Molecular mechanisms of the chromosome condensation and decondensation cycle in mammalian cells

The chromosomes undergo a condensation-decondensation cycle within the life cycle of mammalian cells. Chromosome condensation is a complex and critical event that is necessary for the equal distribution of genetic material between the two daughter cells. Although chromosome condensation-decondensation and segregation is mechanistically complex, it proceeds with high fidelity during the eukaryotic cell division cycle. Cell fusion studies have indicated the presence of chromosome condensation factors in mammalian cells during mitosis. If extracts from mitotic cells are injected into immature oocytes of Xenopus laevis, they induce meiotic maturation (i.e. germinal vesicle breakdown and chromosome condensation) within 2–3 hours. Recently, we showed that the maturation-promoting activity of the mitotic cell extracts is inactivated by certain protein factors present in cells during the G₁ period. The activity of the G₁ factors coincides with the process of chromosome decondensation that begins at telophase and continues throughout the G₁ period. These studies have revealed that the mitotic factors and the G₁ factors play a pivotal role in the regulation of condensation and decondensation of chromosomes. Furthermore, our studies strongly suggest that nonhistone protein phosphorylation and dephosphorylation may mediate chromosome condensation and decondensation, respectively.     

Loss of Drosophila Myb interrupts the progression of chromosome condensation

Completion of chromosome condensation is required before segregation during the mitotic cell cycle to ensure the transmission of genetic material with high fidelity in a timely fashion. In many eukaryotes this condensation is regulated by phosphorylation of histone H3 on Ser 10 (H3S10). This phosphorylation normally begins in the late-replicating pericentric heterochromatin and then spreads to the earlier replicating euchromatin. Here, we show that these phases of condensation are genetically separable in that the absence of Drosophila Myb causes cells to arrest with H3S10 phosphorylation of heterochromatin but not euchromatin. In addition, we used mosaic analysis to demonstrate that although the Myb protein can be removed in a single cell cycle, the failure of chromosome condensation occurs only after many cell divisions in the absence of Myb protein. The Myb protein is normally located in euchromatic but not heterochromatic regions of the nucleus, suggesting that Myb may be essential for a modification of euchromatin that is required for the efficient spread of chromosome condensation.     

Premature chromosome condensation and cell cycle analysis.

The application of the phenomenon of premature chromosome condensation for cell cycle analysis in HeLa and CHO cells has been examined. Random populations of HeLa and CHO cells pulse labelled with H3-TdR were separately fused with mitotic HeLa cells using U.V. inactivated Sendai virus. The resulting prematurely condensed chromosomes (PCC) were scored and classified into G1, S and G2-PCC on the basis of both morphological and autoradiographic data, The results of this study indicated that the G1, S and G2 phase cells are equally susceptible to virus-induced fusion with mitotic cells and subsequent induction into PCC. Hence the PCC method for cell cycle analysis is both practical and accurate. This study also revealed that the process of chromosome decondensation initiated during the telophase of mitosis continues throughout the G1 period reaching an ultimate state of decondensation by the end of G1, at which point the fusion of such cells with those in mitosis yield PCC with the most diffused morphology instead of the discrete single stranded structures characteristic of early G1-PCC. Thus, the decondensation of chromatin during G1 appears to be a prerequisite for the subsequent initiation of DNA synthesis.     

The 'BN2' gene, a regulator for the onset of chromosome condensation

This review deals with the condensation–decondensation cycle of chromatin. This cycle can be analysed in increasing detail because of the availability of well-characterized temperature-sensitive cell-cycle mutants in which the control for condensation is aberrant at the non-permissive temperature. DNA transfection and gene cloning techniques using one such mutant have resulted in the identification of a gene involved in the normal regulation of entry into mitosis.     

Teniposide, a topoisomerase II inhibitor, prevents chromosome condensation and separation but not decondensation in fertilized surf clam (Spisula solidissima) oocytes

DNA topoisomerase II has been implicated in regulating chromosome interactions. We investigated the effects of the specific DNA topoisomerase II inhibitor, teniposide on nuclear events during oocyte maturation, fertilization, and early embryonic development of fertilized Spisula solidissima oocytes using DNA fluorescence. Teniposide treatment before fertilization not only inhibited chromosome separation during meiosis, but also blocked chromosome condensation during mitosis; however, sperm nuclear decondensation was unaffected. Chromosome separation was selectively blocked in oocytes treated with teniposide during either meiotic metaphase I or II indicating that topoisomerase II activity may be required during oocyte maturation. Teniposide treatment during meiosis also disrupted mitotic chromosome condensation. Chromosome separation during anaphase was unaffected in embryos treated with teniposide when the chromosomes were already condensed in metaphase of either first or second mitosis; however, chromosome condensation during the next mitosis was blocked. When interphase two- and four-cell embryos were exposed to topoisomerase II inhibitor, the subsequent mitosis proceeded normally in that the chromosomes condensed, separated, and decondensed; in contrast, chromosome condensation of the next mitosis was blocked. These observations suggest that in Spisula oocytes, topoisomerase II activity is required for chromosome separation during meiosis and condensation during mitosis, but is not involved in decondensation of the sperm nucleus, maternal chromosomes, and somatic chromatin.     

Inhibition of cation-induced DNA condensation by intercalating dyes

Several intercalating dyes are shown to inhibit the cation-induced condensation of λ-DNA when Co³⁺(NH₃)₆ is the condensing agent. The dyes that have been studied are ethidium, propidium, proflavin, quinacrine, and actinomycin D. Earlier work has shown that intercalating dyes inhibit ψ-DNA condensation. [Lerman, L. S. (1971) Prog. Mol. Subcell. Biol. 2 , 382–391; Cheng, S. & Mohr, S. C. (1975) Biopolymers 14 , 663–674.] Dye-induced decondensation of intramolecularly condensed DNA has been studied by making use of conditions in which Co³⁺(NH₃)₆ produces intramolecular condensation without significant aggregation. Some aggregation is caused, however, during dye-induced decondensation. Dye titration curves of DNA decondensation have been measured by excess light scattering to monitor decondensation and by fluorescence to monitor intercalation. All of the dyes studied act as competing cations in displacing the condensing cation Co³⁺(NH₃)₆ from the DNA. Competition occurs both in and below the transition zone for condensation. The effectiveness of a dye as a competing cation increases with its net positive charge. Before decondensation begins, no intercalated dye can be detected, suggesting that intercalation might be incompatible with the proper helix packing needed for cation-induced DNA condensation. To test this last point, methidium–spermine was synthesized: it contains an intercalating methidium head group combined with a polyamine tail. Methidium–spermine is found to cause λ-DNA condensation, but aggregation accompanies condensation, as has been found earlier for spermine and spermidine. Fluorescence and absorption spectra indicate that the methidium group is intercalated when the DNA is condensed, indicating that intercalation need not be incompatible with DNA condensation. The presence of aggregates among the condensed DNA molecules makes this last conclusion tentative.     

Real-time observation of nucleoplasmin-mediated DNA decondensation and condensation reveals its specific functions as a chaperone

Fertilization requires decondensation of promatine-condensed sperm chromatin, a dynamic process serving as an attractive system for the study of chromatin reprogramming. Nucleoplasmin is a key factor in regulating nucleosome assembly as a chaperone during fertilization process. However, knowledge on nucleoplasmin in chromatin formation remains elusive. Herein, magnetic tweezers (MT) and a chromatin assembly system were used to study the nucleoplasmin-mediated DNA decondensation/condensation at the single-molecular level in vitro. We found that protamine induces DNA condensation in a stepwise manner. Once DNA was condensed, nucleoplasmin, polyglutamic acid, and RNA could remove protamine from the DNA at different rates. The affinity binding of the different polyanions with protamine suggests chaperone-mediated chromatin decondensation activity occurs through protein–protein interactions. After decondensation, both RNA and polyglutamic acid prevented the transfer of histones onto the naked DNA. In contrast, nucleoplasmin is able to assist the histone transfer process, even though it carries the same negative charge as RNA and polyglutamic acid. These observations imply that the chaperone effects of nucleoplasmin during the decondensation/condensation process may be driven by specific spatial configuration of its acidic pentamer structure, rather than by electrostatic interaction. Our findings offer a novel molecular understanding of nucleoplasmin in sperm chromatin decondensation and subsequent developmental chromatin reprogramming at individual molecular level.     

Changes in the organization of chromosomes during the cell cycle: response to ultraviolet light.

Fusion between mitotic and interphase cells results in the premature condensation of the interphase chromosomes into a morphology related to the position in the cell cycle at the time of fusion. These prematurely condensed chromosomes (PCC) have been used in conjunction with u.v. irradiation to examine the interphase chromosome condensation cycle of HeLa cells. The following observations have been made: (I) There is a progressive decondensation of the chromosomes during G1 which is accentuated by u.v. irradiation: (2) The chromosomes become more resistant to u.v.-induced decondensation during G2 and mitosis. (3) There is a close correlation between the degree of chromosome decondensation and the amount of unscheduled DNA synthesis induced by u.v. irradiation during G1 and mitosis: (4) Hydroxyurea enhances the ability of u.v. irradiation to promote the decondensation of chromosomes during G1, G2 and mitosis. Hydroxyurea also potentiates the lethal action of u.v. irradiation during mitosis and G1. These data are discussed in relation to the suggestion that chromosomes undergo a progressive decondensation during G1 and condensation during G2.     

Regulation of chromosome condensation and segregation

Regulated and controlled chromosome condensation and segregation is essential for the transmission of genetic information from one generation to the next. A myriad of techniques has been utilized over the last few decades to identify proteins required for the organized compaction of the massive length of a cell's DNA. A full understanding of the components and processes involved relies on further work, exploiting biochemical, genetic, cytological, and proteomics approaches to complete the picture of how a cell packages and partitions its genome during the cell cycle.     

Akt phosphorylates acinus and inhibits its proteolytic cleavage, preventing chromatin condensation

Akt promotes cell survival by phosphorylating and inhibiting components of the intrinsic cell death machinery. Akt translocates into the nucleus upon exposure of cells to survival factors, but little is known about its functions in the nucleus. Here, we show that acinus, a nuclear factor required for apoptotic chromatin condensation, is a direct target of Akt. We demonstrate that Akt phosphorylation of acinus on serine 422 and 573 results in its resistance to caspase cleavage in the nucleus and the inhibition of acinus-dependent chromatin condensation. Abolishing acinus phosphorylation by Akt through mutagenesis accelerates its proteolytic degradation and chromatin condensation. Acinus S422, 573D, a mutant mimicking phosphorylation, resists against apoptotic cleavage and prevents chromatin condensation. Knocking down of acinus substantially decreases chromatin condensation, and depletion of Akt provokes the apoptotic cleavage of acinus. Thus, Akt inhibits chromatin condensation during apoptosis by phosphorylating acinus in the nucleus, revealing a specific mechanism by which nuclear Akt promotes cell survival.     

A physical model for the condensation and decondensation of eukaryotic chromosomes

During the eukaryotic cell cycle, chromatin undergoes several conformational changes, which are believed to play key roles in gene expression regulation during interphase, and in genome replication and division during mitosis. In this paper, we propose a scenario for chromatin structural reorganization during mitosis, which bridges all the different scales involved in chromatin architecture, from nucleosomes to chromatin loops. We build a model for chromatin, based on available data, taking into account both physical and topological constraints DNA has to deal with. Our results suggest that the mitotic chromosome condensation/decondensation process is induced by a structural change at the level of the nucleosome itself.     

水稻淀粉胚乳细胞编程性死亡中细胞核变化特征

应用透射电子显微镜技术 ,观察了水稻 (OryzasativaL .)淀粉胚乳细胞编程性死亡过程中核的变化特征。伴随胚乳的发育进程 ,淀粉胚乳细胞核表现出衰退特征 :核变形、染色质凝缩、核膜多处被降解破坏、核基质外泄等。DNALadder显示核内大片段DNA呈严重的弥散状拖尾现象 ,而核内和胞质中在 14 0～ 180bp处有明显的条带。在核衰退的同时 ,其胞质中的粗面内质网、淀粉质体和线粒体等细胞器具有正常的代谢功能 ,细胞仍在合成并积累营养物质 ,淀粉胚乳细胞一边衰退一边行使其功能 ,直至死亡。这些结果表明 ,水稻淀粉胚乳在核衰退的同时 ,细胞仍在积极合成与积累贮藏产物 ,表现为一种特殊形式的植物细胞编程性死亡现象。此外 ,对淀粉胚乳细胞特有的核质关系、植物细胞编程性死亡过程中细胞核的变化等问题进行了讨论。     

Cis-acting loci involved in the induction and reversal of chromosome condensation in plant prophase,determined by their different replication times

Summary Anoxic UVA irradiation (300–400 nm) of cells in prophase induced their chromatin to return to the interphase decondensed form when their DNA was unifiliarly bromosubstituted. This immediate effect may be related to the incompetence of chromatin with Br-DNA when irradiated to bind proteins which induce its condensation. Hence, inhibition of protein synthesis also causes chromatin decondensation in cells with native DNA.Bromosubstitution of DNA sequences replicated in the last two thirds of the S period was as efficient as bromosubstitution of the whole genome for such an effect to take place in a nucleus. On the other hand, the irradiation accelerated the entrance into prophase of those cells in which only sequences replicated in the first third of S were bromosubstituted. Thus, early replicating loci may act as attachment sites for binding proteins preventing the induction of chromatin condensation. DNA bromosubstitution during portions of S was carried out in synchronous cell populations labelled as binucleate by a previous short caffeine treatment, inAllium cepa L. root meristems.Abbreviations Br-DNA bromosubstituted DNA - BrUdR 5-bromodeoxyuridine - UVA 300–400 nm wavelengths light - p 34 34 kDa protein codified by genecdc 2 inS. pombe or its analogues in other species     

Ion condensation and signal transduction

Many abiotic and other signals are transduced in eukaryotic cells by changes in the level of free calcium via pumps, channels and stores. We suggest here that ion condensation should also be taken into account. Calcium, like other counterions, is condensed onto linear polymers at a critical value of the charge density. Such condensation resembles a phase transition and has a topological basis in that it is promoted by linear as opposed to spherical assemblies of charges. Condensed counterions are delocalised and can diffuse in the so-called near region along the polymers. It is generally admitted that cytoskeletal filaments, proteins colocalised with these filaments, protein filaments distinct from cytoskeletal filaments, and filamentous assemblies of other macromolecules, constitute an intracellular macromolecular network. Here we draw attention to the fact that this network has physicochemical characteristics that enable counterion condensation. We then propose a model in which the feedback relationships between the condensation/decondensation of calcium and the activation of calcium-dependent kinases and phosphatases control the charge density of the filaments of the intracellular macromolecular network. We show how condensation might help mediate free levels of calcium both locally and globally. In this model, calcium condensation/decondensation on the macromolecular network creates coherent patterns of protein phosphorylation that integrate signals. This leads us to hypothesize that the process of ion condensation operates in signal transduction, that it can have an integrative role and that the macromolecular network serves as an integrative receptor.     

The topoisomerase II inhibitor VM-26 induces marked changes in histone H1 kinase activity, histones H1 and H3 phosphorylation, and chromosome condensation in G2 phase and mitotic BHK cells

We have examined the effects of topoisomerase inhibitors on the phosphorylation of histones in chromatin during the G2 and the M phases of the cell cycle. Throughout the G2 phase of BHK cells, addition of the topoisomerase II inhibitor VM-26 prevented histone H1 phosphorylation, accompanied by the inhibition of intracellular histone H1 kinase activity. However, VM-26 had no inhibitory effect on the activity of the kinase in vitro, suggesting an indirect influence on histone H1 kinase activity. Entry into mitosis was also prevented, as monitored by the absence of nuclear lamina depolymerization, chromosome condensation, and histone H3 phosphorylation. In contrast, the topoisomerase I inhibitor, camptothecin, inhibited histone H1 phosphorylation and entry into mitosis only when applied at early G2. In cells that were arrested in mitosis, VM-26 induced dephosphorylation of histones H1 and H3, DNA breaks, and partial chromosome decondensation. These changes in chromatin parameters probably reverse the process of chromosome condensation, unfolding condensed regions to permit the repair of strand breaks in the DNA that were induced by VM- 26. The involvement of growth-associated histone H1 kinase in these processes raises the possibility that the cell detects breaks in the DNA through their effects on the state of DNA supercoiling in constrained domains or loops. It would appear that histone H1 kinase and topoisomerase II work coordinately in both chromosome condensation and decondensation, and that this process participates in the VM-26- induced G2 arrest of the cell.     

Microspectrophotometric measurements of DNA by automated computerized scanning in cycling cells of theChrysanthemum segetum shoot apex

Summary A new technique of exploitation of the data was proposed after DNA scanning microdensitometry. By using all of the measurements obtained from the seriated sections of a single nucleus, this method made it possible to estimate six characteristic parameters during the different phases of the cell cycle in the various shoot apical cells. The cells whose rate of proliferation was the highest showed the biggest variations of their nuclear and nucleolar volumes during the cell cycle. In the axial zone, where the cells have a slow cell cycle and display the longest duration of the G₁ phase, the volume occupied by dispersed DNA was greater than in the cells of the lateral zone and of the rib meristem, where the cell cycle and the G₁ phase were short. No matter what the cell type, the proportion of the dispersed and condensed DNA varied little when the G₁ and G₂ phases were compared. In the Z phase, characterized by a decondensation of the DNA, the mean DNA amount was 3.4 C. The evolution of the nuclear density during the interphase was also estimated. It is demonstrated that the main feature of the shoot apex zonation was the decondensation of the condensed DNA in the axial zone in both the G₁ and G₂ phases.     

Quantitative analysis of condensation/decondensation status of pDNA in the nuclear sub-domains by QD-FRET

Recent studies indicate that controlling the nuclear decondensation and intra-nuclear localization of plasmid DNA (pDNA) would result in an increased transfection efficiency. In the present study, we established a technology for imaging the nuclear condensation/decondensation status of pDNA in nuclear subdomains using fluorescence resonance energy transfer (FRET) between quantum dot (QD)-labeled pDNA as donor, and rhodamine-labeled polycations as acceptor. The FRET-occurring pDNA/polycation particle was encapsulated in a nuclear delivery system; a tetra-lamellar multifunctional envelope-type nano device (T-MEND), designed to overcome the endosomal membrane and nuclear membrane via step-wise fusion. Nuclear subdomains (i.e. heterochromatin and euchromatin) were distinguished by Hoechst33342 staining. Thereafter, Z-series of confocal images were captured by confocal laser scanning microscopy. pDNA in condensation/decondensation status in heterochromatin or euchromatin were quantified based on the pixel area of the signals derived from the QD and rhodamine. The results obtained indicate that modulation of the supra-molecular structure of polyrotaxane (DMAE-ss-PRX), a condenser that is cleaved in a reductive environment, conferred euchromatin-preferred decondensation. This represents the first demonstration of the successful control of condensation/decondensation in specific nuclear sub-domain via the use of an artificial DNA condenser.     

Changes of chromatin condensation in one patient with ataxia telangiectasia disorder: a structural study

Differential scanning calorimetry and quantitative fluorescence microscopy have been employed to characterize the structure and organization of in situ chromatin in lymphoblastoid cells obtained from one ataxia telangiectasia (A-T) patient and one healthy family member. The proven capability of these biophysical techniques to measure changes of chromatin condensation directly inside the cells makes them very powerful in studying the eventual structural changes associated with the appearance of a pleiotropic genetic disorder such as ataxia telangiectasia. A-T syndrome is genetically heterogeneous and can be induced by different mutations of a single gene. The aim of this work is to determine whether the genetic mutation exhibited by the A-T patient of this study may be associated with modifications of chromatin structure and organization. Both the calorimetric and the fluorescence microscopy results acquired on cells from the A-T patient show that the structure and distribution of nuclear chromatin in situ change considerably with respect to the control. A significant decondensation of the nuclear chromatin is in fact associated with the appearance of the A-T disorder in the A-T patient under analysis, together with a rearrangement of the chromatin domains inside the nucleus.     

The Y chromosome of the mouse is decondensed in Sertoli cells

The condensation of the Y chromosome in mouse cells was studied with two repetitive DNA probes, pY353/B and M34. Both DNA probes are specific to the Y chromosome and hybridize in situ along the whole chromosome. Due to the high resolution of the in situ hybridization technique with non-radioactive labeled DNA probes it was possible to observe the degree of condensation of the Y chromosome in the interphase cell nuclei of various somatic tissues and on testes preparations. The Sertoli cells were the only cell type in which the Y chromosome was always observed to be in a highly decondensed state. The decondensation of the Y chromosome in the Sertoli cells supports the view that the genetic activity of the Y chromosome is cell autonomous and opens the way to its molecular analysis.     

Loss of Acinus inhibits oligonucleosomal DNA fragmentation but not chromatin condensation during apoptosis

Chromatin condensation and oligonucleosomal DNA fragmentation are the nuclear hallmarks of apoptosis. A proteolytic fragment of the apoptotic chromatin condensation inducer in the nucleus (Acinus), which is generated by caspase cleavage, has been implicated in mediating apoptotic chromatin condensation prior to DNA fragmentation. Acinus is also involved in mRNA splicing and a component of the apoptosis and splicing-associated protein (ASAP) complex. To study the role of Acinus for apoptotic nuclear alterations, we generated stable cell lines in which Acinus isoforms were knocked down by inducible and reversible RNA interference. We show that Acinus is not required for nuclear localization and interaction of the other ASAP subunits SAP18 and RNPS1; however, knockdown of Acinus leads to a reduction in cell growth. Most strikingly, down-regulation of Acinus did not inhibit apoptotic chromatin condensation either in intact cells or in a cell-free system. In contrast, although apoptosis proceeds rapidly, analysis of nuclear DNA from apoptotic Acinus knockdown cells shows inhibition of oligonucleosomal DNA fragmentation. Our results therefore suggest that Acinus is not involved in DNA condensation but rather point to a contribution of Acinus in internucleosomal DNA cleavage during programmed cell death.