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

Three-dimensional organization of chromosome territories in the human interphase cell nucleus.

The synthesis of proteins, maintenance of structure and duplication of the eukaryotic cell itself are all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. The regulation of genes – their transcription and replication - has been shown to be connected closely to the threedimensional organization of the genome in the cell nucleus. Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown. The nucleus of the cell has for a long time been viewed as a 'spaghetti soup' of DNA bound to various proteins without much internal structure, except during cell division when chromosomes are condensed into separate entities. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in the interphase, i.e. between cell divisions. In an analogy of the Bauhaus principle that "form follows function" we believe that analyzing in which form DNA is organized in these territories will help us to understand genomic function. We use computer models - Monte Carlo and Brownian dynamics simulations - to develop plausible proposals for the structure of the interphase genome and compare them to experimental data. In the work presented here, we simulate interphase chromosomes for different folding morphologies of the chromatin fiber which is organized into loops of 100kbp to 3 Mbp that can be interconnected in various ways. The backbone of the fiber is described by a wormlike-chain polymer whose diameter and stiffness can be estimated from independent measurements. The implementation describes this polymer as a segmented chain with 3000 to 20000 segments for chromosome 15 depending on the phase of the simulation. The modeling is performed on a parallel computer (IBM SP2 with 80 nodes). We also determine genomic marker distributions within the Prader- Willi-Region on chromosome 15q11.2-13.3. For these measurements we use a fluorescence in situ hybridisation method (in collaboration with I. Solovai, J. Craig and T. Cremer, Munich, FRG) conserving the structure of the nucleus. As probes we use 10 kbp long lambda clones (Prof. B. Horsthemke, Essen, FRG) covering genomic marker distances between 8 kbp and 250 kbp. The markers are detected with confocal and standing wavefield light microscopes (in collaboration with J.Rauch, J. Bradl, C. Cremer and E.Stelzer, both Heidelberg, FRG) and using special image reconstruction methods developed solely for this purpose (developed by R. Eils. and W. Jaeger, Heidelberg, FRG). Best agreement between simulations and experiments is reached for a Multi-Loop-Subcompartment model with a loop size of 126 kbp which are forming rosetts and are linked by a chromatin linker of 126 kbp. We also hypothesize a different folding structure for maternal versus paternal chromosome 15. In simulations of whole cell nuclei this modell also leads to distinct chromosome territories and subcompartments. A fractal analysis of the simulations leads to multifractal behavior in good agreement with predictions drawn from porous network research

Three-dimensional organization of chromosome territories in the human interphase nucleus

The synthesis of proteins, maintenance of structure and duplication of the eukaryotic cell itself are all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. The regulation of genes – their transcription and replication - has been shown to be connected closely to the three- dimensional organization of the genome in the cell nucleus. Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown. The nucleus of the cell has for a long time been viewed as a 'spaghetti soup' of DNA bound to various proteins without much internal structure, except during cell division when chromosomes are condensed into separate entities. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in the interphase, i.e. between cell divisions. In an analogy of the Bauhaus principle that "form follows function" we believe that analyzing in which form DNA is organized in these territories will help us to understand genomic function. We use computer models - Monte Carlo and Brownian dynamics simulations - to develop plausible proposals for the structure of the interphase genome and compare them to experimental data. In the work presented here, we simulate interphase chromosomes for different folding morphologies of the chromatin fiber which is organized into loops of 100kbp to 3 Mbp that can be interconnected in various ways. The backbone of the fiber is described by a wormlike-chain polymer whose diameter and stiffness can be estimated from independent measurements. The implementation describes this polymer as a segmented chain with 3000 to 20000 segments for chromosome 15 depending on the phase of the simulation. The modeling is performed on a parallel computer (IBM SP2 with 80 nodes). We also determine genomic marker distributions within the Prader- Willi-Region on chromosome 15q11.2-13.3. For these measurements we use a fluorescence in situ hybridisation method (in collaboration with I. Solovai, J. Craig and T. Cremer, Munich, FRG) conserving the structure of the nucleus. As probes we use 10 kbp long lambda clones (Prof. B. Horsthemke, Essen, FRG) covering genomic marker distances between 8 kbp and 250 kbp. The markers are detected with confocal and standing wavefield light microscopes (in collaboration with J.Rauch, J. Bradl, C. Cremer and E.Stelzer, both Heidelberg, FRG) and using special image reconstruction methods developed solely for this purpose (developed by R. Eils. and W. Jaeger, Heidelberg, FRG). Best agreement between simulations and experiments is reached for a Multi-Loop-Subcompartment model with a loop size of 126 kbp which are forming rosetts and are linked by a chromatin linker of 126 kbp. We also hypothesize a different folding structure for maternal versus paternal chromosome 15. In simulations of whole cell nuclei this modell also leads to distinct chromosome territories and subcompartments. A fractal analysis of the simulations leads to multifractal behavior in good agreement with predictions drawn from porous network research

Three-dimensional organization of the human interphase nucleus: Experiments compared to simulations.

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 fibre 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 fibre. 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. Fluorescence in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labelling 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-euclidean chromatin distribution of the nucleus. The dynamic behaviour 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-modellike chromatin distribution. Simulations of fragment distributions based on double strand breakage after carbonion irradiation differ in different models. Here again a comparison with experiments favours a MLS-model

Diffusion and transport in the human interphase cell nucleus - FCS experiments compared to simulations.

Despite the succesful linear sequencing of the human genome the three-dimensional arrangement of chromatin, functional, and structural components is still largely unknown. Molecular transport and diffusion are important for processes like gene regulation, replication, or repair and are vitally influenced by the structure. With a comparison between fluorescence correlation spectroscopy (FCS) experiments and simulations we show here an interdisciplinary approach for the understanding of transport and diffusion properties in the human interphase cell nucleus. 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 fibre 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 fibre. 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. Fluorescence in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labelling 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-euclidean chromatin distribution of the nucleus. The dynamic behaviour 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. FCS in combination with a scanning device is a suitable tool to study the diffusion characteristics of fluorescent proteins in living cell nuclei with high spatial resolution. Computer simulations of the three-dimensional organization of the human interphase nucleus allows a detailed test of theoretical models in comparison to experiments. Diffusion and transport in the nucleus are most appropriately described with the concept of obstructed diffusion. A large volume fraction of the nucleus seems to contain a cytosol-like liquid with an apparent viscosity 5 times higher than in water. The geometry of particles and structure as well as their interactions influence the mobilities in terms of speed and spatial coverage. A considerable amount of genomic sites is accessible for not too large particles. FCS experiments and simulations based on the polymer model are in a good agreement. Using recently developed in vivo chromatin markers, a detailed study of mobility vs. structure is subject of current work

Three-dimensional organization of the human interphase nucleus

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 fibre 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 fibre. 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. Fluorescence in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labelling 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-euclidean chromatin distribution of the nucleus. The dynamic behaviour 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-modellike chromatin distribution. Simulations of fragment distributions based on double strand breakage after carbonion irradiation differ in different models. Here again a comparison with experiments favours a MLS-model

Three-dimensional organization of the human interphase nucleus.

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 fiber 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

Three-dimensional organization of the human interphase nucleus

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 fiber 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

Three-dimensional organization of the human interphase nucleus

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 fiber 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 agreemen

The three-dimensional organization of a self replicating nano fabrication site: The human cell nucleus.

The eukaryotic cell is a prime example of a functioning nano-machinery. The synthesis of proteins, maintenance of structure and duplication of the machinery itself are all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. Especially the regulation of genes has been shown to be connected closely to the organization of the genome in the nucleus. The nucleus of the cell has for a long time been viewed as a 'spaghetti soup' of DNA bound to various proteins without much internal structure, except during cell division when chromosomes are condensed into separate entities. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in the interphase, i.e. between cell divisions. In an analogy of the Bauhaus principle that "form follows function" we believe that analyzing in which form DNA is organized in these territories will help us to understand genomic function. We use computer models - Monte Carlo and Brownian dynamics simulations - to develop plausible proposals for the structure of the interphase genome and compare them to experimental data. In the work presented here, we simulate interphase chromosomes for different folding morphologies of the chromatin fiber which is organized into loops of 100 kbp to 3 Mbp that can be interconnected in various ways. The backbone of the fiber is described by a wormlike-chain polymer whose diameter and stiffness can be estimated from independent measurements. The implementation describes this polymer as a segmented chain with 3000 to 20000 segments for chromosome 15 depending on the phase of the simulation. The modeling is performed on a parallel computer (IBM SP2 with 80 nodes). Currently we determine genomic marker distributions within the Prader-Willi-Region on chromosome 15q11.2-13.3. For these measurements we use a fluorescence in situ hybridisation method (in collaboration with I. Solovai, J. Crai and T. Cremer, Munich, FRG) conserving the structure of the nucleus. As probes we use 10 kbp long lambda clones (Prof. B. Horsthemke, Essen, FRG) covering genomic marker distances between 8 kbp and 250 kbp. The markers are detected with confocal and standing wavefield light microscopes (in collaboration with J.Rauch, J. Bradl, C. Cremer and E.Stelzer, both Heidelberg, FRG) and using special image reconstruction methods developed solely for this purpose (developed by R. Eils. and W. Jaeger, Heidelberg, FRG). The work is part of the Heidelberg 3D Human Genome Study Group, which is part of the German Human Genome Project