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Molecular Cell Nov 2021Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to...
Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to simultaneously reconstruct cryo-EM structures of protein complexes from interphase or metaphase chromosomes. The reconstructed interphase and metaphase nucleosome structures are on average indistinguishable from canonical nucleosome structures, despite DNA sequence heterogeneity, cell-cycle-specific posttranslational modifications, and interacting proteins. Nucleosome structures determined by a decoy-classifying method and structure variability analyses reveal the nucleosome structural variations in linker DNA, histone tails, and nucleosome core particle configurations, suggesting that the opening of linker DNA, which is correlated with H2A C-terminal tail positioning, is suppressed in chromosomes. High-resolution (3.4-3.5 Å) nucleosome structures indicate DNA-sequence-independent stabilization of superhelical locations ±0-1 and ±3.5-4.5. The linker histone H1.8 preferentially binds to metaphase chromatin, from which chromatosome cryo-EM structures with H1.8 at the on-dyad position are reconstituted. This study presents the structural characteristics of nucleosomes in chromosomes.
Topics: Animals; Cell Communication; Cell Cycle; Cell Division; Chromatin; Chromosomes; Computer Simulation; Cryoelectron Microscopy; DNA; Humans; Hydrophobic and Hydrophilic Interactions; Interphase; Metaphase; Nucleosomes; Protein Conformation; Protein Domains; Protein Processing, Post-Translational; Xenopus
PubMed: 34478647
DOI: 10.1016/j.molcel.2021.08.010 -
Journal of Ovarian Research Feb 2020DCN (decorin) is a proteoglycan known to be involved in regulating cell proliferation, collagen fibril organization and migration. In our global transcriptome...
BACKGROUND
DCN (decorin) is a proteoglycan known to be involved in regulating cell proliferation, collagen fibril organization and migration. In our global transcriptome RNA-sequencing approach to systematically identify new ovulation-associated genes, DCN was identified as one of the highly regulated genes. We therefore hypothesize that DCN may have a role in ovulatory processes such as cell migration and proliferation.
AIM
To characterize the expression, regulation and function of the proteoglycan DCN in the human ovarian follicles during the preovulatory period.
METHODS
The in-vivo expression of DCN mRNA in mural (MGCs) and cumulus (CGCs) granulosa cells was characterized using quantitative RT-PCR and western blot. A signaling study was performed by treating human MGCs cultures with gonadotropins and different stimulators and inhibitors to determine their effect on DCN expression by qRT- PCR and elucidate the pathways regulating these proteins. In a functional study, KGN granulosa cell line was used to study cell migration with a scratch assay.
RESULTS
DCN mRNA expression was significantly higher in MGCs compared to CGCs. DCN mRNA was significantly higher in CGCs surrounding mature metaphase II (MII) oocytes compared to CGCs of germinal vesicle (GV) and metaphase I (MI) oocytes. hCG significantly increased DCN mRNA and protein expression levels in cultured MGCs. Using signal transduction activators and inhibitors, we demonstrated that DCN induction by LH/hCG is carried out via PKA, PKC, ERK/MEK, and PI3K pathways. We showed that DCN expression is also induced in high-density cell cultures, in a dose-dependent pattern. In addition, progesterone induced a significant increase in DCN secretion to the media. MGCs from follicles of endometriosis patients exhibited reduced (about 20% of) mRNA transcriptions levels compared to MGCs follicles of control patients. More significantly, we found that DCN has an inhibiting effect on KGN cell migration.
CONCLUSIONS
Our study indicates that DCN is a unique ovulatory gene. Our findings support the hypothesis that DCN plays an important new role during the preovulatory period and ovulation, and stress its involvement in endometriosis infertility. A better understanding of DCN role in ovulation and endometriosis may provide treatment for some types of infertility.
Topics: Cells, Cultured; Decorin; Female; Humans; Ovarian Follicle; Ovulation; Prospective Studies; RNA, Messenger
PubMed: 32041647
DOI: 10.1186/s13048-020-0612-3 -
Physics in Medicine and Biology Dec 2022. To develop a metaphase chromosome model representing the complete genome of a human lymphocyte cell to support microscopic Monte Carlo (MMC) simulation-based...
. To develop a metaphase chromosome model representing the complete genome of a human lymphocyte cell to support microscopic Monte Carlo (MMC) simulation-based radiation-induced DNA damage studies.. We first employed coarse-grained polymer physics simulation to obtain a rod-shaped chromatid segment of 730 nm in diameter and 460 nm in height to match Hi-C data. We then voxelized the segment with a voxel size of 11 nm per side and connected the chromatid with 30 types of pre-constructed nucleosomes and 6 types of linker DNAs in base pair (bp) resolutions. Afterward, we piled different numbers of voxelized chromatid segments to create 23 pairs of chromosomes of 1-5m long. Finally, we arranged the chromosomes at the cell metaphase plate of 5.5m in radius to create the complete set of metaphase chromosomes. We implemented the model in gMicroMC simulation by denoting the DNA structure in a four-level hierarchical tree: nucleotide pairs, nucleosomes and linker DNAs, chromatid segments, and chromosomes. We applied the model to compute DNA damage under different radiation conditions and compared the results to those obtained with G0/G1 model and experimental measurements. We also performed uncertainty analysis for relevant simulation parameters.. The chromatid segment was successfully voxelized and connected in bps resolution, containing 26.8 mega bps (Mbps) of DNA. With 466 segments, we obtained the metaphase chromosome containing 12.5 Gbps of DNA. Applying it to compute the radiation-induced DNA damage, the obtained results were self-consistent and agreed with experimental measurements. Through the parameter uncertainty study, we found that the DNA damage ratio between metaphase and G0/G1 phase models was not sensitive to the chemical simulation time. The damage was also not sensitive to the specific parameter settings in the polymer physics simulation, as long as the produced metaphase model followed a similar contact map distribution.. Experimental data reveal that ionizing radiation induced DNA damage is cell cycle dependent. Yet, DNA chromosome models, except for the G0/G1 phase, are not available in the state-of-the-art MMC simulation. For the first time, we successfully built a metaphase chromosome model and implemented it into MMC simulation for radiation-induced DNA damage computation.
Topics: Humans; Metaphase; Nucleosomes; DNA Damage; Radiation, Ionizing; DNA; Polymers
PubMed: 36533598
DOI: 10.1088/1361-6560/aca5ea -
PloS One 2020Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated...
Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated legal and ethical issues, and delaying childbearing years. While the outcome of oocyte cryopreservation has recently been improved, currently used vitrification method still suffer from increased biosafety risk and handling issues while slow freezing techniques yield overall low success. Understanding better the mechanism of cryopreservation-induced injuries may lead to development of more reliable and safe methods for oocyte cryopreservation. Using the mouse model, a microarray study was conducted on oocyte cryopreservation to identify cryoinjuries to transcriptionally active genome. To this end, metaphase II (MII) oocytes were subjected to standard slow freezing, and then analyzed at the four-cell stage after embryonic genome activation. Non-frozen four-cell embryos served as controls. Differentially expressed genes were identified and validated using RT-PCR. Embryos produced from the cryopreserved oocytes displayed 200 upregulated and 105 downregulated genes, associated with the regulation of mitochondrial function, protein ubiquitination and maintenance, cellular response to stress and oxidative states, fatty acid and lipid regulation/metabolism, and cell cycle maintenance. These findings reveal previously unrecognized effects of standard slow oocyte freezing on embryonic gene expression, which can be used to guide improvement of oocyte cryopreservation methods.
Topics: Animals; Cryopreservation; Embryo, Mammalian; Embryonic Development; Female; Fertilization in Vitro; Freezing; Gene Expression Regulation, Developmental; Humans; Male; Metaphase; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Oocytes; Protein Interaction Maps; Real-Time Polymerase Chain Reaction; Transcriptome
PubMed: 32251418
DOI: 10.1371/journal.pone.0231108 -
Reproductive Sciences (Thousand Oaks,... May 2024This paper will review a remarkable new approach to in vitro maturation "IVM" of oocytes from ovarian tissue, based on our results with in vitro oogenesis from somatic... (Review)
Review
This paper will review a remarkable new approach to in vitro maturation "IVM" of oocytes from ovarian tissue, based on our results with in vitro oogenesis from somatic cells. As an aside benefit we also have derived a better understanding of ovarian longevity from ovary transplant. We have found that primordial follicle recruitment is triggered by tissue pressure gradients. Increased pressure holds the follicle in meiotic arrest and prevents recruitment. Therefore recruitment occurs first in the least dense inner tissue of the cortico-medullary junction. Many oocytes can be obtained from human ovarian tissue and mature to metaphase 2 in vitro with no need for ovarian stimulation. Ovarian stimulation may only be necessary for removing the oocyte from the ovary, but this can also be accomplished by simple dissection at the time of ovary tissue cryopreservation. By using surgical dissection of the removed ovary, rather than a needle stick, we can obtain many oocytes from very small follicles not visible with ultrasound. A clearer understanding of ovarian function has come from in vitro oogenesis experiments, and that explains why IVM has now become so simple and robust. Tissue pressure (and just a few "core genes" in the mouse) direct primordial follicle recruitment and development to mature oocyte, and therefore also control ovarian longevity. There are three distinct phases to oocyte development both in vitro and in vivo: in vitro differentiation "IVD" which is not gonadotropin sensitive (the longest phase), in vitro gonadotropin sensitivity "IVG" which is the phase of gonadotropin stimulation to prepare for meiotic competence, and IVM to metaphase II. On any given day 35% of GVs in ovarian tissue have already undergone "IVD" and "IVG" in vivo, and therefore are ready for IVM.
Topics: Female; Animals; Oogenesis; Humans; Ovary; In Vitro Oocyte Maturation Techniques; Oocytes; Ovarian Follicle; Mice
PubMed: 38160209
DOI: 10.1007/s43032-023-01427-1 -
Cellular and Molecular Life Sciences :... Mar 2021Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure... (Review)
Review
Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure and not a real nicked break within the DNA molecule. Most facts about fragile sites result from studies in mitotic cells, mainly during metaphase and mainly in lymphocytes. Here, we synthesize facts about the genomic regions that are prone to form gaps and breaks on metaphase chromosomes in the context of interphase. We conclude that nuclear architecture shapes the activity profile of the cell, i.e. replication timing and transcriptional activity, thereby influencing genomic integrity during interphase with the potential to cause fragility in mitosis. We further propose fragile sites as examples of regions specifically positioned in the interphase nucleus with putative anchoring points at the nuclear lamina to enable a tightly regulated replication-transcription profile and diverse signalling functions in the cell. Consequently, fragility starts before the actual display as chromosomal breakage in metaphase to balance the initial contradiction of cellular overgrowth or malfunctioning and maintaining diversity in molecular evolution.
Topics: Animals; Cell Nucleus; Chromosomal Instability; Chromosome Fragile Sites; DNA; DNA Replication; Genome, Human; Humans; Interphase; Mitosis
PubMed: 33219838
DOI: 10.1007/s00018-020-03698-2 -
PLoS Genetics Jan 2021Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases...
Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.
Topics: Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA Topoisomerases, Type II; Drosophila melanogaster; Kinetochores; Larva; Metaphase; Microtubules; Mitosis; Spindle Apparatus; Cohesins
PubMed: 33513180
DOI: 10.1371/journal.pgen.1009304 -
The Journal of Cell Biology Jun 2022Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the...
Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the alignment of all chromosomes by the mitotic spindle at the metaphase plate, and any misalignment must be corrected before anaphase is triggered. The spindle is situated in a membrane-free "exclusion zone"; beyond this zone, endomembranes (mainly endoplasmic reticulum) are densely packed. We investigated what happens to misaligned chromosomes localized beyond the exclusion zone. Here we show that such chromosomes become ensheathed in multiple layers of endomembranes. Chromosome ensheathing delays mitosis and increases the frequency of chromosome missegregation and micronucleus formation. We use an induced organelle relocalization strategy in live cells to show that clearance of endomembranes allows for the rescue of chromosomes that were destined for missegregation. Our findings indicate that endomembranes promote the missegregation of misaligned chromosomes that are outside the exclusion zone and therefore constitute a risk factor for aneuploidy.
Topics: Anaphase; Aneuploidy; Cell Membrane; Chromosome Segregation; Chromosomes; Endoplasmic Reticulum; Humans; Metaphase; Mitosis; Spindle Apparatus
PubMed: 35486148
DOI: 10.1083/jcb.202203021 -
Current Protocols Mar 2023The fluorescent dyes Hoechst (HO) and Chromomycin A3 (CA3) are commonly used for bivariate flow karyotyping to distinguish individual chromosomes from one another based...
The fluorescent dyes Hoechst (HO) and Chromomycin A3 (CA3) are commonly used for bivariate flow karyotyping to distinguish individual chromosomes from one another based on differences in base composition and DNA content. However, analysis of chromosomes using this fluorescent dye combination requires a flow cytometer equipped with lasers of specific wavelengths and higher power than is typical of conventional flow cytometers. This unit presents a chromosome staining technique with a dye combination of DAPI and propidium iodide (PI). Chromosomes stained using this dye combination can be analyzed on conventional flow cytometers equipped with a typical configuration of lasers and optics. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cell culture and metaphase harvest of suspension cell line Alternate Protocol 1: Cell culture and metaphase harvest of adherent cell line Basic Protocol 2: Preparation of chromosome suspension using polyamine isolation buffer Basic Protocol 3: Staining chromosomes with DAPI and propidium iodide Alternate Protocol 2: Staining chromosomes with Hoechst and Chromomycin A3 Basic Protocol 4: Bivariate flow karyotyping on a cell analyzer Basic Protocol 5: Bivariate flow karyotyping on a cell sorter Basic Protocol 6: Purification of flow-sorted chromosomes.
Topics: Chromomycin A3; DNA; Propidium; Chromosomes; Karyotyping; Fluorescent Dyes
PubMed: 36920094
DOI: 10.1002/cpz1.718 -
EMBO Reports Apr 2024Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear....
Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear. Here, we show that CKAP5, a conserved microtubule plus tip protein, regulates CENP-E at kinetochores in human cells. Depletion of CKAP5 impairs CENP-E localization at kinetochores at the metaphase plate and results in increased kinetochore-microtubule stability and attachment errors. Erroneous attachments are also supported by computational modeling. Analysis of CKAP5 knockout cancer cells of multiple tissue origins shows that CKAP5 is preferentially essential in aneuploid, chromosomally unstable cells, and the sensitivity to CKAP5 depletion is correlated to that of CENP-E depletion. CKAP5 depletion leads to reduction in CENP-E-BubR1 interaction and the interaction is rescued by TOG4-TOG5 domain of CKAP5. The same domain can rescue CKAP5 depletion-induced CENP-E removal from the kinetochores. Interestingly, CKAP5 depletion facilitates recruitment of PP1 to the kinetochores and furthermore, a PP1 target site-specific CENP-E phospho-mimicking mutant gets stabilized at kinetochores in the CKAP5-depleted cells. Together, the results support a model in which CKAP5 controls mitotic chromosome attachment errors by stabilizing CENP-E at kinetochores and by regulating stability of the kinetochore-attached microtubules.
Topics: Humans; Kinetochores; Chromosomal Proteins, Non-Histone; Microtubules; Metaphase; Kinesins; HeLa Cells; Mitosis; Chromosome Segregation; Microtubule-Associated Proteins
PubMed: 38424231
DOI: 10.1038/s44319-024-00106-9