Approaching the Three-Dimensional Organization and Dynamics of the Human Genome

Genomes are one of the major foundations of life due to their role in information storage, process regulation and evolution. However, the sequential and three-dimensional structure of the human genome in the cell nucleus as well as its interplay with and embedding into the cell and organism only arise scarcely. To achieve a deeper understanding of the human genome the three-dimensional organization of the human cell nucleus, the structural-, scaling- and dynamic properties of interphase chromosomes and cell nuclei were simulated and combined with the analysis of long-range correlations in completely sequenced genomes as well as the chromatin distribution in vivo. Using Monte Carlo and Brownian Dynamics methods, the 30 nm chromatin fibre was simulated according to the Multi-Loop-Subcompartment (MLS) model, in which ~100 kbp loops form rosettes, connected by a linker, and the Random-Walk/Giant-Loop (RW/GL) topology, in which 1-5 Mbp loops are attached to a flexible backbone. Both the MLS and the RW/GL model form chromosome territories but only the MLS rosettes result in distinct subcompartments visible with light microscopy and low overlap of chromosomes, -arms and subcompartments. The MLS morphology, the size of subcompartments and chromatin density distribution of simulated confocal (CLSM) images agree with the expression of fusionproteins from the histones H1, H2A, H2B, H3, H4 and mH2A1.2 with the auto-fluorescent proteins CFP, GFP, YFP, DsRed-1 and DsRed-2 which also revealed different interphase morphologies for different cell lines. Even small changes of the model parameters induced significant rearrangements of the chromatin morphology. Thus, pathological diagnoses, are closely related to structural changes on the chromatin level. The position of interphase chromosomes depends on their metaphase location, and suggests a possible origin of current experimental findings. The scaling behaviour of the chromatin fibre topology and morphology of CLSM stacks revealed finestructured multi-scaling behaviour in agreement with the model prediction and correlations in the DNA sequence. Review and comparison of experimental to simulated spatial distance measurements between genomic markers as function of their genomic separation also favour an MLS model with loop and linker sizes of 63 to 126 kbp. Simulated and experimental DNA fragment distribution after ion-irradiation revealed also best agreement with such an MLS. Correlation analyses of completely sequenced Archaea, Bacteria and Eukarya chromosomes revealed fine-structured positive long-range correlation due to codon, nucleosomal or block organization of the genomes, allowing classification as well as tree construction. This shows a complex sequential organization of genomes closely connected to their three-dimensional organization. Visual inspection of the morphology reveals also big spaces between the chromatin fibre allowing high accessibility to nearly every spatial location, due to the chromatin occupancy <30% and a mean mesh spacing of 29 to 82 nm for nuclei of 6 to 12 μm diameter. This agrees with a simulated displacement of 10 nm sized particles of ~1 to 2 μm takes place within 10 ms, i.e. a moderately obstructed diffusion of biological molecules in agreement with experiments. Thus, the local, global and dynamic characteristics of cell nuclei are not only tightly interconnected, but also are integrated holisticly to fulfill the overall function of the genome

Three-Dimensional Organization of Chromosome Territories and the Human Cell Nucleus

To study the three-dimensional organization of chromosome territories and the human interphase cell nucleus we developed models, which could be compared to experiments. Despite the successful linear sequencing of the human genome its 3D-organization is widely unknown. Using Monte Carlo and Brownian dynamics simulations we managed to model the chromatin fiber as a wormlike-chain polymer. A typical chromosome consists of 20.000 and a nucleus with all 46 chromosomes of 1.200.000 polymer chain segments. The parallel simulations are performed on a SP2512 and a Cray T3E. With fluorescent in situ hybridization and confocal microscopy we determined genomic marker distributions and chromosome arm overlap. Best agreement between simulations and experiments is reached for a Multi-Loop-Subcompartment model (126 kbp loops connected to rosettes connected by a 126 kbp chromatin linker). A fractal analysis of simulations leads to multi-fractal behaveour in good agreement with porous network research. The formation of chromosome territories was shown as predicted and low overlap of chromosomes and their arms was also reached in contrast to other models

Approaching the three-dimensional organization and dynamics of the human genome

To approach the three-dimensional organization of the human cell nucleus, the structural-, scaling- and dynamic properties of interphase chromosomes and cell nuclei were simulated with Monte Carlo and Brownian Dynamics methods. The 30 nm chromatin fibre was folded according to the Multi-Loop-Subcompartment (MLS) model, in which ~100 kbp loops form rosettes, connected by a linker, and the Random-Walk/Giant-Loop (RW/GL) topology, in which 1-5 Mbp loops are attached to a flexible backbone. Both the MLS and the RW/GL model form chromosome territories but only the MLS rosettes result in distinct subcompartments visible with light microscopy and low overlap of chromosomes, -arms and subcompartments. This morphology and the size of subcompartments agree with the morphology found by expression of histone auto-fluorescent protein fusions and fluorescence in situ hybridization (FISH) experiments. Even small changes of the model parameters induced significant rearrangements of the chromatin morphology. Thus, pathological diagnoses based on this morphology, are closely related to structural changes on the chromatin level. The position of interphase chromosomes depends on their metaphase location, and suggests a possible origin of current experimental findings. The chromatin density distribution of simulated confocal (CLSM) images agrees with the MLS model and with recent experiments. The scaling behaviour of the chromatin fibre topology and morphology of CLSM stacks revealed fine-structured multi-scaling behaviour in agreement with the model prediction. Review and comparison of experimental to simulated spatial distance measurements between genomic markers as function of their genomic separation also favour an MLS model with loop and linker sizes of 63 to 126 kbp. Visual inspection of the morphology reveals also big spaces allowing high accessibility to nearly every spatial location, due to the chromatin occupancy <30% and a mean mesh spacing of 29 to 82 nm for nuclei of 6 to 12 µm diameter. The simulation of diffusion agreed with this structural prediction, since the mean displacement for 10 nm sized particles of ~1 to 2 µm takes place within 10 ms. Therefore, the diffusion of biological relevant tracers is only moderately obstructed, with the degree of obstruction ranging from 2.0 to 4.0 again in experimental agreement

Towards a holistic understanding of the human genome by determination and integration of its sequential and three-dimensional organization

Genomes are one of the major foundations of life due to their role in information storage, process regulation and evolution. However, the sequential and three-dimensional structure of the human genome in the cell nucleus as well as its interplay with and embedding into the cell and organism only arise scarcely from the unknown, despite recent successes e. g. in the linear sequencing efforts and growing evidence for seven genomic organization levels. To achieve a deeper understanding of the human genome the structural, scaling and dynamic properties in the simulation of interphase chromosomes and cell nuclei are determined and combined with the analysis of long-range orrelations in completely sequenced genomes as well as the analysis of the chromatin distribution in vivo: This integrative approach reveals that the chromatin fiber is most likely folded according to the Multi-Loop-Subcompartment (MLS) model in which the chromatin fiber bents into 63–126 kbp big loops aggregated to rosettes connected by again 63–126 kbp linkers. The MLS model exhibits fine-structured multi-scaling and predicts correctly the transport of molecules by moderately obstructed/anomalous diffusion. On the basic sequence level, genomes show fine-structured positive long-range correlations, allowing classification and tree construction. This, DNA fragment distributions after carbon ion irradiation and on the highest structural level, the nuclear morphology visualized by histone autofluorescent protein fusions in vivo, agrees again best with the MLS model. Thus, the local, global and dynamic characteristics of cell nuclei are not only tightly inter-connected, but also are integrated holisticly to fulfill the overall function of the genome

Three-Dimensional Organization of Chromosome Territories and the Human Cell Nucleus: Comparison between simulated Parameters and Experiments

Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fiber within these territories are under debate: In the Random- Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop- Subcompartment (MLS) model loops of around 120 kbp are forming rosettes, which are also interconnected by the chromatin fiber. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescernce in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labeling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as predicted by the MLS-model. The spatial distances between pairs of genomic markers as function of their genomic separation result in a MLS-model with loop and linker sizes around 126 kbp. With the development of GFP-fusion-proteins it is possible to study the chromatin distribution and dynamics resulting from cell cycle, treatment by chemicals or radiation in vivo. The chromatin distributions are similar to those found in the simulation of whole cell nuclei of the MLS-model. Fractal analysis is especially suited to quantify the unordered and non-euklidean chromatin distribution of the nucleus. The dynamic behaveour of the chromatin structure and the diffusion of particles in the nucleus are also closely connected to the fractal dimension. Fractal analysis of the simulations reveal the multi-fractality of chromosomes. First fractal analysis of chromatin distributions in vivo result in significant differences for different morphologies and might favour a MLS-model- like chromatin distribution. Simulations of fragment distributions based on double strand breakage after carbon- ion irradiation differ in different models. Here again a comparison with experiments favours a MLS-model

Approaching the three-dimensional organization and dynamics of the human genome

Genomes are one of the major foundations of life due to their role in information storage, process regulation and evolution. However, the sequential and three-dimensional structure of the human genome in the cell nucleus as well as its interplay with and embedding into the cell and organism only arise scarcely. To achieve a deeper understanding of the human genome the three-dimensional organization of the human cell nucleus, the structural-, scaling- and dynamic properties of interphase chromosomes and cell nuclei were simulated and combined with the analysis of long-range correlations in completely sequenced genomes as well as the chromatin distribution in vivo. Using Monte Carlo and Brownian Dynamics methods, the 30 nm chromatin fibre was simulated according to the Multi-Loop-Subcompartment (MLS) model, in which ~100 kbp loops form rosettes, connected by a linker, and the Random-Walk/Giant-Loop (RW/GL) topology, in which 1-5 Mbp loops are attached to a flexible backbone. Both the MLS and the RW/GL model form chromosome territories but only the MLS rosettes result in distinct subcompartments visible with light microscopy and low overlap of chromosomes, -arms and subcompartments. The MLS morphology, the size of subcompartments and chromatin density distribution of simulated confocal (CLSM) images agree with the expression of fusionproteins from the histones H1, H2A, H2B, H3, H4 and mH2A1.2 with the auto-fluorescent proteins CFP, GFP, YFP, DsRed-1 and DsRed-2 which also revealed different interphase morphologies for different cell lines. Even small changes of the model parameters induced significant rearrangements of the chromatin morphology. Thus, pathological diagnoses, are closely related to structural changes on the chromatin level. The position of interphase chromosomes depends on their metaphase location, and suggests a possible origin of current experimental findings. The scaling behaviour of the chromatin fibre topology and morphology of CLSM stacks revealed finestructured multi-scaling behaviour in agreement with the model prediction and correlations in the DNA sequence. Review and comparison of experimental to simulated spatial distance measurements between genomic markers as function of their genomic separation also favour an MLS model with loop and linker sizes of 63 to 126 kbp. Simulated and experimental DNA fragment distribution after ion-irradiation revealed also best agreement with such an MLS. Correlation analyses of completely sequenced Archaea, Bacteria and Eukarya chromosomes revealed fine-structured positive long-range correlation due to codon, nucleosomal or block organization of the genomes, allowing classification as well as tree construction. This shows a complex sequential organization of genomes closely connected to their three-dimensional organization. Visual inspection of the morphology reveals also big spaces between the chromatin fibre allowing high accessibility to nearly every spatial location, due to the chromatin occupancy <30% and a mean mesh spacing of 29 to 82 nm for nuclei of 6 to 12 μm diameter. This agrees with a simulated displacement of 10 nm sized particles of ~1 to 2 μm takes place within 10 ms, i.e. a moderately obstructed diffusion of biological molecules in agreement with experiments. Thus, the local, global and dynamic characteristics of cell nuclei are not only tightly interconnected, but also are integrated holisticly to fulfill the overall function of the genome

3D Architecture, dynamics as well as functional implications of genome organization of the Prader-Willi/Angelmann syndrome region & the Immunoglobin Heavy-Chain locus

The general 3D architecture of the immunoglobin heavy-chain (Igh) locus was determined by a novel interdisciplinary combination of high-resolution FISH and high-resolution epifluorescence spectral distance microscopy with analytical analysis, computer simulations, as well as trilateration (Cell 133, 265-279, 2008). The Igh locus is organized into distinct regions that contain multiple variable (VH), diversity (DH), joining (JH) and constant (CH) coding elements. Determination of distance distributions between genomic markers across the entire locus showed that the Igh locus is organized into compartments consisting of small loops separated by linkers with in detail dynamic functional relevance: VH, DH, JH, and CH elements showed striking conformational changes involving VH and DH-JH elements during early B cell development, culminating in a merger and juxtaposition of the entire repertoire of VH regions to the DH elements in pro-B cells allowing long-range genomic interactions with relatively high frequency. This is in agreement with our recent study of the Prader- Willi/Angelmann region using a similar approach (Differentiation 76, 66-82, 2008) and in agreement with the Multi-Loop-Subcompartment (MLS) model of chromosome organization predicting 60-150 kbp loop aggregates separated by a similar linker (Knoch, ISBN 3-00-009959-X, 2002). Synopsis with previous spatial distance measurement studies and combination with sequence correlation analysis of the DNA sequence, fine-structure multi-scaling analysis of the chromatin fiber topology or in vivo morphology of entire cell nuclei, electron microscopy of chromosome spreading studies and even the diffusion behaviour within the cell nucleus, are all suggesting such an MLS architecture. This framework reveals a consistent picture of genome organization joining structural and dynamical aspects ranging from the DNA sequence to the entire nuclear morphology level with functional aspects of gene location and regulation. Many previously contradictory viewpoints are resolved by this framework as well. Consequently, the determination of the general 3D architecture of the Igh locus has beyond its major functional relevance, huge implications for the understanding of the entire genome understanding in a holistic system-biological manner.al manner

News from the Pizza-Connection

Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fiber within these territories are under debate: In the Random- Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop- Subcompartment (MLS) model loops of around 120 kbp are forming rosettes, which are also interconnected by the chromatin fiber. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescernce in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labeling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as predicted by the MLS-model. The spatial distances between pairs of genomic markers as function of their genomic separation result in a MLS-model with loop and linker sizes around 126 kbp. With the development of GFP-fusion-proteins it is possible to study the chromatin distribution and dynamics resulting from cell cycle, treatment by chemicals or radiation in vivo. The chromatin distributions are similar to those found in the simulation of whole cell nuclei of the MLS-model. Fractal analysis is especially suited to quantify the unordered and non-euklidean chromatin distribution of the nucleus. The dynamic behaveour of the chromatin structure and the diffusion of particles in the nucleus are also closely connected to the fractal dimension. Fractal analysis of the simulations reveal the multi-fractality of chromosomes. First fractal analysis of chromatin distributions in vivo result in significant differences for different morphologies and might favour a MLS-model- like chromatin distribution. Simulations of fragment distributions based on double strand breakage after carbon- ion irradiation differ in different models. Here again a comparison with experiments favours a MLS-model