromere typical of other chromosomes [1]. They have been described for the first time in cytogenetic experiments dating from 1935 and, since this first observation, the term holocentric chromosome has referred to chromosomes that: i. lack the primary constriction corresponding to centromere observed in monocentric chromosomes [2]; ii. possess multiple kinetochores dispersed along the chromosomal axis so that microtubules bind to chromosomes along their entire length and move broadside to the pole from the metaphase plate [3]. These chromosomes are also termed holokinetic, because, during cell division, chromatids move apart in parallel and do not form the classical V-shaped figures typical of monocentric chromosomes [4-6]. Holocentric chromosomes evolved several times during both animal and plant evolution and are currently reported in about eight hundred diverse species, including plants, insects, arachnids and nematodes [7,8]. As a consequence of their diffuse kinetochores, holocentric chromosomes may stabilize chromosomal fragments favouring karyotype rearrangements [9,10]. However, holocentric chromosome may also present limitations to crossing over causing a restriction of the number of chiasma in bivalents [11] and may cause a restructuring of meiotic divisions resulting in an inverted meiosis [12].

Non-redundant roles in sister chromatid cohesion of the DNA helicase DDX11 and the SMC3 acetyl transferases ESCO1 and ESCO2.

In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.

18F-FDG-induced DNA damage, chromosomal aberrations, and toxicity in V79 lung fibroblast cells.

18F-FDG PET/CT imaging is used in the diagnosis of diseases, including cancers. The principal photons used for imaging are 511 ke V gamma photons resulting from positron annihilation. The absorbed dose varies among body organs, depending on administered radioactivity and biological clearance. We have attempted to evaluate DNA double-strand breaks (DSB) and toxicity induced in V79 lung fibroblast cells in vitro by 18F-FDG, at doses which might result from PET procedures. Cells were irradiated by 18F-FDG at doses (14.51 and 26.86 mGy), comparable to absorbed doses received by critical organs during PET procedures. The biological endpoints measured were formation of γ-H2AX foci, mitochondrial stress, chromosomal aberrations, and cell cycle perturbation. Irradiation induced DSB (γH2AX assay), mitochondrial depolarization, and both chromosome and chromatid types of aberrations. At higher radiation doses, increased aneuploidy and reduced mitotic activity were also seen. Thus, significant biological effects were observed at the doses delivered by the 18F-FDG exposure and the effects increased with dose.

Post-metaphase correction of aberrant kinetochore-microtubule attachments in mammalian eggs.

The accuracy of the two sequential meiotic divisions in oocytes is essential for creating a haploid gamete with a normal chromosomal content. Here, we have analysed the 3D dynamics of chromosomes during the second meiotic division in live mouse oocytes. We find that chromosomes form stable kinetochore-microtubule attachments at the end of prometaphase II stage that are retained until anaphase II onset. Remarkably, we observe that more than 20% of the kinetochore-microtubule attachments at the metaphase II stage are merotelic or lateral. However, < 1% of all chromosomes at onset of anaphase II are found to lag at the spindle equator and < 10% of the laggards missegregate and give rise to aneuploid gametes. Our results demonstrate that aberrant kinetochore-microtubule attachments are not corrected at the metaphase stage of the second meiotic division. Thus, the accuracy of the chromosome segregation process in mouse oocytes during meiosis II is ensured by an efficient correction process acting at the anaphase stage.

Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement.

Many stem cells undergo asymmetric division to produce a self-renewing stem cell and a differentiating daughter cell. Here we show that, similarly to H3, histone H4 is inherited asymmetrically in Drosophila melanogaster male germline stem cells undergoing asymmetric division. In contrast, both H2A and H2B are inherited symmetrically. By combining super-resolution microscopy and chromatin fiber analyses with proximity ligation assays on intact nuclei, we find that old H3 is preferentially incorporated by the leading strand, whereas newly synthesized H3 is enriched on the lagging strand. Using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional replication fork movement in testes-derived chromatin and DNA fibers. Biased fork movement coupled with a strand preference in histone incorporation would explain how asymmetric old and new H3 and H4 are established during replication. These results suggest a role for DNA replication in patterning epigenetic information in asymmetrically dividing cells in multicellular organisms.

Spatiotemporal regulation of Aurora B recruitment ensures release of cohesion during C. elegans oocyte meiosis.

The formation of haploid gametes from diploid germ cells requires the regulated two-step release of sister chromatid cohesion (SCC) during the meiotic divisions. Here, we show that phosphorylation of cohesin subunit REC-8 by Aurora B promotes SCC release at anaphase I onset in C. elegans oocytes. Aurora B loading to chromatin displaying Haspin-mediated H3 T3 phosphorylation induces spatially restricted REC-8 phosphorylation, preventing full SCC release during anaphase I. H3 T3 phosphorylation is locally antagonized by protein phosphatase 1, which is recruited to chromosomes by HTP-1/2 and LAB-1. Mutating the N terminus of HTP-1 causes ectopic H3 T3 phosphorylation, triggering precocious SCC release without impairing earlier HTP-1 roles in homolog pairing and recombination. CDK-1 exerts temporal regulation of Aurora B recruitment, coupling REC-8 phosphorylation to oocyte maturation. Our findings elucidate a complex regulatory network that uses chromosome axis components, H3 T3 phosphorylation, and cell cycle regulators to ensure accurate chromosome segregation during oogenesis.

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

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

Maternal age-dependent APC/C-mediated decrease in securin causes premature sister chromatid separation in meiosis II.

Sister chromatid attachment during meiosis II (MII) is maintained by securin-mediated inhibition of separase. In maternal ageing, oocytes show increased inter-sister kinetochore distance and premature sister chromatid separation (PSCS), suggesting aberrant separase activity. Here, we find that MII oocytes from aged mice have less securin than oocytes from young mice and that this reduction is mediated by increased destruction by the anaphase promoting complex/cyclosome (APC/C) during meiosis I (MI) exit. Inhibition of the spindle assembly checkpoint (SAC) kinase, Mps1, during MI exit in young oocytes replicates this phenotype. Further, over-expression of securin or Mps1 protects against the age-related increase in inter-sister kinetochore distance and PSCS. These findings show that maternal ageing compromises the oocyte SAC-APC/C axis leading to a decrease in securin that ultimately causes sister chromatid cohesion loss. Manipulating this axis and/or increasing securin may provide novel therapeutic approaches to alleviating the risk of oocyte aneuploidy in maternal ageing.

Physical Proximity of Sister Chromatids Promotes Top2-Dependent Intertwining.

Sister chromatid intertwines (SCIs), or catenanes, are topological links between replicated chromatids that interfere with chromosome segregation. The formation of SCIs is thought to be a consequence of fork swiveling during DNA replication, and their removal is thought to occur because of the intrinsic feature of type II topoisomerases (Top2) to simplify DNA topology. Here, we report that SCIs are also formed independently of DNA replication during G2/M by Top2-dependent concatenation of cohesed chromatids due to their physical proximity. We demonstrate that, in contrast to G2/M, Top2 removes SCIs from cohesed chromatids at the anaphase onset. Importantly, SCI removal in anaphase requires condensin and coincides with the hyperactivation of condensin DNA supercoiling activity. This is consistent with the longstanding proposal that condensin provides a bias in Top2 function toward decatenation. A comprehensive model for the formation and resolution of toxic SCI entanglements on eukaryotic genomes is proposed.

A Reversible Association between Smc Coiled Coils Is Regulated by Lysine Acetylation and Is Required for Cohesin Association with the DNA.

Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes.

Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA-DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.

Decrease in expression of maternal effect gene Mater is associated with maternal ageing in mice.

STUDY HYPOTHESIS: What factors in mouse oocytes are involved in the ageing-related decline in oocyte quality? STUDY FINDING: The maternal effect gene Mater is involved in ageing-related oocyte quality decline in mice. WHAT IS KNOWN ALREADY: Premature loss of centromere cohesion is a hallmark of ageing-related oocyte quality decline; the maternal effect gene Mater (maternal antigen that embryos require, also known as Nlrp5) is required for preimplantation embryo development beyond the 2-cell stage, and mRNA expression of Mater decreases with maternal ageing. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Mater protein expression level in mature oocytes from 7 young (5-8 weeks old) to 7 old mice (41-68 weeks old) was compared by immunoblotting analysis. Wild-type and Mater-null mice were used to examine whether Mater is necessary for maintaining normal centromere cohesion by means of cytogenetic karyotyping, time-lapse confocal microscopy and immunofluorescence staining. MAIN RESULTS AND THE ROLE OF CHANCE: Mater protein is decreased in mature oocytes from old versus young mice (P = 0.0022). Depletion of Mater from oocytes leads to a reduction in centromere cohesion, manifested by precocious sister chromatid separation, enlargement of sister centromere distance and misalignment of chromosomes in the metaphase plate during meiosis I and II. LIMITATIONS, REASONS FOR CAUTION: This study was conducted in mice. Whether or not the results are applicable to human remains further elucidation. In addition, we were unable to confirm if the strain of mice (C57BL/6XSv129) at the age of 41-68 weeks old has the 'cohesin-loss' phenotype. WIDER IMPLICATIONS OF THE FINDINGS: Investigating Mater's functional mechanisms could provide fresh insights into understanding how the ageing-related oocyte quality decline occurs. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by the research grant from Chinese NSFC to P.Z. (31071274). We have no conflict of interests to declare.

Stacked thin layers of metaphase chromatin explain the geometry of chromosome rearrangements and banding.

The three-dimensional organization of tightly condensed chromatin within metaphase chromosomes has been one of the most challenging problems in structural biology since the discovery of the nucleosome. This study shows that chromosome images obtained from typical banded karyotypes and from different multicolour cytogenetic analyses can be used to gain information about the internal structure of chromosomes. Chromatin bands and the connection surfaces in sister chromatid exchanges and in cancer translocations are planar and orthogonal to the chromosome axis. Chromosome stretching produces band splitting and even the thinnest bands are orthogonal and well defined, indicating that short stretches of DNA can occupy completely the chromosome cross-section. These observations impose strong physical constraints on models that attempt to explain chromatin folding in chromosomes. The thin-plate model, which consists of many stacked layers of planar chromatin perpendicular to the chromosome axis, is compatible with the observed orientation of bands, with the existence of thin bands, and with band splitting; it is also compatible with the orthogonal orientation and planar geometry of the connection surfaces in chromosome rearrangements. The results obtained provide a consistent interpretation of the chromosome structural properties that are used in clinical cytogenetics for the diagnosis of hereditary diseases and cancers.

An Alternatively Spliced Bifunctional Localization Signal Reprograms Human Shugoshin 1 to Protect Centrosomal Instead of Centromeric Cohesin.

Separation of human sister chromatids involves the removal of DNA embracing cohesin ring complexes. Ring opening occurs by prophase-pathway-dependent phosphorylation and separase-mediated cleavage, with the former being antagonized at centromeres by Sgo1-dependent PP2A recruitment. Intriguingly, prophase pathway signaling and separase's proteolytic activity also bring about centriole disengagement, whereas Sgo1 is again counteracting this licensing step of later centrosome duplication. Here, we demonstrate that alternative splice variants of human Sgo1 specifically and exclusively localize and function either at centromeres or centrosomes. A small C-terminal peptide encoded by exon 9 of SGO1 (CTS for centrosomal targeting signal of human Sgo1) is necessary and sufficient to drive centrosomal localization and simultaneously abrogate centromeric association of corresponding Sgo1 isoforms. Cohesin is shown to be a target of the prophase pathway at centrosomes and protected by Sgo1-PP2A. Accordingly, premature centriole disengagement in response to Sgo1 depletion is suppressed by blocking ring opening of an engineered cohesin.

Inferring Latent States and Refining Force Estimates via Hierarchical Dirichlet Process Modeling in Single Particle Tracking Experiments.

Understanding the basis for intracellular motion is critical as the field moves toward a deeper understanding of the relation between Brownian forces, molecular crowding, and anisotropic (or isotropic) energetic forcing. Effective forces and other parameters used to summarize molecular motion change over time in live cells due to latent state changes, e.g., changes induced by dynamic micro-environments, photobleaching, and other heterogeneity inherent in biological processes. This study discusses limitations in currently popular analysis methods (e.g., mean square displacement-based analyses) and how new techniques can be used to systematically analyze Single Particle Tracking (SPT) data experiencing abrupt state changes in time or space. The approach is to track GFP tagged chromatids in metaphase in live yeast cells and quantitatively probe the effective forces resulting from dynamic interactions that reflect the sum of a number of physical phenomena. State changes can be induced by various sources including: microtubule dynamics exerting force through the centromere, thermal polymer fluctuations, and DNA-based molecular machines including polymerases and protein exchange complexes such as chaperones and chromatin remodeling complexes. Simulations aiming to show the relevance of the approach to more general SPT data analyses are also studied. Refined force estimates are obtained by adopting and modifying a nonparametric Bayesian modeling technique, the Hierarchical Dirichlet Process Switching Linear Dynamical System (HDP-SLDS), for SPT applications. The HDP-SLDS method shows promise in systematically identifying dynamical regime changes induced by unobserved state changes when the number of underlying states is unknown in advance (a common problem in SPT applications). We expand on the relevance of the HDP-SLDS approach, review the relevant background of Hierarchical Dirichlet Processes, show how to map discrete time HDP-SLDS models to classic SPT models, and discuss limitations of the approach. In addition, we demonstrate new computational techniques for tuning hyperparameters and for checking the statistical consistency of model assumptions directly against individual experimental trajectories; the techniques circumvent the need for "ground-truth" and/or subjective information.

[Cohesion inside eu- and heterochromatin in human cells].

It is considered that sister chromatids are held together immediately after replication by special protein complex--cohesin that consists of Smc1--Smc3 core dimer and two additional subunits, Scc1 and Scc3. This process is called cohesion. We have characterized binding of cohesin complex to early- and late-replicated chromatin at different stages of the cell cycle in human cells HeLa and HT1080 using superresolution microscopy (based on Structural ilumination microscopy--SIM) and immunoelectron microscopy. It has been shown that cohesins do not play important role in cohesion of heterochromatic domains, but they provide cohesion and organization of subdomains in euchromatic regions.