19 research outputs found
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
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
Trichostatin A induced histone acetylation causes decondensation of interphase chromatin.
The effect of trichostatin A (TSA)-induced histone
acetylation on the interphase chromatin structure was
visualized in vivo with a HeLa cell line stably expressing
histone H2A, which was fused to enhanced yellow
fluorescent protein. The globally increased histone
acetylation caused a reversible decondensation of dense
chromatin regions and led to a more homogeneous
distribution. These structural changes were quantified by
image correlation spectroscopy and by spatially resolved
scaling analysis. The image analysis revealed that a
chromatin reorganization on a length scale from 200 nm to
>1 mm was induced consistent with the opening of
condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be
assigned to the folding of chromatin during G1 phase by
characterizing the effect of TSA on cell cycle progression
and developing a protocol that allowed the identification of
G1 phase cells on microscope coverslips. An analysis by
flow cytometry showed that the addition of TSA led to a
significant arrest of cells in S phase and induced apoptosis.
The concentration dependence of both processes was
studied
In vivo characterization of protein-protein interactions in the AP1 system with fluorescence correlation spectroscopy (FCS).
The aim of these studies is the quantitative investigation of protein-protein interactions in the AP1 system in
vivo. First results of FCS measurements show an exchange in the nucleus of the proteins Fos-CFP and Jun-YFP
in the stably mono-transfected HeLa-Cells. This is also shown by fitting the bleaching curves measured in the
nucleus with an appropriate model. We obtained dissociation times between 10 and 20 seconds in the nucleus. In
the autocorrelation function a free and an obstructed component of diffusion are shown. For further studies
doubly transfected cells with both proteins, Fos-CFP and Jun-YFP, were prepared. These cells will now be
characterized with FCCS to investigate the protein-protein interactions. In order to obtain the dissociation rates
of the complex in the cell nucleus bleaching curves will be recorded on these cell lines. We also overexpressed
and purified Jun-YFP and Fos-CFP for in vitro studies
Aub and Ago3 Are Recruited to Nuage through Two Mechanisms to Form a Ping-Pong Complex Assembled by Krimper
In Drosophila, two Piwi proteins, Aubergine (Aub) and Argonaute-3 (Ago3), localize to perinuclear “nuage” granules and use guide piRNAs to target and destroy transposable element transcripts. We find that Aub and Ago3 are recruited to nuage by two different mechanisms. Aub requires a piRNA guide for nuage recruitment, indicating that its localization depends on recognition of RNA targets. Ago3 is recruited to nuage independently of a piRNA cargo and relies on interaction with Krimper, a stable component of nuage that is able to aggregate in the absence of other nuage proteins. We show that Krimper interacts directly with Aub and Ago3 to coordinate the assembly of the ping-pong piRNA processing (4P) complex. Symmetrical dimethylated arginines are required for Aub to interact with Krimper, but they are dispensable for Ago3 to bind Krimper. Our study reveals a multi-step process responsible for the assembly and function of nuage complexes in piRNA-guided transposon repression
The detailed 3D multi-loop aggregate/rosette chromatin architecture and functional dynamic organization of the human and mouse genomes
Background: The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function—the storage, expression, and replication of genetic information—is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture (T2C), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. Results: The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30–100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. Conclusions: This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow “architectural sequencing” at a genome mechanics level to understand the inseparable systems genomic properties