Visualization of chromosome territories in interphase nuclei of ovarian nurse cells in Calliphora erythrocephala Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

Visualization of Chromosome Territories in Interphase Nuclei of Ovarian Nurse Cells in <Emphasis Type="Italic">Calliphora erythrocephala</Emphasis> Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

Factors,mechanisms and implications of chromatin condensation and chromosomal structural maintenance through the cell cycle

A series of well-orchestrated events help in the chromatin condensation and the formation of chromosomes. Apart from the formation of chromosomes, maintenance of their structure is important, especially for the cell division. The structural maintenance of chromosome (SMC) proteins, the non-SMC proteins and the SMC complexes are critical for the maintenance of chromosome structure. While condensins have roles for the DNA compaction, organization, and segregation, the cohesin functions in a cyclic manner through the cell cycle, as a “cohesin cycle.” Specific mechanisms maintain the architecture of the centromere, the kinetochore and the telomeres which are in tandem with the cell cycle checkpoints. The presence of chromosomal territories and compactness differences through the length of the chromosomes might have implications on selective susceptibility of specific chromosomes for induced genotoxicity.     

Non-apoptotic chromosome condensation induced by stress: delineation of interchromosomal spaces

Chromosomes are known to occupy distinct territories, suggesting the existence of definite borders. Visualization of these borders requires chromatin condensation like that seen in prophase cells. We developed a novel method to induce chromosome condensation in all cells regardless of cell cycle stage using a complex set of stresses. The cells were not apoptotic, as indicated by the absence of DNA damage, maintenance of the intact lamina and scaffold attachment factor A, and by the continuation of metabolic processes as well as proliferative capacity. That the appearance of chromosome condensation did not represent a premature mitotic event was shown by the absence of fibrillarin and Ki67 envelopment of chromosomes, continued protein synthesis and the reversibility of chromosome condensation. That chromosome condensation was achieved was demonstrated by the removal of chromatin from the nuclear envelope and chromosome painting. Specific genetic sites known to be at the surface of chromosomes retained their positions as shown by in situ hybridization. Stress-induced chromosome condensation was used to prove that specific nuclear domains such as ND10 are interchromosomally located and that green fluorescent protein-tagged ND10-associated proteins are useful markers for chromosomal boundaries after adenovirus 5 track formation in vivo. From these observations we conclude that chromosomal territories appear to have boundaries that exclude developing macromolecular aggregates. Received: 26 November 1999 / Accepted: 29 February 2000     

Condensin II resolves chromosomal associations to enable anaphase I segregation in Drosophila male meiosis

Several meiotic processes ensure faithful chromosome segregation to create haploid gametes. Errors to any one of these processes can lead to zygotic aneuploidy with the potential for developmental abnormalities. During prophase I of Drosophila male meiosis, each bivalent condenses and becomes sequestered into discrete chromosome territories. Here, we demonstrate that two predicted condensin II subunits, Cap-H2 and Cap-D3, are required to promote territory formation. In mutants of either subunit, territory formation fails and chromatin is dispersed throughout the nucleus. Anaphase I is also abnormal in Cap-H2 mutants as chromatin bridges are found between segregating heterologous and homologous chromosomes. Aneuploid sperm may be generated from these defects as they occur at an elevated frequency and are genotypically consistent with anaphase I segregation defects. We propose that condensin II–mediated prophase I territory formation prevents and/or resolves heterologous chromosomal associations to alleviate their potential interference in anaphase I segregation. Furthermore, condensin II–catalyzed prophase I chromosome condensation may be necessary to resolve associations between paired homologous chromosomes of each bivalent. These persistent chromosome associations likely consist of DNA entanglements, but may be more specific as anaphase I bridging was rescued by mutations in the homolog conjunction factor teflon. We propose that the consequence of condensin II mutations is a failure to resolve heterologous and homologous associations mediated by entangled DNA and/or homolog conjunction factors. Furthermore, persistence of homologous and heterologous interchromosomal associations lead to anaphase I chromatin bridging and the generation of aneuploid gametes.     

Higher-order chromatin structure: bridging physics and biology

Advances in microscopy and genomic techniques have provided new insight into spatial chromatin organization inside of the nucleus. In particular, chromosome conformation capture data has highlighted the relevance of polymer physics for high-order chromatin organization. In this context, we review basic polymer states, discuss how an appropriate polymer model can be determined from experimental data, and examine the success and limitations of various polymer models of higher-order interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization.     

Chromatin position in human HepG2 cells: Although being non-random,significantly changed in daughter cells

Mammalian chromosomes occupy chromosome territories within nuclear space the positions of which are generally accepted as non-random. However, it is still controversial whether position of chromosome territories/chromatin is maintained in daughter cells. We addressed this issue and investigated maintenance of various chromatin regions of unknown composition as well as nucleolus-associated chromatin, a significant part of which is composed of nucleolus organizer region-bearing chromosomes. The photoconvertible histone H4-Dendra2 was used to label such regions in transfected HepG2 cells, and its position was followed up to next interphase. The distribution of labeled chromatin in daughter cells exhibited a non-random character. However, its distribution in a vast majority of daughter cells extensively differed from the original ones and the labeled nucleolus-associated chromatin differently located into the vicinity of different nucleoli. Therefore, our results were not consistent with a concept of preservation chromatin position. This conclusion was supported by the finding that the numbers of nucleoli significantly differed between the two daughter cells. Our results support a view that while the transfected daughter HepG2 cells maintain some features of the parental cell chromosome organization, there is also a significant stochastic component associated with reassortment of chromosome territories/chromatin that results in their positional rearrangements.     

Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming

The ability of cloned embryos to sustain full-term development depends on the ability of the recipient ooplasm to reprogram the donor cell genome. As the nuclear architecture has recently emerged as a key-factor in the regulation of gene expression, we questioned whether early embryos obtained from transfer of ES metaphasic chromosomes into mouse ooplasm would adopt the somatic or embryonic type of nuclear organization. We have particularly focused on the arrangement of chromosomal territories with respect to the nucleolar compartment, and the pericentric heterochromatin domains called chromocenters. We found that nuclear transfer triggers profound chromatin rearrangements including the dispersion of the donor cell chromocenters components. These rearrangements lead to a typical 1-cell pronuclear organization, namely a radial arrangement of the chromosome territories with centromeres attached to the nucleoli, which adopt the compact fibrillar structure of nucleolar precursor bodies (NPBs). Subsequently, during the second cycle, the cloned embryos undergo further reorganization with the establishment of new chromocenters, clustered in one part of the nucleus, as during normal embryogenesis. We could also establish that the adequate distribution of chromosomal territories at the pronuclear stage seems important for the development until blastocyst.     

Chromosomal proteins of Drosophila melanogaster and an approach for their localisation on polytene chromosomes

Chromosomal proteins have been prepared from embryos of Drosophila melanogaster and separated into histone and nonhistone fractions by a procedure which completely avoids exposure to extremes of pH. These fractions have been characterised by amino acid analysis and gel electrophoresis. Antisera have been prepared against whole chromatin and against the two chromosomal protein fractions. — A new method is described for the preparation of Drosophila salivary chromosomes. This method employs microdissection techniques and completely avoids the use of acid fixatives. Preservation of fine structure in these preparations is comparable to, if not better than, that in classical acid-fixed preparations. Antisera against embryo chromatin and chromosomal protein fractions react with the salivary chromosome preparations. These reactions exhibit selectivity with different chromosomal structures. Evidence is presented suggesting a specific distribution of protein antigens along the chromosome.     

Topological origins of chromosomal territories

Using freely jointed polymer model we compare equilibrium properties of crowded polymer chains whose segments are either permeable or not permeable for other segments to pass through. In particular, we addressed the question whether non-permeability of long chain molecules, in the absence of excluded volume effect, is sufficient to compartmentalize highly crowded polymer chains, similarly to what happens during formation of chromosomal territories in interphase nuclei. Our results indicate that even polymers without excluded volume compartmentalize and show strongly reduced intermingling when they are mutually non-permeable. Judging from the known fact that chromatin fibres originating from different chromosomes show very limited intermingling in interphase nuclei, we propose that regular chromatin fibres during chromosome decondensation can hardly serve as a substrate of cellular type II DNA topoisomerases.     

Large-scale reconstruction of 3D structures of human chromosomes from chromosomal contact data

Chromosomes are not positioned randomly within a nucleus, but instead, they adopt preferred spatial conformations to facilitate necessary long-range gene–gene interactions and regulations. Thus, obtaining the 3D shape of chromosomes of a genome is critical for understanding how the genome folds, functions and how its genes interact and are regulated. Here, we describe a method to reconstruct preferred 3D structures of individual chromosomes of the human genome from chromosomal contact data generated by the Hi-C chromosome conformation capturing technique. A novel parameterized objective function was designed for modeling chromosome structures, which was optimized by a gradient descent method to generate chromosomal structural models that could satisfy as many intra-chromosomal contacts as possible. We applied the objective function and the corresponding optimization method to two Hi-C chromosomal data sets of both a healthy and a cancerous human B-cell to construct 3D models of individual chromosomes at resolutions of 1 MB and 200 KB, respectively. The parameters used with the method were calibrated according to an independent fluorescence in situ hybridization experimental data. The structural models generated by our method could satisfy a high percentage of contacts (pairs of loci in interaction) and non-contacts (pairs of loci not in interaction) and were compatible with the known two-compartment organization of human chromatin structures. Furthermore, structural models generated at different resolutions and from randomly permuted data sets were consistent.