385 research outputs found
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
Escherichia coli low-copy-number plasmid R1 centromere parC forms a U-shaped complex with its binding protein ParR
The Escherichia coli low-copy-number plasmid R1 contains a segregation machinery composed of parC, ParR and parM. The R1 centromere-like site parC contains two separate sets of repeats. By atomic force microscopy (AFM) we show here that ParR molecules bind to each of the 5-fold repeated iterons separately with the intervening sequence unbound by ParR. The two ParR protein complexes on parC do not complex with each other. ParR binds with a stoichiometry of about one ParR dimer per each single iteron. The measured DNA fragment lengths agreed with B-form DNA and each of the two parC 5-fold interon DNA stretches adopts a linear path in its complex with ParR. However, the overall parC/ParR complex with both iteron repeats bound by ParR forms an overall U-shaped structure: the DNA folds back on itself nearly completely, including an angle of ∼150°. Analysing linear DNA fragments, we never observed dimerized ParR complexes on one parC DNA molecule (intramolecular) nor a dimerization between ParR complexes bound to two different parC DNA molecules (intermolecular). This bacterial segrosome is compared to other bacterial segregation complexes. We speculate that partition complexes might have a similar overall structural organization and, at least in part, common functional properties
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.
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
How proteins squeeze through polymer networks: a Cartesian lattice study
In this paper a lattice model for the diffusional transport of particles in
the interphase cell nucleus is proposed. The dynamic behaviour of single chains
on the lattice is investigated and Rouse scaling is verified. Dynamical dense
networks are created by a combined version of the bond fluctuation method and a
Metropolis Monte Carlo algorithm. Semidilute behaviour of the dense chain
networks is shown. By comparing diffusion of particles in a static and a
dynamical chain network, we demonstrate that chain diffusion does not alter the
diffusion process of small particles. However, we prove that a dynamical
network facilitates the transport of large particles. By weighting the mean
square displacement trajectories of particles in the static chain network data
from the dynamical network can be reconstructed. Additionally, it is shown that
subdiffusive behaviour of particles on short time scales results from trapping
processes in the crowded environment of the chain network. In the presented
model a protein with 30 nm diameter has an effective diffusion coefficient of
1.24E-11 m^2/s in a chromatin fiber network.Comment: submitted to J. Chem. Phy
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
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