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.     

Positioning of human chromosomes in murine cell hybrids according to synteny

Chromosomes occupy non-random spatial positions in interphase nuclei. It remains unclear what orchestrates this high level of organisation. To determine how the nuclear environment influences the spatial positioning of chromosomes, we utilised a panel of stable mouse hybrid cell lines carrying a single, intact human chromosome. Eleven of 22 human chromosomes revealed an alternative location in hybrid nuclei compared to that of human fibroblasts, with the majority becoming more internally localised. Human chromosomes in mouse nuclei position according to neither their gene density nor size, but rather the position of human chromosomes in hybrid nuclei appears to mimic that of syntenic mouse chromosomes. These results suggest that chromosomes adopt the behaviour of their host species chromosomes and that the nuclear environment is an important determinant of the interphase positioning of chromosomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of triple helical COMBO-FISH and standard FISH by means of quantitative microscopic image analysis of abl/bcr positions in cell nuclei

In this study, a novel DNA fluorescence labelling technique, called triple helical COMBO-FISH (Combinatorial Oligo Fluorescence In Situ Hybridisation), was compared to the standard FISH (Fluorescence In Situ Hybridisation by means of commercially available probe kits) by quantitative evaluation of the nuclear position of the hybridisation signals of the Abelson murine leukaemia (abl) region and the breakpoint cluster region (bcr) in 3D-conserved cell nuclei of lymphocytes and CML blood cells. Two sets of 31 homopyrimidine oligonucleotides each, corresponding to co-localising sequences in the abl region of chromosome 9 and in the bcr region of chromosome 22 were synthesised. Probe types and sizes (in bases) as well as the binding mechanisms of both FISH techniques were completely different. In accordance to established findings that cell type specific radial positioning of chromosomes and sub-chromosomal elements is evolutionarily conserved, no significant difference was found between the two FISH techniques for the radial localisation of the barycentre of the analysed genomic loci. Thermal denaturation and hypotonic treatment of cell nuclei subjected to standard FISH, however, led to different absolute radii and volumes of the cell nuclei, in comparison to the quantities determined for the triple helical COMBO-FISH technique; the chromatin appears to shrink in laterally enlarged, flat nuclei. Consequently, the absolute distances of the homologous labelled sites shifted to greater values. For precise quantitative microscopic analysis of genomic loci, fluorescence labelling procedures are recommended that well maintain the native chromatin topology. Triple helical COMBO-FISH may offer such an approach.     

Comparison of triple helical COMBO-FISH and standard FISH by means of quantitative microscopic image analysis of abl/bcr positions in cell nuclei

In this study, a novel DNA fluorescence labelling technique, called triple helical COMBO-FISH (Combinatorial Oligo Fluorescence In Situ Hybridisation), was compared to the standard FISH (Fluorescence In Situ Hybridisation by means of commercially available probe kits) by quantitative evaluation of the nuclear position of the hybridisation signals of the Abelson murine leukaemia (abl) region and the breakpoint cluster region (bcr) in 3D-conserved cell nuclei of lymphocytes and CML blood cells. Two sets of 31 homopyrimidine oligonucleotides each, corresponding to co-localising sequences in the abl region of chromosome 9 and in the bcr region of chromosome 22 were synthesised. Probe types and sizes (in bases) as well as the binding mechanisms of both FISH techniques were completely different. In accordance to established findings that cell type specific radial positioning of chromosomes and sub-chromosomal elements is evolutionarily conserved, no significant difference was found between the two FISH techniques for the radial localisation of the barycentre of the analysed genomic loci. Thermal denaturation and hypotonic treatment of cell nuclei subjected to standard FISH, however, led to different absolute radii and volumes of the cell nuclei, in comparison to the quantities determined for the triple helical COMBO-FISH technique; the chromatin appears to shrink in laterally enlarged, flat nuclei. Consequently, the absolute distances of the homologous labelled sites shifted to greater values. For precise quantitative microscopic analysis of genomic loci, fluorescence labelling procedures are recommended that well maintain the native chromatin topology. Triple helical COMBO-FISH may offer such an 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.     

Non-random organization of the Biomphalaria glabrata genome in interphase Bge cells and the spatial repositioning of activated genes in cells co-cultured with Schistosomamansoni

Biomphalaria glabrata is a major intermediate host for the parasitic trematode Schistosoma mansoni, a causative agent of human schistosomiasis. To decipher the molecular basis of this host-parasite interaction, the Bge embryonic cell line provides a unique in vitro model system to assess whether interactions between the snail and parasite affect the cell and genome biology in either organism. The organization of the B. glabrata genome in Bge cells was studied using image analysis through positioning territories of differently sized chromosomes within cell nuclei. The snail chromosome territories are similar in morphology as well as in non-random radial positioning as those found in other derived protostome and deuterostome organisms. Specific monitoring of four gene loci, piwi, BgPrx, actin and ferritin, revealed non-random radial positioning of the genome. This indicates that specific parts of the snail genome reside in reproducible nuclear addresses. To determine whether exposure to parasite is reflected in genome organization, the interphase spatial positioning of genes was assessed after co-culturing Bge cells with either normal or irradiation attenuated miracidia for 30 min to 24 h. The loci of actin and ferritin, genes that are up-regulated in the snail when subjected to infection, were visualized by fluorescence in situ hybridisation (FISH) and their radial nuclear positions i.e. their position in the interphase nucleus with respect to the nuclear edge/envelope, mapped. Interestingly, large scale gene repositioning correlated to temporal kinetics of gene expression levels in Bge cells co-cultured with normal miracidia while irradiated parasites failed to elicit similar gene expression or gene loci repositioning as demonstrated using the ferritin gene. This indicates that normal but not attenuated schistosomes provide stimuli that evoke host responses that are reflected in the host’s nuclear architecture. We believe that this is not only the first time that gene-repositioning studies have been attempted in a mollusc but also demonstrates a parasite influencing the interphase genome organization of its host.     

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.     

Local gene density predicts the spatial position of genetic loci in the interphase nucleus

Specific chromosomal translocations are hallmarks of many human leukemias. The basis for these translocation events is poorly understood, but it has been assumed that spatial positioning of genes in the nucleus of hematopoietic cells is a contributing factor. Analysis of the nuclear 3D position of the gene MLL, frequently involved in chromosomal translocations and five of its translocation partners (AF4, AF6, AF9, ENL and ELL), and two control loci revealed a characteristic radial distribution pattern in all hematopoietic cells studied. Genes in areas of high local gene density were found positioned towards the nuclear center, whereas genes in regions of low gene density were detected closer to the nuclear periphery. The gene density within a 2 Mbp window was found to be a better predictor for the relative positioning of a genomic locus within the cell nucleus than the gene density of entire chromosomes. Analysis of the position of MLL, AF4, AF6 and AF9 in cell lines carrying chromosomal translocations involving these genes revealed that the position of the normal genes was different from that of the fusion genes, and this was again consistent with the changes in local gene density within a 2 Mbp window. Thus, alterations in gene density directly at translocation junctions could explain the change in the position of affected genes in leukemia cells.     

Localization of genetic elements of intact and derivative chromosome 11 and 22 territories in nuclei of Ewing sarcoma cells

Recurring chromosomal abnormalities are associated with specific tumour types. The EWSR1 and FLI1 genes are involved in balanced translocation t(11;22)(q24;q12), which is present in more than 85% of Ewing sarcomas. In our previous study, we have found that the fusion genes pertaining to both derivative chromosomes 11 and 22 in Ewing sarcoma cell nuclei are shifted to the midway nuclear position between the native EWSR1 and FLI1 genes. In this contribution we focused our attention at nuclear positioning of other genetic elements of chromosomes 11 and 22 in order to find if the whole derivative chromosomes or only their translocated parts change their nuclear positions in comparison with the native chromosomes. Using repeated fluorescence in situ hybridization and high-resolution cytometry, 2D radial positions of EWSR1, BCR, FLI1, BCL1 genes and fluorescence weight centres of chromosome territories were compared for intact and derivative chromosomes 11 and 22 in nuclei of three Ewing sarcoma samples. Significant radial shift was obtained for the derivative EWSR1, FLI1 and BCL1 genes and for the derivative chromosome 11 compared with the intact ones and not very significant for chromosome 22 and the BCR gene. Our results also suggest that the mean nuclear positions of fusion genes are determined by the final structure of the derivative chromosomes and do not depend on the location of the translocation event.     

Exceptional LINE Density at V1R Loci: The Lyon Repeat Hypothesis Revisited on Autosomes

The mammalian olfactory system utilizes three large receptor families: the olfactory receptors (ORs) of the main nose and the vomeronasal type-1 and type-2 receptor genes (V1Rs and V2Rs) of the vomeronasal organ. We find that these loci are among the most long interspersed nuclear element (LINE)-dense regions of mammalian genomes. We investigate two evolutionary models to account for this cohabitation. First, we investigate an adaptive selection model, in which LINEs have contributed to expansions of mouse V1R repertoires. We find that even evolutionarily stable V1R loci are exceptionally LINE-rich compared to other genome loci, including loci containing other large gene clusters. Also, a more detailed analysis of specific V1R duplications does not reveal LINE patterns predicted by common LINE-mediated duplication mechanisms. Next, we investigate neutral models, in which LINEs were tolerated by, but not advantageous for, surrounding V1R genes. We find that V1R loci are exceptionally LINE-rich compared to other regions of similar AT base composition, and that duplicated V1R gene blocks are generally depleted of LINE elements, suggesting that these loci did not become densely populated with LINEs simply as a consequence of targeted integration or passive multiplication along with the genes. Finally, we show that individual LINE repeats of a given age at V1R, V2R, and OR loci exhibit a significantly longer average length than at other autosomal loci, suggesting a reduced tendency for these LINEs to be disrupted. We speculate that LINEs at V1R, V2R, and OR loci might be selectively retained because they contribute to allelic regulation of these three gene families. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Altered chromosomal positioning, compaction, and gene expression with a lamin A/C gene mutation

Background

Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.

Methods/Findings

To investigate the hypothesis that mutant lamin A/C changes the lamina''s ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.

Conclusions

These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered.