Conservation of relative chromosome positioning in normal and cancer cells

Chromosomes exist in the interphase nucleus as individual chromosome territories. It is unclear to what extent chromosome territories occupy particular positions with respect to each other and how structural rearrangements, such as translocations, affect chromosome organization within the cell nucleus. Here we analyze the relative interphase positioning of chromosomes in mouse lymphoma cells compared to normal splenocytes. We show that in a lymphoma cell line derived from an ATM(-/-) mouse, two translocated chromosomes are preferentially positioned in close proximity to each other. The relative position of the chromosomes involved in these translocations is conserved in normal splenocytes. Relative positioning of chromosomes in normal splenocytes is not due to their random distribution in the interphase nucleus and persists during mitosis. These observations demonstrate that the relative arrangement of chromosomes in the interphase nucleus can be conserved between normal and cancer cells and our data support the notion that physical proximity facilitates rearrangements between chromosomes.     

Chromosome positioning in the interphase nucleus

Chromosomes occupy distinct territories in the interphase cell nucleus. These chromosome territories are non-randomly arranged within the nuclear space. We are only just uncovering how chromosome territories are organized, what determines their position and how their spatial organization affects the expression of genes and genomes. Here, we discuss emerging models of non-random nuclear chromosome organization and consider the functional implications of chromosome positioning for gene expression and genome stability.     

Chromosome painting reveals asynaptic full alignment of homologs and HIM-8-dependent remodeling of X chromosome territories during Caenorhabditis elegans meiosis

During early meiotic prophase, a nucleus-wide reorganization leads to sorting of chromosomes into homologous pairs and to establishing associations between homologous chromosomes along their entire lengths. Here, we investigate global features of chromosome organization during this process, using a chromosome painting method in whole-mount Caenorhabditis elegans gonads that enables visualization of whole chromosomes along their entire lengths in the context of preserved 3D nuclear architecture. First, we show that neither spatial proximity of premeiotic chromosome territories nor chromosome-specific timing is a major factor driving homolog pairing. Second, we show that synaptonemal complex-independent associations can support full lengthwise juxtaposition of homologous chromosomes. Third, we reveal a prominent elongation of chromosome territories during meiotic prophase that initiates prior to homolog association and alignment. Mutant analysis indicates that chromosome movement mediated by association of chromosome pairing centers (PCs) with mobile patches of the nuclear envelope (NE)-spanning SUN-1/ZYG-12 protein complexes is not the primary driver of territory elongation. Moreover, we identify new roles for the X chromosome PC (X-PC) and X-PC binding protein HIM-8 in promoting elongation of X chromosome territories, separable from their role(s) in mediating local stabilization of pairing and association of X chromosomes with mobile SUN-1/ZYG-12 patches. Further, we present evidence that HIM-8 functions both at and outside of PCs to mediate chromosome territory elongation. These and other data support a model in which synapsis-independent elongation of chromosome territories, driven by PC binding proteins, enables lengthwise juxtaposition of chromosomes, thereby facilitating assessment of their suitability as potential pairing partners.     

Chromosome Architecture and Genome Organization

How the same DNA sequences can function in the three-dimensional architecture of interphase nucleus, fold in the very compact structure of metaphase chromosomes and go precisely back to the original interphase architecture in the following cell cycle remains an unresolved question to this day. The strategy used to address this issue was to analyze the correlations between chromosome architecture and the compositional patterns of DNA sequences spanning a size range from a few hundreds to a few thousands Kilobases. This is a critical range that encompasses isochores, interphase chromatin domains and boundaries, and chromosomal bands. The solution rests on the following key points: 1) the transition from the looped domains and sub-domains of interphase chromatin to the 30-nm fiber loops of early prophase chromosomes goes through the unfolding into an extended chromatin structure (probably a 10-nm “beads-on-a-string” structure); 2) the architectural proteins of interphase chromatin, such as CTCF and cohesin sub-units, are retained in mitosis and are part of the discontinuous protein scaffold of mitotic chromosomes; 3) the conservation of the link between architectural proteins and their binding sites on DNA through the cell cycle explains the “mitotic memory” of interphase architecture and the reversibility of the interphase to mitosis process. The results presented here also lead to a general conclusion which concerns the existence of correlations between the isochore organization of the genome and the architecture of chromosomes from interphase to metaphase.     

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     

Fluorescent in situ hybridization (FISH) analysis of the relationship between chromosome location and nuclear morphology in human neutrophils

Human neutrophil nuclei typically consist of three of four large heterochromatic lobes joined by thin, DNA-containing filaments. In addition, some lobes exhibit appendages of various sizes and shapes. Classical genetic and cytological studies suggest that some appendages contain specific chromsomes. The studies reported here provide the first detailed analysis of the spatial relationship between individual chromosomes and recognizable structures in neutrophil nuclei using fluorescent in situ hybridization. Analysis of DNA sequences in chromosomes 2, 18, X, and Y demonstrate that specific lobes in a population of neutrophil nuclei do not have a fied chromosome content. This result implies that chromosomes partition randomly among lobes during neutrophil differentiation. However, neutrophil nuclear topography is not entirely fortuitous. For instance, none of the sequences probed in this study mapped to a filament and most centromeres lie in clusters near the nuclear periphery. In addition, one of the X chromosome centromeres in females and the Y chromosome centromere in males consistently associate with specific nuclear appendages found in a subset of neutrophil nuclei. Chromosomes 2 and 18 occupy discrete nd separate territories within individual lobes and neither territory ever extends into a filament. Surprisingly, the sizes of these territories are not proportional to chromosome length, suggesting that individual neutrophil chromosomes vary in their degree of compaction. These results are discussed in the light of models that attempt to explain nuclear morphology in terms of chromosome spatial organization. Received: 10 April 1997 / Accepted: 14 April 1997     

Unscheduled DNA synthesis after partial UV irradiation of the cell nucleus. Distribution in interphase and metaphase.

Cells of an euploid strain of the Chinese hamster synchronized in the G1 phase were microirradiated in the nucleus with a laser UV microbeam (λ = 257 nm) and pulse-labelled with [³H]thymidine. In autoradiographs of cells fixed immediately after the pulse unscheduled DNA synthesis (UDS) was found restricted to the microirradiated part of the nucleus. The rate of UDS varied with the UV energy applied and the post-irradiation incubation time. In other experiments chromosome preparations were established after an additional chase and a subsequent growth period. In 28 mitotic cells autoradiographic label was found concentrated on a few chromosomes which lay adjacent to each other in one part of the metaphase plate. The distribution of label on the chromosomes could clearly be distinguished from patterns which originate from semi-conservative DNA synthesis within S phase. The label on chromosomes of microirradiated cells thus represents UDS. Our findings support the following ideas on the arrangement of interphase chromosomes: (1) Decondensed interphase chromosomes may occupy rather compact territories. (2) Chromosomes do not necessarily exhibit a close and permanent association with their respective homologues.     

Reorganization of inter‐chromosomal interactions in the 2q37‐deletion syndrome

Chromosomes occupy distinct interphase territories in the three‐dimensional nucleus. However, how these chromosome territories are arranged relative to one another is poorly understood. Here, we investigated the inter‐chromosomal interactions between chromosomes 2q, 12, and 17 in human mesenchymal stem cells (MSCs) and MSC‐derived cell types by DNA‐FISH. We compared our findings in normal karyotypes with a three‐generation family harboring a 2q37‐deletion syndrome, featuring a heterozygous partial deletion of histone deacetylase 4 (HDAC4) on chr2q37. In normal karyotypes, we detected stable, recurring arrangements and interactions between the three chromosomal territories with a tissue‐specific interaction bias at certain loci. These inter‐chromosomal interactions were confirmed by Hi‐C. Interestingly, the disease‐related HDAC4 deletion resulted in displaced inter‐chromosomal arrangements and altered interactions between the deletion‐affected chromosome 2 and chromosome 12 and/or 17 in 2q37‐deletion syndrome patients. Our findings provide evidence for a direct link between a structural chromosomal aberration and altered interphase architecture that results in a nuclear configuration, supporting a possible molecular pathogenesis.     

Organization of early and late replicating DNA in human chromosome territories

It has been suggested that DNA organized into replication foci during S-phase remains stably aggregated in non-S-phase cells and that these stable aggregates provide fundamental units of nuclear or chromosome architecture [C. Meng and R. Berezney (1991) J. Cell Biol. 115, 95a; E. Sparvoli et al. (1994) J. Cell Sci. 107, 3097-3103; D. A. Jackson and A. Pombo (1998) J. Cell Biol. 140, 1285-1295; D. Zink et al. (1998) Hum. Genet. 112, 241-251]. To test this hypothesis, early and late replicating DNA of human diploid fibroblasts was labeled specifically by incorporating two different thymidine analogs [J. Aten (1992) Histochem. J. 24, 251-259; A. E. Visser (1998) Exp. Cell Res. 243, 398-407], during distinct time segments of S-phase. On mitotic chromosomes the amount and spatial distribution of early and late replicating DNA corresponded to R/G-banding patterns. After labeling cells were grown for several cell cycles. During this growth period individual replication labeled chromosomes were distributed into an environment of unlabeled chromosomes. The nuclear territories of chromosomes 13 and 15 were identified by additional chromosome painting. The distribution of early and late replicating DNA was analyzed for both chromosomes in quiescent (G0) cells or at G1. Early and late replicating DNA occupied distinct foci within chromosome territories, displaying a median overlap of only 5-10%. There was no difference in this regard between G1 and G0 cells. Chromosome 13 and 15 territories displayed a similar structural rearrangement in G1 cells compared to G0 cells resulting in the compaction of the territories. The findings demonstrate that early and late replicating foci are maintained during subsequent cell cycles as distinctly separated units of chromosome organization. These findings are compatible with the hypothesis that DNA organized into replicon clusters remains stably aggregated in non-S-phase cells.     

Sperm Nuclear Architecture Is Locally Modified in Presence of a Robertsonian Translocation t(13;17)

In mammals, the non-random organization of the sperm nucleus supports an early function during embryonic development. Altering this organization may interfere with the zygote development and reduce fertility or prolificity. Thus, rare studies on sperm cells from infertile patients described an altered nuclear organization that may be a cause or a consequence of their respective pathologies. Thereby, chromosomal rearrangements and aneuploidy can be studied not only for their adverse effects on production of normal/balanced gametes at meiosis but also for their possible impact on sperm nuclear architecture and the epigenetic consequences of altered chromosome positioning. We decided to compare the global architecture of sperm nuclei from boars, either with a normal chromosome composition or with a Robertsonian translocation involving chromosomes 13 and 17. We hypothesized that the fusion between these chromosomes may change their spatial organization and we examined to what extend it could also modify the global sperm nuclear architecture. Analysis of telomeres, centromeres and gonosomes repartition does not support a global nuclear disorganization. But specific analysis of chromosomes 13 and 17 territories highlights an influence of chromosome 17 for the positioning of the fused chromosomes within the nucleus. We also observed a specific clustering of centromeres depending of the chromosome subtypes. Altogether our results showed that chromosome fusion does not significantly alter sperm nucleus architecture but suggest that centromere remodelling after chromosome fusion locally impacts chromosome positioning.     

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.     

T antigen banding on chromosomes of simian virus 40 infected muntjac cells.

Chromosomes were prepared from mitotic munjac cells 48 to 72 h after infection with SV40 virus. When stained for SV40 T antigen by indirect immunofluorescence, all chromosomes within an infected cell were fluorescent, indicating the presence of T antigen. Furthermore, the chromosomes were not uniformly stained but appeared to have regions of high and low fluorescence intensity. A variety of controls showed that the banding patterns are specific and highly reproducible and may indeed reflect the binding sites of T antigen. The bright, fluorescent bands T antigen were found to correspond to bands visualized by trypsin-Giesma staining (G-bands) and also by quinacrine staining (Q-bands). Current knowledge of chromosome banding indicates that Q-bands reflect the distribution of AT-rich regions along the chromosome. From the DNA sequence of SV40, it is known that one of the T antigen binding sites contains AT-rich sequences; thus, T antigen banding might be due to the base-specific binding of T antigen to chromatin. In addition, these bands have been implicated as centers for chromosome condensation and units in control of DNA replication. While the functional significance of T antigen binding has yet to be determined, the SV40-muntjac system provides an unusual opportunity to study the interaction of a known regulatory protein with mammalian chromosomes.     

Cytochemical studies of metaphase chromosomes by flow cytometry

The cytochemical properties of metaphase chromosomes from Chinese hamster and human cells were studied by flow cytometry. This technique allows precise quantitation of the fluorescence properties of individual stained chromosome types. Chromosomes were stained with the following fluorescent DNA stains: Hoechst 33258, DAPI, chromomycin A₃, ethidium bromide, and propidium iodide. The relative fluorescence of individual chromosome types varied depending on the stain used, demonstrating that individual chromosome types differ in chemical properties. Flow measurements were performed as a function of stain and chromosome concentration to characterize the number and distribution of stain binding sites. Flow analysis of double stained chromosomes show that bound stains interact by energy transfer with little or no binding competition. For most hamster chromosomes, there is a strong correlation between relative fluorescence and stain base preference suggesting that staining differences may be determined primarily by differences in average base composition. A few hamster chromosome types exhibit anomalous staining which suggests that some other property, such as repetitive DNA sequences, also may be an important determinant of chromosomal staining.     

Ultrastructure of the mitotic chromosomes in pig embryonic kidney cells during their reversible artificial decondensation in vivo

Using methods of in vivo observation and ultrathin sectioning, it is shown that chromosomes of metaphase PE cells, previously treated with diluted Henk's solutions (70, 30 and 15%), undergo some structural transitions resulting in the formation of micronuclei. At the early stages of hypotonic treatment chromosomes are seen considerably swollen and losing the higher levels of organization, including the chromonema and chromomeres. The chromosomal bodies are formed by DNP fibers 10-25 nm in diameter making loops radiating from the central part of the chromatids. Chromosomes are capable of recondensing from this state by consecutive reconstitution of G-bands, chromomeres and the chromonema. The subsequent secondary decondensation of chromosomes is analogous to telophase decondensation at the normal mitosis, but it results in the formation of a great number of small nuclei (micronuclei). The chromatin structure in micronuclei as well as their ability to synthesize RNA and to replicate DNA show these effects to be reversible. It has been suggested that the loop organization of DNP may be essential for sustaining the structural integrity of the mitotic chromosome.     

Structure of isolated protein-depleted chromosomes of plants

Chromosomes from poppy (Papaver somniferum L.) and wheat (Triticum monococcum L.) were obtained from cell suspension cultures using a mass isolation procedure. Protein-depleted isolated chromosomes were produced using different modes of extraction (e.g., sodium chloride, dextran sulphate-heparin) and examined by protein electrophoresis as well as light and electron microscopy. The results are discussed as they relate to the reported structure of protein-depleted animal chromosomes. With respect to the scaffold model of mitotic chromosomes we conclude that i) nonhistone proteins seem to play a fundamental role in plant chromosome architecture; ii) DNA is a structural component of protein-depleted chromosomes; iii) centromeric regions may be of structural importance for the higher order organization of chromosomes; iv) the existence of a 2M NaCl resistant scaffold appears not to be a common feature to both plant and animal chromosomes; v) despite the absence of a typical scaffold in plant chromosomes our results suggest that the higher order organization of plant and animal chromosomes is similar if not the same.     

Mechanics and Dynamics of X-Chromosome Pairing at X Inactivation

At the onset of X-chromosome inactivation, the vital process whereby female mammalian cells equalize X products with respect to males, the X chromosomes are colocalized along their Xic (X-inactivation center) regions. The mechanism inducing recognition and pairing of the X's remains, though, elusive. Starting from recent discoveries on the molecular factors and on the DNA sequences (the so-called “pairing sites”) involved, we dissect the mechanical basis of Xic colocalization by using a statistical physics model. We show that soluble DNA-specific binding molecules, such as those experimentally identified, can be indeed sufficient to induce the spontaneous colocalization of the homologous chromosomes but only when their concentration, or chemical affinity, rises above a threshold value as a consequence of a thermodynamic phase transition. We derive the likelihood of pairing and its probability distribution. Chromosome dynamics has two stages: an initial independent Brownian diffusion followed, after a characteristic time scale, by recognition and pairing. Finally, we investigate the effects of DNA deletion/insertions in the region of pairing sites and compare model predictions to available experimental data.     

Chromosome length and DNA loop size during early embryonic development of Xenopus laevis

The looped organization of the eukaryotic genome mediated by a skeletal framework of non-histone proteins is conserved throughout the cell cycle. The radial loop/scaffold model envisages that the higher order architecture of metaphase chromosomes relies on an axial structure around which looped DNA domains are radially arranged through stable attachment sites. In this light we investigated the relationship between the looped organization and overall morphology of chromosomes. In developing Xenopus laevis embryos at gastrulation, the bulk of the loops associated with histone-depleted nuclei exhibit a significant size increase, as visualized by fluorescence microscopy of the fully extended DNA halo surrounding high salt treated, ethidium bromide stained nuclei. This implies a reduction in the number of looped domains anchored to the supporting nucleoskeletal structure. The cytological analysis of metaphase plates from acetic acid fixed whole embryos, carried out in the absence of drugs inducing chromosome condensation, reveals a progressive thickening and shortening of metaphase chromosomes during development. We interpret these findings as a strong indication that the size and number of DNA loops influence the thickness and length of the chromosomes, respectively. The quantitative analysis of chromosome length distributions at different developmental stages suggests that the shortening is timed differently in different embryonic cells.