In eukaryotic cell nuclei genomic DNA is packaged into chromatin fibres. Properties of the chromatin fibres can be changed by modification of its constituent histone proteins and through interaction with structural non-histone proteins. These modifications contribute to packaging of the chromatin into higher-order structures, from the level of chromatin loop domains via chromosomal territories (CTs) to suprachromosomal organization of the genome.
Chromatin mediates gene expression in response to external or internal signals. These signals induce complex patterns of enzyme-catalyzed chromatin modifications, such as DNA methylation by DNA methylases, histone phospohorylation by kinases, acetylation/ deacetylation of histone tails by histone- acetyltransferases and deacetylases, methylation by histone-methyltransferases, ubiquitination by Ub-ligases, etc. These epigenetic modifications lead to complex changes of the physical-chemical properties of chromatin, including steric effects on the chromatin structure and formation of recognition sites for other proteins. Distinct histone tail modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins, which in turn dictate dynamic transitions between transcriptionally active or transcriptionally silent chromatin states. The combinatorial nature of histone amino-terminal modifications has led to the concept of a "histone code" that considerably extends the information potential of the genetic code. These results support the hypothesis that epigenetic chromatin modifications and concomitant changes of the 3D structure of chromatin are responsible for the transition of the transcriptionally silent chromatin into active chromatin states and vice versa.
Constitutive heterochromatin is virtually free of protein coding genes and mostly located in peri- and paracentromeric chromosomal subregions. Constitutive heterochromatin in general may play a role in nuclear architecture and gene silencing. Facultative heterochromatin contains silent genes. The presence of different chromocentres in cell nuclei (spatial associations of centromeric heterochromatin), ?myeloid? (in monocytes and granulocytes) and ?lymphoid? (in lymphocytes), was detected by Alcobia et al.
Recruitment of genes into the close neighbourhood of constitutive heterochromatin or packaging into facultative heterochromatic chromatin domains represents an important mechanism of epigenetic regulation of gene silencing. For example, the distances between genes and centromeric heterochromatin correlate with specific gene activity measured during myeloid cell differentiation. On the other hand, tissue-specific enhancers and locus control regions (LCRs) prevent active genes from being included in a region of transcriptional inactive condensed chromatin (heterochromatin) that forms during cell maturation.
The main pathway of heterochromatin formation is apparently related to histone H3 lysine (K) methyltransferases (HMTases), stably modifying histones H3 by methylating lysine at 9 position. Thus, a central role of histone-lysine methylation in epigenetic organisation of eukaryotic genomes has been strongly established. H3-K9 methylation creates a binding site for the chromatin organisation modifier (chromo) domain of heterochromatic HP1 proteins. These findings have suggested existence of a biochemical mechanism for induction and propagation of subdomains of facultative heterochromatin. Heterochromatic domains are maintained by highly dynamic HP1 binding and, consequently, silent genes are easily accessible to individual regulatory factors, although it is not clear to which extent the diffusion of larger protein complexes into heterochromatin foci may be impaired or inhibited.
Several mechanisms were considered as an explanation of structural modification of gene activity. For example Ikaros, a DNA-binding protein localized in the discrete foci of nuclei of murine B-lymphocytes, is in close association with centromeric heterochromatin.
A strong correlation was found between these foci and the location of transcriptionally inactive genes. In addition, in the context of differentiation of human lymphocytes a discovery was made that the promoter-specific binding factor of Ikaros mediates association of cell-type-specific genes with centromeric heterochromatin. Ikaros regulates movement of the genes towards centromeric heterochromatin, whereas activated genes are released. Thus, gene positioning on the periphery of the chromosome territory could facilitate not only access to the transcriptional machinery (enabling gene activation), but also access to the factors inhibiting genetic expression (e.g., clusters of centromeric heterochromatin).
Orderliness and Randomness in the Global Structure of the Human Genome
Visualization of CTs by in situ fluorescence hybridization (FISH) in mammalian and plant cells lead to intensive investigations of the structure of human genome. Studies of the arrangement of human genome and CTs have been performed for more than 15 years using 2D or 3D FISH in fixed cells. Recently, experiments using incorporation of labelled precursors or GFP tagged proteins binding DNA (e.g. histone H2B-GFP) provided additional information on the structure and dynamics of human genome.
3D Structure of the Human Genome is not Random
The currently available data support the view that the cell nucleus is far from being a randomly arranged bag of molecules, rather functioning as an integrated and highly ordered machine. On the other hand, the high degree of variability observed among nuclei with stained genetic elements leads to the conclusion that order in nuclear organisation might be manifested through statistical regularities. Two fundamental organizational principles have been distinguished: Retention of mitotic chromosome geometry, as reflected by polarization of the nucleus along a centromere to telomere axis and separation of chromosomes into non-overlapping territories; and organization through specific contacts with the nuclear envelope. The latter principle is found in mammalian cells.
It has been shown for many different genetic elements that the positions in the cell nucleus where these elements are located form non-random radial distributions between the centre of the nucleus and the membrane. Genes of highly expressed CT regions are localized in the central parts of the cell nucleus (A); genes or other sequences of regions with low expression are found preferentially near the nuclear periphery (B). The density of points represents the probability density per volume unit of the genetic element occurrence in a given position. The average centre of the nucleus-to-element distances are element-specific, largely maintained in different cell types and evolutionary conserved. Highly expressed chromosomal sub-regions are found close to the centre of the nucleus on the inner sides of CTs; while sub-regions with low expression are localised close to the nuclear membrane on the opposite sides of the territories. It could be shown in yeast that the tethering or targeting of a silencer-flanked reporter gene to the nuclear envelope facilitates its repression. On the other hand, association with the nuclear periphery is not sufficient if the reporter construct has no silencer element.
The Randomness in CTs Neighbourhood
Mutual positioning of CTs in HG is highly variable. This fact is evident from the observation of mutual positions of two pairs of CTs painted by different fluorochromes. More exact testing can be based on the determination of fluorescence weight centres of CTs in 3D space and calculation of the angle CT1-centre-CT2 in the plane defined by these 3 points. Random mutual positioning of CTs is reflected in the angular distribution corresponding to the sine function. In 3D space, the most frequent angle is 90o owing to the fact that the number of possible CT positions corresponding to this angle is the largest. Obviously the number of free positions for CTs will be proportional to the length of the circle perpendicular to the plane of the image corresponding to angle a, that is 2p.cos(a). Angular distributions reminding of the sine function have been found for a number of different homologous and heterologous pairs of genetic elements, which suggests that in most cases CT mutual positions are random. This type of randomness in the location of CTs is largely responsible for the variability of cell nuclei.
Dynamics of the Human Genome Structure
These experiments showed a high degree of stability of the interphase chromatin arrangement from G1 to G2 stages of the cell cycle and, to some extent, transmission of chromosome positions from mother to daughter cells. The authors admit that some intermixing that occurs within one cell division may lead to randomizing of CTs positions over several cell cycles. Constrained chromatin motion due to the likely association with nuclear compartments in human cells was shown using lacO integrant cell lines. The loci at nucleoli or the nuclear periphery were significantly less mobile than other, more nucleoplasmic loci.
Both 3D FISH and live cell approaches have their specific advantages and limitations and it is important to explore both approaches in parallel. For example, the 3D FISH approach is particularly suited to study the topology of a large set of active and inactive genes with respect to higher order euchromatic and heterochromatic compartments. A potential drawback of 3D FISH is represented by the fact that considerable chromatin damage produced by this procedure was detected at the EM level. However, the level of preservation of the nuclear topography even after the heat denaturation step is sufficient to study the large scale chromatin topology.
Structure and Orientation of CTs in the Cell Nucleus
The arrangement of interphase chromosomes into separate territories provides a framework for investigation of the relationship between the higher-order chromatin structure and function. The basic question is whether gene expression is determined, at least in part, by the structure of chromosome territory. The studies trying to resolve this issue are aimed at determining whether the organization of CTs is random, whether particular genomic sequences occupy special positions within chromosome territories, whether these positions differ according to the transcriptional activity of the sequences and whether genomic regions or whole individual chromosomes occupy particular compartments within the cell nucleus.
The first systematic quantitative studies of the topology of genetic elements in cell nuclei lead to the conclusion that CTs could be represented by randomly walking polymers. The authors measured average spatial distances between two genetic elements (Ds) with known molecular distance (Dm) and showed linear dependence Ds2(Dm), with the simplest explanation with a random flight polymer. In a later model of the interphase chromosome two levels of randomness were distinguished: Randomly walking loops of DNA (level 1) attached to a flexible and randomly walking backbone (level 2). This model explained behaviour of the two components of the dependence Ds2(Dm). Experiments were performed very carefully with high statistics using methanol 2D fixation as well as 3D paraformaldehyde fixation. In spite of the fact that the analysed chromosome 4 and 15 territories are relatively gene poor and not highly expressed, these studies represent a basis for further thought.
CTs are Polar and Oriented in Cell Nuclei
Investigation of the higher-order compartmentalization of chromatin according to its replication timing suggested a polar orientation of early and late replication sub-regions of chromosomes, with transcription competent and active chromatin located within the nuclear interior. Recent discoveries have demonstrated existence of an important factor influencing nuclear location of a genetic element, which is concentration of highly expressed genes in the element molecular environment on the chromosome. Density of highly expressed genes in the environment can be established according to Caron et al. If a genetic element is located in a region rich in highly expressed genes, its nuclear location is close to the nuclear centre. If it is located in a region poorly populated with expressed genes, its nuclear position is more peripheral.
Regions of high expression that protrude from the more condensed parts of the chromosome located in the proximity of the nuclear membrane to the nuclear centre determine the polar character of CTs which can be directly shown by measurements of 3D positions of at least 3 genetic elements along the territory, e.g. a centromere and both telomeres. Polar nature of CTs has been shown for HSA 3, 8, 9, and 19, where centromeres were localized on one side of the territory and both telomeres on the other side. Chromosomes are polar independently of their positions inside cell nuclei, i.e., regardless of whether they are located near the membrane or in the centre of the cell nucleus. In addition, a majority of the polar chromosome territories are oriented in the cell nucleus with the centromere localized near the nuclear periphery and both telomeres placed in the interior of the cell nucleus. Only 5-10% of chromosome 8 and 9 territories showed the opposite orientation.
Chromosome polarity and orientation can also be deduced from experiments with induced transcription performed in fixed cells or in living cells. Targeting the VP16 acidic activation domain (AAD) to an engineered chromosome site resulted in its transcriptional activation and redistribution from a predominantly peripheral to a more interior nuclear localization. Direct visualization in vivo revealed that the chromosome site normally moves into the nuclear interior transiently in the early G1 and again in the early S phase. In contrast, VP16 AAD targeting induced this sites permanent interior localization in the early G1. These results show that at least active CTs must be polar and oriented in cell nuclei.
Repeated FISH allows identification of genetic elements stained in the first hybridization using the same fluorochrome and identification of elements pertaining to different homologous CTs. In this way the number of the investigated genetic elements can be increased. The method used for the evaluation of the results is based on minimization of mutual distances between genetic elements of the same type inside their clusters. The large spheres represent positions of the cluster centres and their sizes represent standard deviations of elements inside the clusters. As demonstrated, the positions of the elements are variable, but still separated enough to outline the general structure of the CT. We conclude that CT can have not only polarity but also really organized 3D shape.
Dynamics of Genetic Elements inside CTs
Although CTs in the cell nucleus are relatively immobile, certain restricted movements or imprecise transitions through mitosis have been observed. Movements inside CTs have also been observed in living cells due to transcriptional activation. Consequently, the question arises how firmly genetic elements are attached to CTs (or the nucleus) during these movements and what is the proportion of movements of CTs as a whole and movements of genetic elements inside the territories.
A visualization of several genetic elements inside CTs has shown that their positioning can be either dependent or independent on the positioning of CTs. After rotation of the cell nucleus and transition of the weight centres of the investigated painted territories to a single point territorial distribution of genetic elements can be seen. The width of these distributions is usually narrower as compared with radial nuclear distributions. This means that the elements adhere to the territory. For example territorial distribution of q-telomeres of HSA 19 in Go-lymphocytes is narrower in comparison with nuclear distribution, which means that the q-telomere is quite firmly attached to the chromosome territory. The opposite possibility is a broader territorial distribution, which means that the corresponding genetic element, not adhering to the territory; may be attached to some other nuclear structures. This extreme possibility is represented by the behaviour of the p-telomere of HSA 19 in stimulated lymphocytes which shows full independence in relation to its own chromosomal territory. These findings show that elements may be attached either to the territory or to the nucleus. This may be explained by the fact that telomeres and telomere-specific binding proteins may associate with the nuclear matrix and participate in anchoring chromosomes. In addition, the de-condensation of chromatin related to high concentrations of expressed genes may cause extension of the distances between genetic elements in the cell nucleus and contribute to the relative independence of an element in relation to its territory.
Tethering of Chromosome Territories
Non-random angular distribution of homologous and heterologous elements was found in some cases. Shorter distances than predicted by random distribution were found between BCR/BCR genes located on homologous acrocentric chromosomes or between BCR/PML belonging to heterologous acrocentric chromosomes. Acrocentric chromosomes participate in the formation of the nucleolus and this common function may influence their nuclear location and lead in some cases to mutual proximity.
Tethering of CTs is well known for centromeres that are frequently localized in chromocentres whose number per cell nucleus is substantially smaller than 46. Centromeres are most frequently localised near the nuclear periphery or near the nucleoli. Nucleoli are thought to represent another example of inner nuclear surface where chromosomes can be attached. Association of centromeres is thought to play an important role in formation of heterochromatic foci and in gene silencing (see the section on heterochromatin).
Physiological telomere associations were found frequent in interphase nuclei of human fibroblasts and less frequent in cycling cells. This was a reason to assume that telomeric associations may be involved in the maintenance of chromosome positional stability in the interphase nucleus, especially in cells that are proliferating slowly, replicatively quiescent, or terminally differentiated. The authors thus conclude that the number of telomere associations in interphase nuclei depends on the cycling status of the cell, rather than on the individual telomeres length and telomerase activity. Using specific DNA probes, telomere association of CTs 8, 9, and 19 was investigated in Amrichová et al. No association between heterologous telomeres was found. On the other hand, homologous telomeres of CT 19 were often close to each other and signals of both telomeres (p-p or q-q) could often be identified as a single spot. This phenomenon was highly prevalent but did not depend on the stage of the cell in the cell cycle.
Experimental distributions of minimum distances between ABL-BCR in human lymphocytes differ from theoretical predictions; distribution of these distances is shifted to lower values. In about 10-25% of Go-lymphocytes of 5 different healthy individuals the minimum distance between ABL and BCR genes was less than 1 mm. No translocation between these genes was found in metaphases of stimulated lymphocytes from these individuals. The shift of distance distributions for the ABL and BCR genes was not observed for stimulated lymphocytes and HL-60 cells, even though tethering was observed for CD34+ progenitor cells. Proximity of specific chromosome regions can lead to their mutual rearrangement under some conditions, as was shown for RET/H4. Our results obtained in 2D or 3D show very close proximity of ABL/BCR genes (< 1 mm) in about 15-20% of Go-lymphocytes. Proximity of these regions might be one of the reasons for their interchanges and the formation of the Philadelphia chromosome typical of chronic myeloid leukaemia. The high frequency of interchanges induced by fast neutrons between chromosomes involved in translocations leading to most frequent haematologic malignancies also indicates the non-ranodom arrangement of some chromosomes in cell nuclei.
Changes of the Genome and Territory Structure Under Different Conditions
The Cell Cycle
CTs do not move (or very slowly) during interphase showing very local Brownian motion (Chubb at al., 2002) which reflects attachment of CTs to some anchoring points. In experiments using the FRAP technique, changes of the large-scale nuclear arrangement were not observed during interphase. After cell division, the chromatin arrangement in the daughter cells somewhat differed in comparison with the mother cell. These experiments confirmed earlier observations using fixed cells where stable higher-order chromatin structure was found in different stages of interphase. FISH experiments showed conserved radial distributions during interphase. In addition, mirror images of daughter cells obtained after FISH lead to the conclusion that the mobility of chromatin in interphase cells is rather restricted. An engineered chromosome site allowing transcriptional activation showed transient movement into the nuclear interior in the early G1 and the early S phase. These results show that stable global interphase arrangement found by other groups may be perturbed by central movement of relatively small chromosome regions.
In prometaphase, chromosome rosettes are formed by centromeres joining together and forming a central ring. Nagele et al. found a precise arrangement of CTs along the ring with homologous CTs being localized on the opposite sides of the ring. These results were not confirmed by Allison and Nestor who found random positioning of CTs in metaphase rosettes. In our experiments using nocodazole to block HL-60 cells in prometaphase we were not able to reproduce the precise chromosome order described by Nagele et al., even though the positions of centromeres were not random, rather showing a trend towards a preferential order.
Spatial and functional dynamics of selected genetic elements was studied during human granulocytic cell differentiation with the activity of a number of genes changed. The role of the chromatin structure in regulation of the studied gene expression was tested. The following three hypotheses were verified: (i) Activated (silenced) genes change their location in the cell nucleus, (ii) Activity of genes correlates with their location within the corresponding chromosome territory, and (iii) Gene expression is regulated by the association of genes with centromeric heterochromatin.
It was found that in the process of cell differentiation genetic elements are shifted to the periphery of the cell nucleus, which, however, does not correlate with genetic expression. Independently of gene expression, genetic elements are located closer to the corresponding fluorescence intensity centre of chromosome territory after differentiation, which rather reflects condensation of the CTs (similar shift to the centre of CTs is also observed for centromeres). Genes were located on the periphery of CTs, unlike centromeres, found closer to the bary centre of CTs, which is in agreement with observations of Kurz et al., who found either active or inactive genes preferentially located on the periphery of CTs. The distributions of distances between the genes and the nearest centromeric heterochromatin revealed a correlation with gene activity. A correlation between transcriptional activities of some tissue-specific genes and their association with pericentromeric heterochromatic regions has been found in several other studies in mammalian cells.
Topography of different genetic loci in human peripheral blood granulocytes was investigated in Bártová et al. Nuclei of granulocytes are characterised by a segmented shape consisting of two to five lobes that are in many cases connected by a thin filament containing DNA. Different topographic types of granulocytes were distinguished on the basis of the pattern of CTs or genetic element segregation into individual lobes. Using dual colour FISH in two-lobed nuclei, five topographic types could be distinguished. Segregation of four sets of genetic structures showed a large number of topographic types. In all these experiments organised distribution of chromosomes into nuclear lobes was found. Painting of the same type of chromosome in two-lobed nuclei showed a prevalence of symmetric topographic types (the homolog segregated in one lobe each). The results of the analysis of five topographic types (defined by two CTs pairs in two-lobed nuclei) have shown that symmetric topographic types for both chromosomes are significantly more frequent than predicted. Repeated hybridisation experiments have confirmed that the occurrence of certain patterns of chromosome segregation is much higher than that predicted from the combination of probabilities. Both genes and centromeres were observed on filaments joining different lobes. The significance of individual topographic types, particularly of those observed with much higher probability then expected, is unclear.
Ionizing radiation (repair of radiation damage)
Rearrangement of human cell homologous CTs in response to ionizing radiation was observed by Dolling et al. In this study, homologous CTs were found closer to each other after irradiation, and the authors proposed that the process of CTs pairing to facilitate re-combinational repair of DNA DSBs may exist. In addition, radial movement of genetic elements was observed after irradiation of several cell lines. The spatial relationships between genetic elements returned to that of the non-irradiated controls during several hours of incubation after irradiation. The authors speculate that the changes of the large-scale chromatin structure might be related to repair processes, however, they exclude repair of DSBs by processes involving homologous recombination, because the angular distributions of homologous sequences remained random after irradiation. Radial movement was also observed by Tumbar and Belmont in live cell experiments using transcriptional activator of artificially engineered chromosome sites.
Nuclear architecture of selected CTs was investigated in apoptotic nuclei of human leukaemia-affected cells. Apoptotic disorganization of chromosome territories was irregular, leading mainly to chromosomal segments of different sizes and, consequently, chromosomal disassembly was not observed at specific sites. In comparison to the control group an increased number of centromeric FISH signals were observed in prolonged confluence-treated K-562 cells induced to apoptosis. This finding can be explained either as a consequence of apoptosis or by poly-ploidization. Sequential staining of the same apoptotic nuclei by the FISH and TUNEL techniques has revealed that chromosome territory segmentation precedes the formation of nuclear apoptotic bodies.
Haemoblastoses and Cancer
Radial and angular distributions have been measured in both normal and tumour cell lines with similar results. Distributions of ABL and BCR are very similar in bone marrow cells, in Go and stimulated lymphocytes, HL-60 cells, HT-29 colon cancer cells and also in nuclei of colon tissue and CML patients. Radial distributions of EWSR1 and FLI1 genes are similar for Go and stimulated lymphocytes as well as for Ewing sarcoma cells. In mouse lymphoma cells two translocated CTs were preferentially positioned in close proximity to each other. The relative positions of the chromosomes involved in these translocations are close even in normal splenocytes. These observations demonstrate the fact that relative arrangement of CTs in the interphase nucleus can be conserved between normal and cancer cells.
Specific translocations that are (casually) related to some types of leukaemia provide ?new? chromosomes and their nuclear location can be investigated. For example, in Ewing sarcoma cells, radial positions were measured for EWSR1, FLI1 and fusion genes. The radial positions of both fusion genes are shifted compared with the radial positions of non-aberrant EWSR1 and FLI1 genes. While HSA 11 fusion gene is shifted more centrally, HSA 22 fusion gene lies towards the periphery. Thus, both fusion genes are located approximately midway between EWSR1 and FLI1 genes in Ewing sarcoma cells. The different location of the fusion genes might be explained by the substitution of a small part of HSA 11 for a larger part of HSA 22 and vice versa. The central nuclear location of HSA 22 correlates with its high gene density. Thus, the transfer of a part of HSA 22 with high gene density to HSA 11 causes relocation in the central direction of the translocation neighbourhood of chimeric HSA 11. On the other hand, the translocation neighbourhood of chimeric HSA 22 is shifted towards the nuclear periphery.
Potential influence of heterochromatin on RB1 gene silencing was investigated in differentiated human retinoblastoma tumour cells with (X;13) translocation. Spreading or proximity of heterochromatic and methylated X chromosome to the q arm of chromosome 13 was suspected to induce functional monosomy of RB1 gene. Thus the regulation of gene activity during important cellular processes such as differentiation or carcinogenesis seems to be realized through heterochromatin-mediated gene silencing.
For the purpose of finding the influence of increased gene expression and amplification in colorectal carcinoma on the chromatin structure nuclear distances between two BAC clones with short genomic separation (1-2 Mb) were measured (using the method called spectral microscopy) and compared between tumour and parallel epithelial cells of 6 patients. Larger nuclear distances were found for tumour as compared with epithelial cells for the same genomic separation. The ratio of the mean nuclear distance between the loci in tumour and epithelium decreased with the mean degree of amplification of genetic loci. Similarly, induction of the fusion PML/RARa protein in PIR9 cells leads to changes in gene expression and corresponding (de)condensation of chromatin detectable by spectral microscopy.