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Nature Structural & Molecular Biology Oct 2019Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA...
Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT ('facilitates chromatin transcription') in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding.
Topics: Cell Cycle Proteins; Chromatin; Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; High Mobility Group Proteins; Protein Interaction Maps; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcriptional Elongation Factors; Cohesins
PubMed: 31582854
DOI: 10.1038/s41594-019-0307-x -
Acta Neuropathologica Communications Apr 2020Although abnormal mitosis with disarranged metaphase chromosomes or many micronuclei in astrocytes (named "Alzheimer I type astrocytes" and later "Creutzfeldt-Peters...
Although abnormal mitosis with disarranged metaphase chromosomes or many micronuclei in astrocytes (named "Alzheimer I type astrocytes" and later "Creutzfeldt-Peters cells") have been known for nearly 100 years, the origin and mechanisms of this pathology remain elusive. In experimental brain insults in rats, we show that abnormal mitoses that are not followed by cytokinesis are typical for reactive astrocytes. The pathology originates due to the inability of the cells to form normal mitotic spindles with subsequent metaphase chromosome congression, which, in turn may be due to shape constraints aggravated by cellular enlargement and to the accumulation of large amounts of cytosolic proteins. Many astrocytes escape from arrested mitosis by producing micronuclei. These polyploid astrocytes can survive for long periods of time and enter into new cell cycles.
Topics: Animals; Astrocytes; Brain Diseases; Gliosis; Mitosis; Rats; Rats, Sprague-Dawley; Rats, Wistar
PubMed: 32293551
DOI: 10.1186/s40478-020-00919-4 -
Theriogenology Jan 2023In oocyte cryopreservation programs, vitrification has overthrown conventional slow freezing both in veterinary and human medicine. In animals, its feasibility in field... (Review)
Review
In oocyte cryopreservation programs, vitrification has overthrown conventional slow freezing both in veterinary and human medicine. In animals, its feasibility in field conditions makes it the preferred technique for the safeguard of genetic resources from zoo or wild animals, including threatened felids, for which the domestic cat is an excellent model. However, many cellular injuries, such as cytoskeleton, mitochondria and meiotic spindle alterations, DNA damage, zona pellucida hardening and cumulus cell loss, might occur following vitrification. After warming, although the exact mechanisms are still unclear, degeneration is a frequent outcome for cat vitrified oocytes. For immature (germinal vesicle) gametes, in vitro maturation after warming is a challenge, and cleavage after fertilization barely reaches 15-30%, while for mature (metaphase II) cryopreserved gametes it can get to 30-50%. Anyway, the progression to late embryos stages is often impaired, and improvements are needed. Standard cryopreservation protocol and the use of conventional in vitro culture systems after warming may not be enough for vitrified oocytes to recover and demonstrate their full developmental potential. Physical or chemical factors applied to oocytes undergoing vitrification, as an enrichment to the vitrification step, or to the culture microenvironment, could create more favorable conditions and promote vitrified oocyte survival and development. From the use of three-dimensional culture systems to the regulation of metabolic activities and cellular pathways, this review aims to explore all the possibilities employed so far, including the studies performed by our own lab, and the future perspectives, to present the most effective strategies for cat oocyte vitrification and the best time for their application (i.e., before, during, or after vitrification-warming).
Topics: Animals; Cats; Vitrification; Oocytes
PubMed: 36442286
DOI: 10.1016/j.theriogenology.2022.11.027 -
Frontiers in Endocrinology 2023The non-growing, meiotically-arrested oocytes housed within primordial follicles are exquisitely sensitive to genotoxic insults from endogenous and exogenous sources....
INTRODUCTION
The non-growing, meiotically-arrested oocytes housed within primordial follicles are exquisitely sensitive to genotoxic insults from endogenous and exogenous sources. Even a single DNA double-strand break (DSB) can trigger oocyte apoptosis, which can lead to accelerated depletion of the ovarian reserve, early loss of fertility and menopause. Therefore, repair of DNA damage is important for preserving the quality of oocytes to sustain fertility across the reproductive lifespan. This study aimed to evaluate the role of KU80 (encoded by the XRCC5 gene) - an essential component of the non-homologous end joining (NHEJ) pathway - in the repair of oocyte DNA DSBs during reproductive ageing, and following insult caused by the DNA-damaging chemotherapies cyclophosphamide and cisplatin.
METHODS
To investigate the importance of KU80 following endogenous and exogenous DNA damage, ovaries from conditional oocyte-specific knockout ( cKO) and wildtype (WT) mice that were aged or exposed to DNA damage-inducing chemotherapy were compared. Ovarian follicles and oocytes were quantified, morphologically assessed and analysed via immunohistochemistry for markers of DNA damage and apoptosis. In addition, chemotherapy exposed mice were superovulated, and the numbers and quality of mature metaphase- II (MII) oocytes were assessed.
RESULTS
The number of healthy follicles, atretic (dying) follicles, and corpora lutea were similar in Xrcc5 cKO and WT mice at PN50, PN200 and PN300. Additionally, primordial follicle number and ovulation rates were similar in young adult Xrcc5 cKO and WT mice following treatment with cyclophosphamide (75mg/kg), cisplatin (4mg/kg), or vehicle control (saline). Furthermore, KU80 was not essential for the repair of exogenously induced DNA damage in primordial follicle oocytes.
DISCUSSION
These data indicate that KU80 is not required for maintenance of the ovarian reserve, follicle development, or ovulation during maternal ageing. Similarly, this study also indicates that KU80 is not required for the repair of exogenously induced DSBs in the prophase-arrested oocytes of primordial follicles.
Topics: Animals; Female; Mice; Cisplatin; Cyclophosphamide; DNA; Oocytes; Ovarian Follicle; Prophase; Ku Autoantigen
PubMed: 37900135
DOI: 10.3389/fendo.2023.1268009 -
Nature Communications Apr 2021Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of...
Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of experimental telomere crisis identified in previous studies. We examine the spectrum of structural variants (SVs) instigated by natural telomere crisis. Eight spontaneous post-crisis clones did not show prominent patterns of BFB cycles or chromothripsis. Their crisis-induced genome rearrangements varied from infrequent simple SVs to more frequent and complex SVs. In contrast, BFB cycles and chromothripsis occurred in MRC5 fibroblast clones that escaped telomere crisis after CRISPR-controlled telomerase activation. This system revealed convergent evolutionary lineages altering one allele of chromosome 12p, where a short telomere likely predisposed to fusion. Remarkably, the 12p chromothripsis and BFB events were stabilized by independent fusions to chromosome 21. The data establish that telomere crisis can generate a wide spectrum of SVs implying that a lack of BFB patterns and chromothripsis in cancer genomes does not indicate absence of past telomere crisis.
Topics: Cell Line; Chromosomal Instability; Chromothripsis; Fibroblasts; Genome; Genomic Instability; Humans; Lung; Metaphase; Models, Biological; Neoplasms; Telomere
PubMed: 33828097
DOI: 10.1038/s41467-021-21933-7 -
Cell Division Jan 2021Cancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs)...
BACKGROUND
Cancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs) have an increased metastatic potential compared to isolated circulating tumor cells. Several widely used chemotherapeutic agents that target the cytoskeleton microtubules and cause cell cycle arrest at mitosis have been reported to modulate CTC number or the size of CTC clusters.
RESULTS
In this study, we investigated in vitro the impact of mitotic arrest on the ability of breast tumor cells to form clusters. By using live imaging and quantitative image analysis, we found that MCF-7 cancer cell aggregation is compromised upon incubation with paclitaxel or vinorelbine, two chemotherapeutic drugs that target microtubules. In line with these results, we observed that MCF-7 breast cancer cells experimentally synchronized and blocked in metaphase aggregated poorly and formed loose clusters. To monitor clustering at the single-cell scale, we next developed and validated an in vitro assay based on live video-microscopy and custom-designed micro-devices. The study of cluster formation from MCF-7 cells that express the fluorescent marker LifeAct-mCherry using this new assay allowed showing that substrate anchorage-independent clustering of MCF-7 cells was associated with the formation of actin-dependent highly dynamic cell protrusions. Metaphase-synchronized and blocked cells did not display such protrusions, and formed very loose clusters that failed to compact.
CONCLUSIONS
Altogether, our results suggest that mitotic arrest induced by microtubule-targeting anticancer drugs prevents cancer cell clustering and therefore, could reduce the metastatic potential of circulating tumor cells.
PubMed: 33514388
DOI: 10.1186/s13008-021-00070-z -
Genes Jan 2022Oligo-fluorescence in situ hybridization (FISH) facilitates precise chromosome identification and comparative cytogenetic analysis. Detection of autosomal chromosomes of...
Oligo-fluorescence in situ hybridization (FISH) facilitates precise chromosome identification and comparative cytogenetic analysis. Detection of autosomal chromosomes of has not been achieved using oligonucleotide sequences. Here, the chromosomes of five taxa in the mitotic metaphase and mitotic metaphase to anaphase were detected using the oligo-FISH probes (AGT), 5S rDNA, and (TTG). In total, 24 small chromosomes were clearly observed in the mitotic metaphase (0.89-3.03 μm), whereas 24-48 small chromosomes were observed in the mitotic metaphase to anaphase (0.94-3.10 μm). The signal number and intensity of (AGT), 5S rDNA, and (TTG) in the mitotic metaphase to anaphase chromosomes were nearly consistent with those in the mitotic metaphase chromosomes when the two split chromosomes were integrated as one unit. Of note, 14 chromosomes (there is a high chance that sex chromosomes are included) were exclusively identified by (AGT), 5S rDNA, and (TTG). The other 10 also showed a terminal signal with (AGT). Moreover, these oligo-probes were able to distinguish one wild taxon from four taxa. These chromosome identification and taxa differentiation data will help in elucidating visual and elaborate physical mapping and guide breeders' utilization of wild resources of .
Topics: Chromosomes, Plant; DNA, Ribosomal; Hippophae; In Situ Hybridization, Fluorescence; RNA, Ribosomal, 5S
PubMed: 35205242
DOI: 10.3390/genes13020195 -
3 Biotech Mar 2023An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in...
An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in vitro-developed shoots (tips) were treated individually with 0.1%, 0.25% and 0.5% (/) colchicine solutions for 4, 6, 8, and 12 h. The colchicine-treated shoot tips were then inoculated on Murashige and Skoog (MS) medium fortified with 1.5 mg/l -Topolin for multiple shoot proliferation and later transferred into 1.5 mg/l indole-3-acetic acid-fortified MS medium for rooting of shoots. The ploidy levels of the colchicine-treated and regenerated plantlets along with the non-treated ones were confirmed via flow cytometry analysis and metaphasic chromosome count. The highest frequency of tetraploid plantlets (50%) were obtained when shoot tips were treated with 0.1% colchicine for 4 h. Morphological observations revealed that induced tetraploid plantlets exhibited delayed fresh shoot initiation, fewer but longer shoots, as well as fewer but broader leaves. Likewise, the study of stomata revealed that in comparison to their diploid counterparts, the tetraploid plantlets exhibited less frequent yet significantly larger stomata, and higher number of chloroplasts. The tetraploids were recorded with significantly higher chlorophyll, carotenoid, and anthocyanin content during the photosynthetic pigment analyses. During ex vitro acclimatization and field growth, the tetraploid plants exhibited delayed proliferation but with higher vigor and thickened broad leaves. The genetic uniformity among the diploid and the tetraploid plants was confirmed using conserved DNA-derived polymorphism (CDDP), directed amplification of minisatellite-region DNA (DAMD), inter simple sequence repeats (ISSR), and start codon targeted (SCoT) polymorphism marker systems. The tetraploids developed in the present study would be of immense importance for the genetic improvement of gerbera as far as its ornamental values are concerned.
PubMed: 36748015
DOI: 10.1007/s13205-022-03457-z -
Frontiers in Cell and Developmental... 2021In female mammals, meiotic prophase one begins during fetal development. Oocytes transition through the prophase one substages consisting of leptotene, zygotene, and... (Review)
Review
In female mammals, meiotic prophase one begins during fetal development. Oocytes transition through the prophase one substages consisting of leptotene, zygotene, and pachytene, and are finally arrested at the diplotene substage, for months in mice and years in humans. After puberty, luteinizing hormone induces ovulation and meiotic resumption in a cohort of oocytes, driving the progression from meiotic prophase one to metaphase two. If fertilization occurs, the oocyte completes meiosis two followed by fusion with the sperm nucleus and preparation for zygotic divisions; otherwise, it is passed into the uterus and degenerates. Specifically in the mouse, oocytes enter meiosis at 13.5 days post coitum. As meiotic prophase one proceeds, chromosomes find their homologous partner, synapse, exchange genetic material between homologs and then begin to separate, remaining connected at recombination sites. At postnatal day 5, most of the oocytes have reached the late diplotene (or dictyate) substage of prophase one where they remain arrested until ovulation. This review focuses on events and mechanisms controlling the progression through meiotic prophase one, which include recombination, synapsis and control by signaling pathways. These events are prerequisites for proper chromosome segregation in meiotic divisions; and if they go awry, chromosomes mis-segregate resulting in aneuploidy. Therefore, elucidating the mechanisms regulating meiotic progression is important to provide a foundation for developing improved treatments of female infertility.
PubMed: 34095134
DOI: 10.3389/fcell.2021.667306 -
Cell Cycle (Georgetown, Tex.) 2022Mammalian oocytes undergo two rounds of developmental arrest during maturation: at the diplotene of the first meiotic prophase and metaphase of the second meiosis. These... (Review)
Review
Mammalian oocytes undergo two rounds of developmental arrest during maturation: at the diplotene of the first meiotic prophase and metaphase of the second meiosis. These arrests are strictly regulated by follicular cells temporally producing the secondary messengers, cAMP and cGMP, and other factors to regulate maturation promoting factor (composed of cyclin B1 and cyclin-dependent kinase 1) levels in the oocytes. Out of these normally appearing developmental arrests, permanent arrests may occur in the oocytes at germinal vesicle (GV), metaphase I (MI), or metaphase II (MII) stage. This issue may arise from absence or altered expression of the oocyte-related genes playing key roles in nuclear and cytoplasmic maturation. Additionally, the assisted reproductive technology (ART) applications such as ovarian stimulation and culture conditions both of which harbor various types of chemical agents may contribute to forming the permanent arrests. In this review, the molecular determinants of developmental and permanent arrests occurring in the mammalian oocytes are comprehensively evaluated in the light of current knowledge. As number of permanently arrested oocytes at different stages is increasing in ART centers, potential approaches for inducing permanent arrests to obtain competent oocytes are discussed.
Topics: Animals; Mammals; Meiosis; Meiotic Prophase I; Metaphase; Oocytes
PubMed: 35072590
DOI: 10.1080/15384101.2022.2026704