57 research outputs found
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
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
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
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
Mapping eGFP Oligomer Mobility in Living Cell Nuclei
Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers
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