The endosperm is at the center of successful seed formation in flowering plants. Being itself a product of fertilization, it is devoted to nourish the developing embryo and typically possesses a triploid genome consisting of two maternal and one paternal genome complement. Interestingly, endosperm development is controlled by epigenetic mechanisms conferring parent-of-origin-dependent effects that influence seed development. In the model plant Arabidopsis thaliana, we have previously described an endosperm-specific heterochromatin fraction, which increases with higher maternal, but not paternal, genome dosage. Here, we report a detailed analysis of chromosomal arrangement and association frequency in endosperm nuclei. We found that centromeric FISH signals in isolated nuclei show a planar alignment that may results from a semi-rigid, connective structure between chromosomes. Importantly, we found frequent pairwise association of centromeres, chromosomal segments, and entire arms of chromosomes in 3C endosperm nuclei. These associations deviate from random expectations predicted by numerical simulations. Therefore, we suggest a non-random chromosomal organization in the triploid nuclei of Arabidopsis endosperm. This contrasts with the prevailing random arrangement of chromosome territories in somatic nuclei. Based on observations on a series of nuclei with varying parental genome ratios, we propose a model where chromosomes associate pairwise involving one maternal and one paternal complement. The functional implications of this predicted chromosomal arrangement are discussed.

The spatial organisation of the genome in the cell nucleus has emerged as a key element to understand gene function. A wealth of molecular and microscopic information has been accumulated, resulting in a variety of sometimes contradictory models of nuclear architecture. So far, however, a large part of this structural information and in consequence also the models derived from them are 'qualitative'. In this overview, a brief introduction will be given into quantitative experimental and modelling approaches to large scale nuclear genome architecture in human cells As a biomedical application example, the use of a quantitative computer model, of the 3D architecture allowed to explore different implications of nuclear structure on chromosomal aberrations. In addition, we shall present two novel examples for quantitative computer modelling. (1) The impact of SC 35 splicing domains on nuclear genome structure; (2) The dynamics of large scale nuclear genome structure in a Brownian motion model Finally, we shall discuss some perspectives to extend quantitative nuclear structure analysis to the nanoscale

A present model of the higher-order chromosome organization suggests the organization of chromosome built up by loops. Here we focus on a single rosette-like part of the fiber and analyse the diffusion behaviour of small particles (corresponding to single proteins/protein complexes) and the accessibility of such particles in relation to the dynamic rosette structure. Surprisingly, although the diffusion pattern of the diffusing particles revealed free diffusion, an area of about 6-12 kbp in the innermost part of these domains becomes visible which is inaccessible even for small particles (corresponding to single proteins/protein complexes). A localisation of a promotor sequence in this area might silence the respective gene by the physical inaccessibility of this area for transcription factors. We conclude that the compartmentalisation of chromatin in domains of a specific dynamical three-dimensional (3D) structure might be of high functional importance.

The hypothesis that distinct chromatin domains expand and are remodelled differently when they undergo transcription, replication or cell cycle processes is well accepted. The condensation changes by which chromosomes are transformed at the metaphase-interphase transition are especially interesting and therefore extensively studied by light microscopy; however, quantitative information of the size on specific small chromatin domains during the cell cycle is scarce. In this respect, a serious problem is the determination of structural features close to the resolution limit. In this report we use a novel approach to quantify the lateral extent of the 8q24/c-myc gene domain and the centromeric region of chromosome 8 in doubly labelled normal human foreskin fibroblasts using confocal laser scanning microscopy (CLSM). The domains were analysed in both metaphase spreads and interphase nuclei. These high precision measurements revealed a somewhat smaller (few 10s of nm) lateral extension of the centromere region in metaphase compared to interphase. Surprisingly, within the same cells the lateral extension of the 8q24/c-myc region was significantly smaller in interphase than in metaphase. For comparison the centromere size was more condensed in metaphase than in interphase. This implies a different folding behaviour for specific chromatin domains with opposite condensation behaviour.

For the understanding of radiation action on biological systems like cellular macromolecules (e.g., DNA in its higher structures) a synergistic approach of experiments and quantitative modelling of working hypotheses is necessary. Further on, the influence on calculated results of certain assumptions in such working hypotheses must critically be evaluated. In the present work, this issue is highlighted in two aspects for the case of DNA damage in single cells. First, yields of double-strand breaks and frequency distributions of DNA fragment lengths after ion irradiation were calculated using different assumptions on the DNA target model. Compared to a former target model now a moderate effect due to the inclusion of a spherical chromatin domain model has been found. Second, the influence of assumptions on particular geometric chromosome models on calculated chromosome aberration data is illustrated with two target-modelling approaches for this end point.

A quantitative computer model was applied to simulate the three-dimensional (3D) spatial organization of chromatin in human cell nuclei under defined conditions of virtual irradiation to explore the implications of spatial organization on chromosome aberrations. To calibrate the virtual irradiation algorithm, a dose-dependent spectrum of radiation-induced chromosome aberrations such as dicentrics, translocations and centric rings was calculated for low-LET radiation doses ranging from 0.5 to 5 Gy. This was compared with the results from experimental studies. While the dose-response curves calculated from model simulations agree well with experimental dose-response curves for dicentrics and translocations, centric rings are significantly more frequent in the model simulation than in experiments despite taking into account exclusive arm territories in the applied Spherical 1 Mbp Chromatin Domain (SCD) computer model explicitly. Taking into account the non-random positioning of chromosome territories observed in lymphocyte cell nuclei (a so-called gene density-correlated arrangement of chromosome territories), aberration frequencies were calculated with the calibrated irradiation algorithm to investigate the impact of chromosome territory neighborhood effects (proximity effects). The absolute frequencies of pairwise exchanges agree well with those found in an experimental study. In conclusion, the results obtained using the computer model approach presented here based on only a few adjustable parameters correlated well with those of experimental studies of chromosome aberration frequencies. Thus the model may be a useful tool in radiation-induced cancer risk estimates in combination with epidemiological studies. (c) 2007 by Radiation Research Society.

Three-dimensional (3D) chromatin structure is closely related to genome function, in particular transcription. However, the folding path of the chromatin fiber in the interphase nucleus is unknown. Here, we systematically measured the 3D physical distance between pairwise labeled genomic positions in gene-dense, highly transcribed domains and gene-poor less active areas on chromosomes 1 and 11 in G1 nuclei of human primary fibroblasts, using fluorescence in situ hybridization. Interpretation of our results and those published by others, based on polymer physics, shows that the folding of the chromatin fiber can be described as a polymer in a globular state (GS), maintained by intra-polymer attractive interactions that counteract self-avoidance forces. The GS polymer model is able to describe chromatin folding in as well the highly expressed domains as the lowly expressed ones, indicating that they differ in Kuhn length and chromatin compaction. Each type of genomic domain constitutes an ensemble of relatively compact globular folding states, resulting in a considerable cellto- cell variation between otherwise identical cells. We present evidence for different polymer folding regimes of the chromatin fiber on the length scale of a few mega base pairs and on that of complete chromosome arms (several tens of Mb). Our results present a novel view on the folding of the chromatin fiber in interphase and open the possibility to explore the nature of the intra-chromatin fiber interactions.

The method of Spectral Precision Distance Microscopy (SPDM) has been used to determine distances between two FISH (Fluorescence-in situ-Hybridization)-labeled gene regions on chromosome 9. To this end we applied methods to correct for chromatic aberrations of the microscope optics alone and also of the sample induced aberrations due to mismatch of the refractive indices. Using a confocal microscope and a threshold based position determination algorithm, positions could be measured with an accuracy of about 65 nm inside of fixed cell nuclei. Distances obtained from the measurements have been verified using a 3D computer model of the cell nucleus. In principle, this SPDM approach could be combined with novel fluorescence microscopes to obtain structural information well below the optical resolution. At present the precision limit of the distance measurements is set by variations of the refractive index throughout the specimens.

Specific mammalian genes functionally and dynamically associate together within the nucleus. Yet, how an array of many genes along the chromosome sequence can be spatially organized and folded together is unknown. We investigated the 3D structure of a well-annotated, highly conserved 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene “deserts.” In nuclei, this region forms multiple, nonrandom “higher order” structures. These structures are based on the gene distribution pattern in primary sequence and are marked by preferential associations among multiple gene clusters. Associating gene clusters represent expressed chromatin, but their aggregation is not simply dependent on ongoing transcription. In chromosomes with aggregated gene clusters, gene deserts preferentially align with the nuclear periphery, providing evidence for chromosomal region architecture by specific associations with functional nuclear domains. Together, these data suggest dynamic, probabilistic 3D folding states for a contiguous megabase-scale chromosomal region, supporting the diverse activities of multiple genes and their conserved primary sequence organization.

In contrast to the situation described for mammals and Drosophila, chromosome territory (CT) arrangement and somatic homologous pairing in interphase nuclei of Arabidopsis thaliana (n = 5) are predominantly random except for a more frequent association of the chromosomes bearing a homologous nucleolus organizer region. To find out whether this chromosome arrangement is also characteristic for other species of the genus Arabidopsis, we investigated Arabidopsis lyrata ssp. lyrata (n = 8), one of the closest relatives of A. thaliana. First, we determined the size of each chromosome and chromosome arm, the sequence type of centromeric repeats and their distribution between individual centromeres and the position of the 5S/45S rDNA arrays in A. lyrata. Then we demonstrated that CT arrangement, homologous pairing and sister chromatid alignment of distinct euchromatic and/or heterochromatic regions within A. lyrata interphase nuclei are similar to that in A. thaliana nuclei. Thus, the arrangement of interphase chromosomes appears to be conserved between both taxa that diverged about 5 million years ago. Since the chromosomes of A. lyrata resemble those of the presumed ancestral karyotype, a similar arrangement of interphase chromosomes is also to be expected for other closely related diploid species of the Brassicaceae family.

In the present study a model for the compactification of the 30 nm chromatin fibre into higher order structures is suggested. The idea is that basically every condensing agent (HMG/SAR, HP1, cohesin, condensin, DNA–DNA interaction …) can be modeled as an effective attractive potential of specific chain segments. This way the formation of individual 1 Mbp sized rosettes from a linear chain could be observed. We analyse how the size of these rosettes depends on the number of attractive segments and on the segment length. It turns out that 8–20 attractive segments per 1 Mbp domain produces rosettes of 300–800 nm in diameter. Furthermore, our results show that the size of the rosettes is relatively insensitive to the segment length.

Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes-independently of their gene density-were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding.

The non-random positioning of chromosome territories (CTs) in lymphocyte cell nuclei has raised the question whether systematic chromosome-chromosome associations exist which have significant influence on interchange rates. In such a case the spatial proximity of certain CTs or even of clusters of CTs is expected to increase the respective exchange yields significantly, in comparison to a random association of CTs. In the present study we applied computer simulated arrangements of CTs to calculate interchange frequencies between all heterologous CT pairs, assuming a uniform action of the molecular repair machinery. For the positioning of CTs in the virtual nuclear volume we assumed a) a statistical, and b) a gene density-correlated arrangement. The gene density-correlated arrangement regards the more experimentally observed interior localization of gene-rich and the more peripheral positioning of gene-poor CTs. Regarding one-chromosome yields, remarkable differences for single CTs were observed taking into account the gene density-correlated distribution of CTs.

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.