Evolutionarily conserved,cell type and species-specific higher order chromatin arrangements in interphase nuclei of primates

Several studies demonstrated a gene-density-correlated radial organization of chromosome territories (CTs) in spherically shaped nuclei of human lymphocytes or lymphoblastoid cells, while CT arrangements in flat-ellipsoidal nuclei of human fibroblasts are affected by both gene density and chromosome size. In the present study, we performed fluorescence in situ hybridization (FISH) experiments to three-dimensionally preserved nuclei (3D-FISH) from human and nonhuman primate cultured lymphoblastoid cells and fibroblasts. We investigated apes, Old, and New World monkeys showing either evolutionarily conserved karyotypes, multiple translocations, fusions, or serial fissions. Our goal was to test whether cell type specific differences of higher order chromatin arrangements are evolutionarily conserved in different primate lineages. Whole genome painting experiments and further detailed analyses of individual chromosomes indicate a gene-density-correlated higher order organization of chromatin in lymphoblastoid cell nuclei of all studied primate species, despite evolutionary chromosome reshuffling. In contrast, in primate fibroblast nuclei evolutionary translocations, fissions and fusions resulted in positional shifts of orthologous chromosome segments, thus arguing against a functional role of chromosome size-dependent spatial chromatin arrangements and for geometrical constraints in flat-ellipsoidal fibroblast nuclei. Notably, in both cell types, regions of rearranged chromosomes with distinct differences in gene density showed polarized arrangements with the more gene-dense segment oriented towards the nuclear interior. Our results indicate that nonrandom breakage and rejoining of preferentially gene-dense chromosomes or chromosome segments may have occurred during evolution. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Radial arrangement of chromosome territories in human cell nuclei: a computer model approach based on gene density indicates a probabilistic global positioning code

Numerous investigations in the last years focused on chromosome arrangements in interphase nuclei. Recent experiments concerning the radial positioning of chromosomes in the nuclear volume of human and primate lymphocyte cells suggest a relationship between the gene density of a chromosome territory (CT) and its distance to the nuclear center. To relate chromosome positioning and gene density in a quantitative way, computer simulations of whole human cell nuclear genomes of normal karyotype were performed on the basis of the spherical 1 Mbp chromatin domain model and the latest data about sequence length and gene density of chromosomes. Three different basic assumptions about the initial distribution of chromosomes were used: a statistical, a deterministic, and a probabilistic initial distribution. After a simulated decondensation in early G1, a comparison of the radial distributions of simulated and experimentally obtained data for CTs Nos. 12, 18, 19, and 20 was made. It was shown that the experimentally observed distributions can be fitted better assuming an initial probabilistic distribution. This supports the concept of a probabilistic global gene positioning code depending on CT sequence length and gene density.     

Inter- and intra-specific gene-density-correlated radial chromosome territory arrangements are conserved in Old World monkeys

Recently it has been shown that the gene-density correlated radial distribution of human 18 and 19 homologous chromosome territories (CTs) is conserved in higher primates in spite of chromosomal rearrangements that occurred during evolution. However, these observations were limited to apes and New World monkey species. In order to provide further evidence for the evolutionary conservation of gene-density-correlated CT arrangements, we extended our previous study to Old World monkeys. They comprise the remaining species group to be analyzed in order to obtain a comprehensive overview of the nuclear topology of human 18 and 19 homologous CTs in higher primates. In the present study we investigated four lymphoblastoid cell lines from three species of Old World monkeys by three-dimensional fluorescence in situ hybridization (3D-FISH): two individuals of Japanese macaque (Macaca fuscata), crab-eating macaque (Macaca fascicularis), and an interspecies hybrid individual between African green monkey (Cercopithecus aethiops) and Patas monkey (Erythrocebus patas). Our data demonstrate that gene-poor human 18 homologous CTs are located preferentially close to the nuclear periphery, whereas gene-dense human 19 homologous CTs are oriented towards the nuclear center in all cell lines analyzed. The gene-density-correlated positioning of human 18 and 19 homologous CTs is evolutionarily conserved throughout all major higher primate lineages, despite chromosomal inversions, fusions, fissions or reciprocal translocations that occurred in the course of evolution in these species. This remarkable preservation of a gene-density-correlated chromatin arrangement gives further support for a functionally relevant higher-order chromatin architecture.     

Inheritance of gene density-related higher order chromatin arrangements in normal and tumor cell nuclei

A gene density-related difference in the radial arrangement of chromosome territories (CTs) was previously described for human lymphocyte nuclei with gene-poor CT #18 located toward the nuclear periphery and gene-dense CT #19 in the nuclear interior (Croft, J.A., J.M. Bridger, S. Boyle, P. Perry, P. Teague, and W.A. Bickmore. 1999. J. Cell Biol. 145:1119-1131). Here, we analyzed the radial distribution of chromosome 18 and 19 chromatin in six normal cell types and in eight tumor cell lines, some of them with imbalances and rearrangements of the two chromosomes. Our findings demonstrate that a significant difference in the radial distribution of #18 and #19 chromatin is a common feature of higher order chromatin architecture in both normal and malignant cell types. However, in seven of eight tumor cell lines, the difference was less pronounced compared with normal cell nuclei due to a higher fraction of nuclei showing an inverted CT position, i.e., a CT #18 located more internally than a CT #19. This observation emphasizes a partial loss of radial chromatin order in tumor cell nuclei.     

Non-random radial arrangements of interphase chromosome territories: evolutionary considerations and functional implications

In the nucleus of animal and plant cells individual chromosomes maintain a compartmentalized structure. Chromosome territories (CTs), as these structures were named by Theodor Boveri, are essential components of the higher-order chromatin architecture. Recent studies in mammals and non-mammalian vertebrates indicate that the radial position of a given CT (or segments thereof) is correlated with its size, its gene-density and its replication timing. As a representative case, chicken cell nuclei show highly consistent radial chromatin arrangements: gene-rich, early replicating microchromosomes are clustered within the nuclear interior, while gene-poor, later replicating macrochromosomes are preferentially located at the nuclear periphery. In humans, chromosomes 18 and 19 (HSA18 and 19) territories that are of similar size show a distinctly different position in the cell nuclei of lymphocytes and lymphoblastoid cells: the gene-rich and early replicating HSA19 CTs are typically found close to the nuclear center, while the gene-poor and later replicating HSA18 CTs are preferentially located at the nuclear periphery. Recent comparative maps between human and chicken chromosomes revealed that the chicken macrochromosomes 2 and Z contain the genes homologous to HSA18, while the genes on HSA19 are located onto the chicken microchromosomes. These data lend tentative support to the hypothesis that differences in the radial nuclear positions of gene-rich, early replicating and gene-poor, later replicating chromatin have been evolutionarily conserved during a period of more than 300 million years irrespective of the evolution of highly divergent karyotypes between humans and chicken.     

Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures]

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.     

Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages

Whether chromosomes maintain their nuclear positions during interphase and from one cell cycle to the next has been controversially discussed. To address this question, we performed long-term live-cell studies using a HeLa cell line with GFP-tagged chromatin. Positional changes of the intensity gravity centers of fluorescently labeled chromosome territories (CTs) on the order of several microm were observed in early G1, suggesting a role of CT mobility in establishing interphase nuclear architecture. Thereafter, the positions were highly constrained within a range of approximately 1 microm until the end of G2. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase, whereas the contiguity of unbleached chromosome segments was lost to a variable extent, when the metaphase plate was formed. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus. We conclude that CT arrangements were stably maintained from mid G1 to late G2/early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. The variability of CT neighborhoods during clonal growth was further confirmed by chromosome painting experiments.     

Spatial organization of chromosome territories in the interphase nucleus of trisomy 21 cells

In the interphase cell nucleus, chromosomes adopt a conserved and non-random arrangement in subnuclear domains called chromosome territories (CTs). Whereas chromosome translocation can affect CT organization in tumor cell nuclei, little is known about how aneuploidies can impact CT organization. Here, we performed 3D-FISH on control and trisomic 21 nuclei to track the patterning of chromosome territories, focusing on the radial distribution of trisomic HSA21 as well as 11 disomic chromosomes. We have established an experimental design based on cultured chorionic villus cells which keep their original mesenchymal features including a characteristic ellipsoid nuclear morphology and a radial CT distribution that correlates with chromosome size. Our study suggests that in trisomy 21 nuclei, the extra HSA21 induces a shift of HSA1 and HSA3 CTs out toward a more peripheral position in nuclear space and a higher compaction of HSA1 and HSA17 CTs. We posit that the presence of a supernumerary chromosome 21 alters chromosome compaction and results in displacement of other chromosome territories from their usual nuclear position.     

Distribution of chromosome 18 and X centric heterochromatin in the interphase nucleus of cultured human cells

In situ hybridization of human chromosome 18 and X-specific alphoid DNA-probes was performed in combination with three dimensional (3D) and two dimensional (2D) image analysis to study the interphase distribution of the centric heterochromatin (18c and Xc) of these chromosomes in cultured human cells. 3D analyses of 18c targets using confocal laser scanning microscopy indicated a nonrandom disposition in 73 amniotic fluid cell nuclei. The shape of these nuclei resembled rather flat cylinders or ellipsoids and targets were preferentially arranged in a domain around the nuclear center, but close to or associated with the nuclear envelope. Within this domain, however, positionings of the two targets occurred independently from each other, i.e., the two targets were observed with similar frequencies at the same (upper or lower) side of the nuclear envelope as those on opposite sides. This result strongly argues against any permanent homologous association of 18c. A 2D analytical approach was used for the rapid evaluation of 18c positions in over 4000 interphase nuclei from normal male and female individuals, as well as individuals with trisomy 18 and Bloom's syndrome. In addition to epithelially derived amniotic fluid cells, investigated cell types included in vitro cultivated fibroblastoid cells established from fetal lung tissue and skin-derived fibroblasts. In agreement with the above 3D observations 18c targets were found significantly closer (P less than 0.01) to the center of the 2D nuclear image (CNI) and to each other in all these cultures compared to a random distribution derived from corresponding ellipsoid or cylinder model nuclei. For comparison, a chromosome X-specific alphoid DNA probe was used to investigate the 2D distribution of chromosome X centric heterochromatin in the same cell types. Two dimensional Xc-Xc and Xc-CNI distances fit a random distribution in diploid normal and Bloom's syndrome nuclei, as well as in nuclei with trisomy X. The different distributions of 18c and Xc targets were confirmed by the simultaneous staining of these targets in different colors within individual nuclei using a double in situ hybridization approach.     

The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

In the nuclei of human lymphocytes, chromosome territories are distributed according to the average gene density of each chromosome. However, chromosomes are very heterogeneous in size and base composition, and can contain both very gene-dense and very gene-poor regions. Thus, a precise analysis of chromosome organisation in the nuclei should consider also the distribution of DNA belonging to the chromosomal bands in each chromosome. To improve our understanding of the chromatin organisation, we localised chromosome 7 DNA regions, endowed with different gene densities, in the nuclei of human lymphocytes. Our results showed that this chromosome in cell nuclei is arranged radially with the gene-dense/GC-richest regions exposed towards the nuclear interior and the gene-poorest/GC-poorest ones located at the nuclear periphery. Moreover, we found that chromatin fibres from the 7p22.3 and the 7q22.1 bands are not confined to the territory of the bulk of this chromosome, protruding towards the inner part of the nucleus. Overall, our work demonstrates the radial arrangement of the territory of chromosome 7 in the lymphocyte nucleus and confirms that human genes occupy specific radial positions, presumably to enhance intra- and inter-chromosomal interaction among loci displaying a similar expression pattern, and/or similar replication timing.     

The 4D Nucleome: Genome Compartmentalization in an Evolutionary Context

4D nucleome research aims to understand the impact of nuclear organization in space and time on nuclear functions, such as gene expression patterns, chromatin replication, and the maintenance of genome integrity. In this review we describe evidence that the origin of 4D genome compartmentalization can be traced back to the prokaryotic world. In cell nuclei of animals and plants chromosomes occupy distinct territories, built up from ~1 Mb chromatin domains, which in turn are composed of smaller chromatin subdomains and also form larger chromatin domain clusters. Microscopic evidence for this higher order chromatin landscape was strengthened by chromosome conformation capture studies, in particular Hi-C. This approach demonstrated ~1 Mb sized, topologically associating domains in mammalian cell nuclei separated by boundaries. Mutations, which destroy boundaries, can result in developmental disorders and cancer. Nucleosomes appeared first as tetramers in the Archaea kingdom and later evolved to octamers built up each from two H2A, two H2B, two H3, and two H4 proteins. Notably, nucleosomes were lost during the evolution of the Dinoflagellata phylum. Dinoflagellate chromosomes remain condensed during the entire cell cycle, but their chromosome architecture differs radically from the architecture of other eukaryotes. In summary, the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals suggests the existence of conserved, but still unknown mechanism(s) controlling this architecture. Notwithstanding this conservation, a comparison of metazoans and protists also demonstrates species-specific structural and functional features of nuclear organization.     

Higher-order chromatin structure of human granulocytes

The structural organisation of chromatin in eukaryotes plays an important role in a number of biological processes. Our results provide a comprehensive insight into the nuclear topography of human peripheral blood granulocytes, mainly neutrophils. The nuclei of granulocytes are characterised by a segmented shape consisting of two to five lobes that are in many cases connected by a thin DNA-containing filament. The segregation of chromosomes into the nuclear lobes was studied using fluorescence in situ hybridisation (FISH). We were able to distinguish different topographic types of granulocytes on the basis of the pattern of segregation. Five topographic types were detected using dual-colour FISH in two-lobed nuclei. The segregation of four sets of genetic structures could be studied with the aid of repeated FISH and a large number of topographic types were observed. In all these experiments a non-random distribution of chromosomes into nuclear lobes was found. The painting of a single type of chromosome in two-lobed nuclei showed the prevalence of symmetric topographic types (on average in 65.5% of cases) with significant variations among individual chromosomes. The results of analysis of five topographic types (defined by two chromosomes in two-lobed nuclei) showed that the symmetric topographic types for both chromosomes are significantly more frequent than predicted. Repeated hybridisation experiments confirmed that the occurrence of certain patterns of chromosome segregation is much higher than that predicted from the combination of probabilities. The frequency of symmetric topographic types for chromosome domains was systematically higher than for genes located on these chromosomes. It appears that the prevalence of symmetric segregation patterns is more probable for large objects such as chromosome domains than for genes located on chromatin loops extending outwards from the surface of the domain defined by specific chromosome paints. This means that one chromosome domain may occur in different lobes of granulocytic nuclei. This observation is supported by the fact that both genes and centromeres were observed on filaments joining different lobes. For all chromosomes, the distances between the membrane and fluorescence gravity centre of the chromosome were measured and correlated with the segregation patterns. A higher percentage of symmetric topographic types was found in those chromosomes that were located closer to the nuclear membrane. Nuclear positioning of all genetic elements in granulocytic nuclei was studied in two-dimensional projection; however, the results were verified using three-dimensional analysis.     

Three-dimensional positioning of genes in mouse cell nuclei

To understand the regulation of the genome, it is necessary to understand its three-dimensional organization in the nucleus. We investigated the positioning of eight gene loci on four different chromosomes, including the β-globin gene, in mouse embryonic stem cells and in in vitro differentiated macrophages by fluorescence in situ hybridization on structurally preserved nuclei, confocal microscopy, and 3D quantitative image analysis. We found that gene loci on the same chromosome can significantly differ from each other and from their chromosome territory in their average radial nuclear position. Radial distribution of a given gene locus can change significantly between cell types, excluding the possibility that positioning is determined solely by the DNA sequence. For the set of investigated gene loci, we found no relationship between radial distribution and local gene density, as it was described for human cell nuclei. We did find, however, correlation with other genomic properties such as GC content and certain repetitive elements such as long terminal repeats or long interspersed nuclear elements. Our results suggest that gene density itself is not a driving force in nuclear positioning. Instead, we propose that other genomic properties play a role in determining nuclear chromatin distribution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.     

Replication labeling patterns and chromosome territories typical of mammalian nuclei are conserved in the early metazoan<Emphasis Type="Italic"> Hydra</Emphasis>

To investigate the evolutionary conservation of higher order nuclear architecture previously described for mammalian cells we have analyzed the nuclear architecture of the simple polyp Hydra. These diploblastic organisms have large nuclei (8–10 m) containing about 3×10⁹ bp of DNA organized in 15 chromosome pairs. They belong to the earliest metazoan phylum and are separated from mammals by at least 600 million years. Single and double pulse labeling with halogenated nucleotides (bromodeoxyuridine, iododeoxyuridine and chlorodeoxyuridine) revealed striking similarities to the known sequence of replication labeling patterns in mammalian nuclei. These patterns reflect a persistent nuclear arrangement of early, mid-, and late replicating chromatin foci that could be identified during all stages of interphase over at least 5–10 cell generations. Segregation of labeled chromatids led after several cell divisions to nuclei with single or a few labeled chromosome territories. In such nuclei distinct clusters of labeled chromatin foci were separated by extended nuclear areas with non-labeled chromatin, which is typical of a territorial arrangement of interphase chromosomes. Our results indicate the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals and suggest the existence of conserved mechanism(s) controlling this architecture.Abbreviations CT Chromosome territory - BrdU Bromodeoxyuridine - IdU Iododeoxyuridine - CldU Chlorodeoxyuridine Communicated by E.A. Nigg