Role In Chromosome Condensation Research Articles

Insight into magnesium ions effect on chromosome banding and ultrastructure.

Magnesium ion (Mg2+ ) plays a fundamental role in chromosome condensation which is important for genetic material segregation. Studies about the effects of Mg2+ on the overall chromosome structure have been reported. Nevertheless, its effects on the distribution of heterochromatin and euchromatin region have yet to be investigated. The aim of this study was to evaluate the effects of Mg2+ on the banding pattern and ultrastructure of the chromosome. Chromosome analysis was performed using the synchronized HeLa cells. The effect of Mg2+ was evaluated by subjecting the chromosomes to three different solutions, namely XBE5 (containing 5 mM Mg2+ ) as a control, XBE (0 mM Mg2+ ), and 1 mM EDTA as cations-chelator. Chromosome banding was carried out using the GTL-banding technique. The ultrastructure of the chromosomes treated with and without Mg2+ was further obtained using SEM. The results showed a condensed chromosome structure with a clear banding pattern when the chromosomes were treated with a buffer containing 5 mM Mg2+ . In contrast, chromosomes treated with a buffer containing no Mg2+ and those treated with a cations-chelator showed an expanded and fibrous structure with the lower intensity of the banding pattern. Elongation of the chromosome caused by decondensation resulted in the band splitting. The different ultrastructure of the chromosomes treated with and without Mg2+ was obvious under SEM. The results of this study further emphasized the role of Mg2+ on chromosome structure and gave insights into Mg2+ effects on the banding distribution and ultrastructure of the chromosome.

Topoisomerase II dysfunction causes metaphase I arrest by activating Aurora B, SAC and MPF and prevents PB1 abscission in mouse oocytes†.

Oocyte aneuploidy is caused mainly by chromosome nondisjunction and/or unbalanced sister chromatid pre-division. Although studies in somatic cells have shown that topoisomerase II (TOP2) plays important roles in chromosome condensation and timely separation of centromeres, little is known about its role during oocyte meiosis. Furthermore, because VP-16, which is a TOP2 inhibitor and induces DNA double strand breaks, is often used for ovarian cancer chemotherapy, its effects on oocytes must be studied for ovarian cancer patients to recover ovarian function following chemotherapy. This study showed that inhibiting TOP2 with either ICRF-193 or VP-16 during meiosis I impaired chromatin condensation, chromosome alignment, TOP2α localization, and caused metaphase I (MI) arrest and first polar body (PB1) abscission failure. Inhibiting or neutralizing either spindle assembly checkpoint (SAC), Aurora B or maturation-promoting factor (MPF) significantly abolished the effect of ICRF-193 or VP-16 on MI arrest. Treatment with ICRF-193 or VP-16 significantly activated MPF and SAC but the effect disappeared when Aurora B was inhibited. Most of the oocytes matured in the presence of ICRF-193 or VP-16 were arrested at MI, and only 11-27% showed PB1 protrusion. Furthermore, most of the PB1 protrusions formed in the presence of ICRF-193 or VP-16 were retracted after further culture for 7h. In conclusion, TOP2 dysfunction causes MI arrest by activating Aurora B, SAC, and MPF, and it prevents PB1 abscission by promoting chromatin bridges.

SMC4 knockdown inhibits malignant biological behaviors of endometrial cancer cells by regulation of FoxO1 activity

Structural maintenance of chromosomes 4 (SMC4) has an important role in chromosome condensation and segregation, which is involved in regulating multiple tumor development. However, the role of SMC4 in endometrial cancer is uncertain. The expression and prognostic value of SMC4 were predicted by UALCAN, Gene Expression Omnibus (GEO), The Human Protein Atlas and Kaplan Meier plotter tools. SMC4-related genes were analyzed by LinkedOmics, Gene Ontology (GO) annotations, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Forkhead box protein O1 (FoxO1) activity was suppressed by AS1842856 (AS). SMC4, Ki67, B-cell lymphoma-2(Bcl-2), Bcl-2 associated X protein (Bax), FoxO1, phosphorylated FoxO1 (p-FoxO1), and p27 protein levels were detected by Western blotting. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) analyses. Cell apoptosis was measured using TUNEL analysis. SMC4 abundance was increased in endometrial cancer, and predicted a worse overall survival. SMC4 knockdown repressed proliferative ability of endometrial cancer cells and promoted cell apoptosis. SMC4 knockdown promoted FoxO1 transactivation by decreasing its phosphorylated level. Addition of AS inhibited FoxO1 activity by increasing the phosphorylated level of FoxO1. The inhibition of FoxO1 activity reversed the effect of SMC4 silencing on cell proliferation and apoptosis. In conclusion, SMC4 silencing restrained cell proliferation and facilitated apoptosis in endometrial cancer via regulating FoxO1 activity.

Inhibition of HDAC6 by tubastatin A disrupts mouse oocyte meiosis via regulating histone modifications and mRNA expression.

Histone deacetylase 6 (HDAC6) participates in mouse oocyte maturation by deacetylating α-tubulin. However, how HDAC6 expression is regulated in oocytes remains unknown. In the present study, we discovered that mouse oocytes had a high level of HDAC6 expression and a low level of DNA methylation status in theirpromoter region. Then, a selective HDAC6 inhibitor, tubastatin A (Tub-A) was chosen to investigate the role of HDAC6 in oocyte maturation. Our results revealed that inhibition of HDAC6 caused meiotic progression arrest, disturbed spindle/chromosome organization, and kinetochore-microtubule attachments without impairing spindle assembly checkpoint function. Moreover, inhibition of HDAC6 not only increased the acetylation of α-tubulin but also elevated the acetylation status of H4K16 and decreased the phosphorylation level of H3T3 and H3S10. Conversely, depressed H3T3 phosphorylation by its kinase inhibitor increased the acetylation level of H4K16. Finally, single cell RNA-seq analysis revealed that the cell cycle-related genes CCNB1, CDK2, SMAD3, YWHAZ and the methylation-related genes DNMT1 and DNMT3B were strongly repressed in Tub-A treated oocytes. Taken together, our results indicate that HDAC6 plays important roles in chromosome condensation and kinetochore function via regulating several key histone modifications and messenger RNA transcription during oocyte meiosis.

Distinct Roles for Condensin's Two ATPase Sites in Chromosome Condensation.

SummaryCondensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin’s ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture.

3D observation of chromosome scaffold structure using a 360° electron tomography sample holder

The chromosome scaffold is considered to be a key structure of the mitotic chromosome. It plays a vital role in chromosome condensation, shaping the X-shaped structure of the mitotic chromosome, and also provides flexibility for chromosome movement during cell division. However, it remains to be elucidated how the chromosome scaffold organizes the mitotic chromosome and how it supports shaping the structure of the chromosome during metaphase. Here we present a new technique that enables the observation of the chromosome scaffold structure in metaphase chromosomes from any direction, by transferring an isolated chromosome to a 360° rotational holder for electron tomography (ET). The chromosome was stained with immunogold-labeled condensin complex, one of the major chromosome scaffold proteins and then observed in three dimensions using ET. Using the locations of gold nanoparticles to visualize the underlying structure, the tomograms we obtained reveal the patterns of chromosome scaffold organization, which appears to consist of a helical structure that serves to organize chromatin loops into the metaphase chromosome.

Plk1 phosphorylation of CAP-H2 triggers chromosome condensation by condensin II at the early phase of mitosis

Condensin complexes play crucial roles in chromosome condensation that is a fundamental process to establish the “rod-like” shape of chromosome structure in mitosis. Failure of the chromosome assembly causes chromosome segregation errors and subsequent genomic instability. However, a molecular mechanism that controls condensin function for the chromosomal organization has not been fully understood. Here, we show that the abundance of CAP-H2, one of the condensin II subunits, is fluctuated during the cell cycle in accordance with Plk1 kinase activity. Inhibition of Plk1 leads to Cdc20-mediated degradation of CAP-H2 in mitosis. Plk1 phosphorylation of CAP-H2 at Ser288 is required for the accumulation of CAP-H2 and accurate chromosomal condensation during prophase. These findings suggest that Plk1 phosphorylation regulates condensin II function by modulating CAP-H2 expression levels to facilitate proper mitotic chromosome organization.

Recent advances in cohesin biology

Sister chromatids are tethered together from the time they are formed in S-phase until they separate at anaphase. A protein complex called cohesin is responsible for holding the sister chromatids together and serves important roles in chromosome condensation, gene regulation, and the repair of DNA damage. Cohesin contains an open central pore and becomes topologically engaged with its DNA substrates. Entrapped DNA can be released either by the opening of a gate in the cohesin ring or by proteolytic cleavage of a component of the ring. This review summarizes recent research that provides important new insights into how DNA enters and exits the cohesin ring and how the rings behave on entrapped DNA molecules to provide functional cohesion.

Visualizing the Assembly of DNA Condensation Clusters by SMC using Single-Molecule Microscopy

SMC (structural maintenance of chromosomes) family members play essential roles in chromosome condensation, sister chromatid cohesion, and DNA repair. Despite intensive efforts, it is still unclear how SMCs are loaded onto DNA and structure chromosomes. We employed single-molecule fluorescence microscopy to visualize how individual Bacillus subtilis SMC (BsSMC) dimers interact with flow-stretched DNAs. We find that BsSMC can vary its initial interaction with DNA, switching between static binding and sliding. Its diffusive properties are insensitive to the presence of ATP, suggesting that the initial loading of BsSMC onto DNA is ATP-independent. At higher concentrations, BsSMCs form distinct clusters that condense DNA by both wrapping and bridging distal DNA segments. SMC-mediated DNA compaction occurs in a weakly ATP-dependent manner. However, ATP increases the apparent cooperativity of DNA condensation, demonstrating that multiple BsSMCs can interact cooperatively through their ATPase head domains. Consistent with these results, BsSMC mutants that alter the ATPase cycle compact DNA more slowly than wild-type BsSMC in the presence of ATP, and an ATPase headless construct compacts DNA non-cooperatively with substantially lower compaction rate. Our results suggest that transiently static BsSMC can nucleate the formation of functionally active clusters that locally condense the chromosome while forming long-range DNA bridges.

Multistep assembly of DNA condensation clusters by SMC.

SMC (structural maintenance of chromosomes) family members play essential roles in chromosome condensation, sister chromatid cohesion and DNA repair. It remains unclear how SMCs structure chromosomes and how their mechanochemical cycle regulates their interactions with DNA. Here we used single-molecule fluorescence microscopy to visualize how Bacillus subtilis SMC (BsSMC) interacts with flow-stretched DNAs. We report that BsSMC can slide on DNA, switching between static binding and diffusion. At higher concentrations, BsSMCs form clusters that condense DNA in a weakly ATP-dependent manner. ATP increases the apparent cooperativity of DNA condensation, demonstrating that BsSMC can interact cooperatively through their ATPase head domains. Consistent with these results, ATPase mutants compact DNA more slowly than wild-type BsSMC in the presence of ATP. Our results suggest that transiently static BsSMC molecules can nucleate the formation of clusters that act to locally condense the chromosome while forming long-range DNA bridges.

Shaping of interphase chromosomes by the microtubule network

It is well established that microtubule dynamics play a major role in chromosome condensation and localization during mitosis. During interphase, however, it is assumed that the metazoan nuclear envelope presents a physical barrier, which inhibits interaction between the microtubules located in the cytoplasm and the chromatin fibers located in the nucleus. In recent years, it has become apparent that microtubule dynamics alter chromatin structure and function during interphase as well. Microtubule motor proteins transport several transcription factors and exogenous DNA (such as plasmid DNA) from the cytoplasm to the nucleus. Various soluble microtubule components are able to translocate into the nucleus, where they bind various chromatin elements leading to transcriptional alterations. In addition, microtubules may apply force on the nuclear envelope, which is transmitted into the nucleus, leading to changes in chromatin structure. Thus, microtubule dynamics during interphase may affect chromatin spatial organization, as well as transcription, replication and repair.

Aurora Kinase Inhibition As an Anticancer Strategy

Article Tools UNDERSTANDING THE PATHWAY Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/JCO.2013.50.7988 Journal of Clinical Oncology - published online before print September 16, 2013 PMID: 24043748 Aurora Kinase Inhibition As an Anticancer Strategy John F. HiltonxJohn F. HiltonSearch for articles by this author , Geoffrey I. ShapiroxGeoffrey I. ShapiroSearch for articles by this author Show More Dana-Farber Cancer Institute, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA https://doi.org/10.1200/JCO.2013.50.7988 First Page Full Text PDF Figures and Tables © 2013 by American Society of Clinical Oncology

SCFSlimb ubiquitin ligase suppresses condensin II-mediated nuclear reorganization by degrading Cap-H2.

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF(Slimb) ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF(Slimb) function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF(Slimb)-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.

Single-Molecule Studies of Chromosome Organization by SMC

The structural maintenance of chromosomes complex (SMC) plays an essential role in chromosome condensation, sister chromatid cohesion, and recombinational repair. SMCs possess a unique structural architecture: SMC monomers dimerize through interactions within their hinge domains while long coiled-coil arms bring together the N- and C-termini which form an ATPase head domain. Despite its cellular importance and extensive efforts, the role of the ATPase domain of SMC still remains mysterious. How ATP binding and hydrolysis is coupled to DNA remodeling or how the mechanochemical cycle of SMC modulates the SMC-DNA interaction is unclear. Using Bacillus Subtilis SMC (BsSMC) as a model we are utilizing single-molecule microscopy to visualize how SMCs interact with flow stretched DNAs. In an approach we have termed DNA motion capture, we site-specifically label DNAs with quantum dots which allow us to track how DNA segments under differing tensions are condensed by SMC. We find that BsSMC is able to compact individual DNAs even in the absence of ATP although ATP greatly enhances the rate of DNA compaction. This supports a picture in which ATP does not provide a powerstroke for DNA organization but instead acts to bias BsSMC conformations that facilitate chromosome remodeling. To better understand the role of ATP in the mechanochemistry of BsSMC we examined how two mutants, E1118Q which slows the rate of ATP hydrolysis and S1090R which blocks head-head engagement, alter the kinetics of BsSMC-mediated DNA condensation. Our single-molecule assay shows that the E1118Q mutant compacts DNA more slowly than the wild-type in the presence of ATP, and significant compaction rate reduction was observed with S1090R mutant. These results suggest a role for both ATP hydrolysis and SMC head reengagement in SMC-mediated DNA condensation.

Drosophila Psf2 has a role in chromosome condensation

The condensation state of chromosomes is a critical parameter in multiple processes within the cell. Failures in the maintenance of appropriate condensation states may lead to genomic instability, mis-expression of genes, and a number of disease states. During cell proliferation, replication of DNA represents an ongoing challenge for chromosome packaging as DNA must be unpackaged for replication and then faithfully repackaged. An integral member of the DNA replication machinery is the GINS complex which has been shown to stabilize the CMG complex which is required for processivity of the Mcm2-7 helicase complex during S phase. Through examination of the phenotypes associated with a null mutation in Psf2, a member of the evolutionarily conserved GINS complex, we find that Drosophila Psf2 likely has a role in establishing chromosome condensation and that the defects associated with this mis-condensation impact M phase progression, genomic stability, and transcriptional regulation.

Abstract 3000: MIIP regulates histone H3 phosphorylation at serine 10 and securin degradation during mitosis in colon cancer cells

Abstract Chromosome condensation and segregation are highly orchestrated during mitosis. In early mitosis, dramatic structural changes occur to produce metaphase chromosomes, each consisting of a pair of compacted sister chromatids (condensation), At anaphase onset, a signal is produced to disrupt the linkage between sister chromatids (segregation), allowing them to be pulled apart to two daughter cells. Recently, we discovered that the MIIP protein, encoded by the Migration and invasion inhibitory protein gene located on chromosome 1p36, plays a critical role in mitotic transition through negatively regulating APC/CCdc20 activity and stabilizing cyclin B1 in glioma cells. Here we report that MIIP inhibits chromosome condensation and segregation resulting in G2/M arrest and polyploidy cell formation in colon cancer cells. Gain-of-function studies showed that the MIIP expression inhibited colony formation of isogenic HCT116 cells independent of the TP53 and PUMA status. To understand the potential role of MIIP in colon cancer cells, a panel of six colon cancer cell lines (DLD1, SW620, SW480, HCT116, NCI-747 and SW837) was used to elevate MIIP expression by adenovirus expression vector (Ad-MIIP). The G2/M arrest and polyploidy cell formation were observed in all the six cell lines we tested irrespective of the status of microsatellite stability and TP53 mutation. By using double thymidine block strategy, we synchronized HCT116 cells at G1/S boundary and released the cells in fresh medium to enrich the cell population in different stage during the cell cycle. We found that the expression of MIIP is cell cycle dependent and peaks at mitosis. Two major mitotic regulators, cyclin B1 and securin, were induced by MIIP. The cycloheximide (CHX) chase experiments showed that MIIP blocked cyclin B1 and securin degradation, which is consistent with our previous finding that MIIP directly interacted with Cdc20 and inhibited APC/CCdc20 activity. We further found that MIIP inhibited histone H3 phosphorylation at serine 10. Taken together, our studies provide evidence that MIIP plays a critical role in chromosome condensation and segregation in mitosis. Because chromosome 1p36 region is commonly deleted in colon cancer cells, the MIIP gene may represent a novel negative cell cycle regulator that is abnormally altered thus contributing to genomic instability in colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2011-3000

Manipulation of Single Knotted DNA Molecules with Topoisomerase II by using Atomic Force Microscopy

Although the hereditary information is encoded in the primary sequence of the genetic material, much of the regulatory information of DNA is hidden in its topology and geometry in the cell. DNA supercoiling and knotting are important for DNA packaging within all cells, however this impairs the well behavior of multiples cellular processes such as replication and transcription, which involve helical winding, strand separation, and movement along the DNA. The steric and topological problems associated with supercoiling can be studied with numerous different tools among which scanning probe microscopy is a very promising one because of its capability to image on line and with a high resolution, biological samples in nearly physiological conditions.In this work we studied the way enzymes and drugs interact with DNA molecules by using Atomic Force Microscopy (AFM). Particularly, we have centered our studies on human type II DNA topoisomerases (Top2), which are essential and ubiquitous enzymes that perform important intracellular roles in chromosome condensation and segregation, and in regulating DNA supercoiling. We will present real time movies showing how the Top2 relaxes DNA. In addition we will present AFM images showing the way DNA changes its topology after being exposed to intercalating agents. We hope that this new methodology will unveil some of the still poorly known DNA-proteins and drugs interaction.

Mutations in the Chromosomal Passenger Complex and the Condensin Complex Differentially Affect Synaptonemal Complex Disassembly and Metaphase I Configuration in Drosophila Female Meiosis

Production of haploid gametes relies on the specially regulated meiotic cell cycle. Analyses of the role of essential mitotic regulators in meiosis have been hampered by a shortage of appropriate alleles in metazoans. We characterized female-sterile alleles of the condensin complex component dcap-g and used them to define roles for condensin in Drosophila female meiosis. In mitosis, the condensin complex is required for sister-chromatid resolution and contributes to chromosome condensation. In meiosis, we demonstrate a role for dcap-g in disassembly of the synaptonemal complex and for proper retention of the chromosomes in a metaphase I-arrested state. The chromosomal passenger complex also is known to have mitotic roles in chromosome condensation and is required in some systems for localization of the condensin complex. We used the QA26 allele of passenger component incenp to investigate the role of the passenger complex in oocyte meiosis. Strikingly, in incenp(QA26) mutants maintenance of the synaptonemal complex is disrupted. In contrast to the dcap-g mutants, the incenp mutation leads to a failure of paired homologous chromosomes to biorient, such that bivalents frequently orient toward only one pole in prometaphase and metaphase I. We show that incenp interacts genetically with ord, suggesting an important functional relationship between them in meiotic chromosome dynamics. The dcap-g and incenp mutations cause maternal effect lethality, with embryos from mutant mothers arrested in the initial mitotic divisions.

Analysis of the eukaryotic topoisomerase II DNA gate: a single-molecule FRET and structural perspective

Type II DNA topoisomerases (topos) are essential and ubiquitous enzymes that perform important intracellular roles in chromosome condensation and segregation, and in regulating DNA supercoiling. Eukaryotic topo II, a type II topoisomerase, is a homodimeric enzyme that solves topological entanglement problems by using the energy from ATP hydrolysis to pass one segment of DNA through another by way of a reversible, enzyme-bridged double-stranded break. This DNA break is linked to the protein by a phosphodiester bond between the active site tyrosine of each subunit and backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this enzymatic cleavage/religation of the backbone. This reversible DNA cleavage reaction is the target of a number of anticancer drugs, which can elicit DNA damage by affecting the cleavage/religation equilibrium. Because of its clinical importance, many studies have sought to determine the manner in which topo II interacts with DNA. Here we highlight recent single-molecule fluorescence resonance energy transfer and crystallographic studies that have provided new insight into the dynamics and structure of the topo II DNA gate.

Specificity of DNA Binding and Dimerization by CspE from Escherichia coli

The CspE protein from Escherichia coli K12 is a single-stranded nucleic acid-binding protein that plays a role in chromosome condensation in vivo. We report here that CspE binds to single-stranded DNA containing 6 or more contiguous dT residues with high affinity (K(D) < 30 nM). The interactions are predominantly through base-specific contacts. When an oligonucleotide contains fewer than 6 contiguous dT residues, the CspE interactions with single-stranded DNA are primarily electrostatic. The minimal length of single-stranded DNA to which CspE binds in a salt-resistant manner is eight nucleotides. We also show that CspE exists as a dimer in solution. We present a possible mechanism to explain the role of CspE in chromosome condensation in vivo by CspE binding to distant DNA regions in the chromosome and dimerizing, thereby condensing the intervening DNA.