scholarly journals pEg7, a New Xenopus Protein Required for Mitotic Chromosome Condensation in Egg Extracts

1998 ◽  
Vol 143 (6) ◽  
pp. 1437-1446 ◽  
Author(s):  
Fabien Cubizolles ◽  
Vincent Legagneux ◽  
René Le Guellec ◽  
Isabelle Chartrain ◽  
Rustem Uzbekov ◽  
...  
Keyword(s):  
Mitotic Chromosome ◽  
Cultured Cells ◽  
Sperm Chromatin ◽  
Mitotic Chromosomes ◽  
Maternal Mrna ◽  
Early Embryos ◽  
Egg Extracts ◽  
Cytostatic Factor ◽  
Cell Expression ◽  
Mitotic Chromosome Condensation

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.

scholarly journals Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts.

1993 ◽  
Vol 120 (3) ◽  
pp. 601-612 ◽  
Author(s):  
T Hirano ◽  
T J Mitchison
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Morphology ◽  
Sperm Chromatin ◽  
Mitotic Chromosomes ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Topo Ii ◽  
Xenopus Egg Extracts

We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.

scholarly journals Centromeric Localization of Dispersed Pol III Genes in Fission Yeast

2010 ◽  
Vol 21 (2) ◽  
pp. 254-265 ◽  
Author(s):  
Osamu Iwasaki ◽  
Atsunari Tanaka ◽  
Hideki Tanizawa ◽  
Shiv I.S. Grewal ◽  
Ken-ichi Noma
Keyword(s):  
Genome Organization ◽  
Gene Locus ◽  
Mitotic Chromosome ◽  
Three Dimensional ◽  
Rrna Genes ◽  
Mitotic Chromosomes ◽  
Gene Encoding ◽  
5S Rrna Genes ◽  
Pol Iii ◽  
Mitotic Chromosome Condensation

The eukaryotic genome is a complex three-dimensional entity residing in the nucleus. We present evidence that Pol III–transcribed genes such as tRNA and 5S rRNA genes can localize to centromeres and contribute to a global genome organization. Furthermore, we find that ectopic insertion of Pol III genes into a non-Pol III gene locus results in the centromeric localization of the locus. We show that the centromeric localization of Pol III genes is mediated by condensin, which interacts with the Pol III transcription machinery, and that transcription levels of the Pol III genes are negatively correlated with the centromeric localization of Pol III genes. This centromeric localization of Pol III genes initially observed in interphase becomes prominent during mitosis, when chromosomes are condensed. Remarkably, defective mitotic chromosome condensation by a condensin mutation, cut3-477, which reduces the centromeric localization of Pol III genes, is suppressed by a mutation in the sfc3 gene encoding the Pol III transcription factor TFIIIC subunit, sfc3-1. The sfc3-1 mutation promotes the centromeric localization of Pol III genes. Our study suggests there are functional links between the process of the centromeric localization of dispersed Pol III genes, their transcription, and the assembly of condensed mitotic chromosomes.

scholarly journals Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei.

1996 ◽  
Vol 133 (5) ◽  
pp. 955-969 ◽  
Author(s):  
J Fang ◽  
R M Benbow
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
Nuclear Dna ◽  
Cultured Cells ◽  
Sperm Chromatin ◽  
Quiescent State ◽  
Cell Nuclei ◽  
A6 Cells ◽  
Egg Extracts ◽  
Liver Nuclei

Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery.

scholarly journals Topoisomerase IIα is essential for maintenance of mitotic chromosome structure

2020 ◽  
Vol 117 (22) ◽  
pp. 12131-12142 ◽  
Author(s):  
Christian F. Nielsen ◽  
Tao Zhang ◽  
Marin Barisic ◽  
Paul Kalitsis ◽  
Damien F. Hudson
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Chromosome Structure ◽  
Chromatin Condensation ◽  
Chromosome Condensation ◽  
Mitotic Chromosomes ◽  
Topoisomerase Iiα ◽  
Mitotic Chromosome Condensation

Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.

scholarly journals The TFIIH Complex is Required to Establish and Maintain Mitotic Chromosome Structure

2021 ◽  
Author(s):  
Julian Haase ◽  
Richard Chen ◽  
Mary Kate Bonner ◽  
Lisa M Miller Jenkins ◽  
Alexander E Kelly
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Structure ◽  
Complete Loss ◽  
General Transcription Factor ◽  
Egg Extracts ◽  
Nucleosome Density ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Mitotic Chromosome Condensation

Condensins compact chromosomes to promote their equal segregation during mitosis, but the mechanism of condensin engagement with and action on chromatin is incompletely understood. Here, we show that the general transcription factor TFIIH complex is continuously required to establish and maintain a compacted chromosome structure in transcriptionally silent Xenopus egg extracts. Inhibiting the DNA-dependent ATPase activity of the TFIIH complex subunit XPB prevents the enrichment of condensins I and II, but not topoisomerase II, on chromatin. In addition, TFIIH inhibition reversibly induces a complete loss of chromosome structure within minutes, prior to the loss of condensins from chromatin. Reducing nucleosome density through partial histone depletion restores chromosome structure and condensin enrichment in the absence of TFIIH activity. We propose that the TFIIH complex promotes mitotic chromosome condensation by dynamically altering chromatin structure to facilitate condensin loading and condensin-dependent loop extrusion.

scholarly journals A Human Condensin Complex Containing hCAP-C–hCAP-E and CNAP1, a Homolog of Xenopus XCAP-D2, Colocalizes with Phosphorylated Histone H3 during the Early Stage of Mitotic Chromosome Condensation

2000 ◽  
Vol 20 (18) ◽  
pp. 6996-7006 ◽  
Author(s):  
John A. Schmiesing ◽  
Heather C. Gregson ◽  
Sharleen Zhou ◽  
Kyoko Yokomori
Keyword(s):  
Mammalian Cells ◽  
Structural Changes ◽  
Mitotic Chromosome ◽  
Early Stage ◽  
Histone H3 ◽  
Chromosome Condensation ◽  
Early Prophase ◽  
Mitotic Chromosomes ◽  
Condensin Complex ◽  
Mitotic Chromosome Condensation

ABSTRACT Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Previously, we identified two human SMC family proteins, hCAP-C and hCAP-E, which form a heterodimeric complex (hCAP-C–hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C–hCAP-E was determined to be the human ortholog of theXenopus SMC complex, XCAP-C–XCAP-E. XCAP-C–XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C–hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C–hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous toXenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G2/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3.

scholarly journals Identification of a Chromosome-Targeting Domain in the Human Condensin Subunit CNAP1/hCAP-D2/Eg7

2002 ◽  
Vol 22 (16) ◽  
pp. 5769-5781 ◽  
Author(s):  
Alexander R. Ball, ◽  
John A. Schmiesing ◽  
Changcheng Zhou ◽  
Heather C. Gregson ◽  
Yoshiaki Okada ◽  
...  
Keyword(s):  
Mitotic Chromosome ◽  
Mitotic Chromosomes ◽  
Family Protein ◽  
Localization Signal ◽  
The Individual ◽  
Mitotic Chromosome Condensation ◽  
Structural Maintenance Of Chromosomes

ABSTRACT CNAP1 (hCAP-D2/Eg7) is an essential component of the human condensin complex required for mitotic chromosome condensation. This conserved complex contains a structural maintenance of chromosomes (SMC) family protein heterodimer and three non-SMC subunits. The mechanism underlying condensin targeting to mitotic chromosomes and the role played by the individual condensin components, particularly the non-SMC subunits, are not well understood. We report here characterization of the non-SMC condensin component CNAP1. CNAP1 contains two separate domains required for its stable incorporation into the complex. We found that the carboxyl terminus of CNAP1 possesses a mitotic chromosome-targeting domain that does not require the other condensin components. The same region also contains a functional bipartite nuclear localization signal. A mutant CNAP1 missing this domain, although still incorporated into condensin, was unable to associate with mitotic chromosomes. Successful chromosome targeting of deletion mutants correlated with their ability to directly bind to histones H1 and H3 in vitro. The H3 interaction appears to be mediated through the H3 histone tail, and a subfragment containing the targeting domain was found to interact with histone H3 in vivo. Thus, the CNAP1 C-terminal region defines a novel histone-binding domain that is responsible for targeting CNAP1, and possibly condensin, to mitotic chromosomes.

scholarly journals Axial contraction and short-range compaction of chromatin synergistically promote mitotic chromosome condensation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Tom Kruitwagen ◽  
Annina Denoth-Lippuner ◽  
Bryan J Wilkins ◽  
Heinz Neumann ◽  
Yves Barral
Keyword(s):  
Short Range ◽  
Mitotic Chromosome ◽  
Chromosome Condensation ◽  
Yeast Cells ◽  
Histone H4 ◽  
Mitotic Chromosomes ◽  
Chromatin Compaction ◽  
Early Anaphase ◽  
Local Compaction ◽  
Mitotic Chromosome Condensation

The segregation of eukaryotic chromosomes during mitosis requires their extensive folding into units of manageable size for the mitotic spindle. Here, we report on how phosphorylation at serine 10 of histone H3 (H3 S10) contributes to this process. Using a fluorescence-based assay to study local compaction of the chromatin fiber in living yeast cells, we show that chromosome condensation entails two temporally and mechanistically distinct processes. Initially, nucleosome-nucleosome interaction triggered by H3 S10 phosphorylation and deacetylation of histone H4 promote short-range compaction of chromatin during early anaphase. Independently, condensin mediates the axial contraction of chromosome arms, a process peaking later in anaphase. Whereas defects in chromatin compaction have no observable effect on axial contraction and condensin inactivation does not affect short-range chromatin compaction, inactivation of both pathways causes synergistic defects in chromosome segregation and cell viability. Furthermore, both pathways rely at least partially on the deacetylase Hst2, suggesting that this protein helps coordinating chromatin compaction and axial contraction to properly shape mitotic chromosomes.

scholarly journals Condensins under the microscope

2018 ◽  
Vol 217 (7) ◽  
pp. 2229-2231 ◽  
Author(s):  
Kazuhiro Maeshima ◽  
Kayo Hibino ◽  
Damien F. Hudson
Keyword(s):  
Quantitative Data ◽  
Mitotic Chromosome ◽  
State Of The Art ◽  
Imaging Techniques ◽  
Mechanistic Model ◽  
Chromosome Condensation ◽  
Mitotic Chromosomes ◽  
Cell Biol ◽  
Key Players ◽  
Mitotic Chromosome Condensation

Condensins are key players in mitotic chromosome condensation. Using an elegant combination of state-of-the-art imaging techniques, Walther et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201801048) counted the number of Condensins, examined their behaviors on human mitotic chromosomes, and integrated the quantitative data to propose a new mechanistic model for chromosome condensation.

scholarly journals Mitotic Chromosome Condensation Driven by a Volume Phase Transition

2021 ◽  
Author(s):  
Andrew J Beel ◽  
Pierre-Jean Matteï ◽  
Roger D Kornberg
Keyword(s):  
Phase Transition ◽  
Mitotic Chromosome ◽  
Chromosome Morphology ◽  
Central Axis ◽  
Volume Phase Transition ◽  
Mitotic Chromosomes ◽  
Volume Phase ◽  
Change Of State ◽  
Physico Chemical ◽  
Mitotic Chromosome Condensation

Procedures were devised for the reversible decondensation and recondensation of purified mitotic chromosomes. Computational methods were developed for the quantitative analysis of chromosome morphology in high throughput, enabling the recording of condensation behavior of thousands of individual chromosomes. Established physico-chemical theory for ionic hydrogels was modified for application to chromosomal material and shown to accurately predict the observed condensation behavior. The theory predicts a change of state (a "volume phase transition") in the course of condensation, and such a transition was shown to occur. These findings, together with classical cytology showing loops of chromatin, lead to the description of mitotic chromosome structure in terms of two simple principles: contraction of length of chromatin fibers by the formation of loops, radiating from a central axis; and condensation of the chromosomal material against the central axis through a volume phase transition.

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