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| Invited Talks
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Andy Andrews (Department
of Biochemistry and Molecular Biology, Colorado State University) |
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A
coupled equilibrium approach for measuring nucleosome thermodynamics |
Previously we have shown that
nucleosome thermodynamics cannot be studied by the standard approach of
either direct titration or by dilution (Thastrom et al., 2004). To
circumvent this problem, we have employed the histone chaperone yNap1
to prevent non-canonical histone-DNA interactions. yNap1 assembles and
disassembles nucleosomes in a thermodynamic manner (Mazurkiewicz et
al., 2006; Park and Luger, 2008), making yNap1 mediated nucleosome
formation reversible and amenable to thermodynamic studies. We have
developed both theoretical and experimental methods to exploit this
coupled equilibrium of histones between histone chaperones and the
nucleosome to measure nucleosome thermodynamics. This first look at
nucleosome thermodynamics is providing valuable insight into the role
of histone chaperones and the role of DNA in nucleosome stability.
References:
Mazurkiewicz, J., Kepert, J.F., and Rippe, K. (2006). On the mechanism
of nucleosome assembly by histone chaperone NAP1. The Journal of
biological chemistry 281, 16462-16472.
Park, Y.J., and Luger, K. (2008). Histone chaperones in nucleosome
eviction and histone exchange. Curr Opin Struct Biol.
Thastrom, A., Gottesfeld, J.M., Luger, K., and Widom, J. (2004).
Histone-DNA Binding Free Energy Cannot Be Measured in Dilution-Driven
Dissociation Experiments. Biochemistry 43, 736-741.
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Thomas C. Bishop
(Center for
Computational Science, Tulane University ) |
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Development
of a Nucleosome Energy Level Diagram for Chromatin Folding |
The structure of chromatin and
its interactions with proteins is
nontrivial. A conceptual model that captures essential features and
that allows for known complexities is needed. Taking the view that
nucleosomes are discrete entities we propose an energy level diagram
similar to an electron configuration diagram as a conceptual framework
for the analysis of nucleosome stability. The framework allows for the
sequence dependent nature of nucleosome stability as well as different
states of chemical modification and
histone association. Like an electron configuration diagram, a
nucleosome energy level diagram specifies the energetics
of individual nucleosomes. For lengths of DNA that may contain many
nucleosomes the diagram provides a simple footprint representation of
the organization of nucleosome arrays and chromatin. Molecular modeling
and graphics tools for creating and analyzing the corresponding 3D
structures allow for the investigation of non-local interactions in the
folding of chromatin. We investigate the organization of the mouse
mammary tumor virus promoter as a sample application.
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Peter R. Cook
(The Sir
William Dunn School of Pathology, Oxford University) |
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A model
for all genomes: the role of transcription factories |
A model for the 3D structure of
all genomes will be presented; it is based on the structure of the
bacterial nucleoid, where active RNA polymerases cluster into
"factories" to loop the intervening DNA. Essentially all transcription
takes place in factories where the local concentration of polymerases
is so much higher than in the soluble pool. Aspects of this
organization in mammalian cells will be described (a collaboration with
Davide Marenduzzo):
- An entropic "depletion attraction" can
drive looping.
- Monte Carlo simulations indicate how
proximity to a factory affects
the frequency that a promoter contacts a factory (and so initiates);
barriers, silencers, and enhancers - which encode active transcription
units and/or binding sites for transcription factors - regulate
activity by tethering target genes at relevant distances from
appropriate factories.
- Monte Carlo simulations (of strings of
beads confined in a sphere
representing chromosomes in the nucleus) also indicate that an
"entropic centrifuge" can resolve sometimes conflicting forces to
position whole chromosome territories: it can (i) position
stiff/compact polymers at the periphery (gene-poor
transcriptionally-inactive chromosomes are often peripheral), (ii)
force thick polymers to the periphery (heterochromatin tends to be
peripheral), (iii) create "territories" with the shape of oblate
ellipsoids from looped fibres (as seen experimentally), and (vi) drive
large terminal beads to the periphery (centromeric heterochromatin
often aggregates peripherally).
Reference:
Marenduzzo D, Faro-Trindade I, Cook PR (2007). What are the
molecular ties that maintain genomic loops? Trends Genet. 23, 126-133.
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Dieter W. Heermann
(Institute
for
Theoretical Physics, Heidelberg University) |
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The
Relation between the Gene Network, Gene Expression and the Physical
Structure of Chromosomes |
Remarkably little is known
about the
higher-order folding motifs of the chromatin fibre inside the cell
nucleus during interphase. Folding depends among others on local gene
density and transcriptional activity and plays an important role in
gene regulation. Strikingly, at fibre lengths above 5 to 10 Mb the
measured mean square distance R2 between any two points
on the
chromosome fibre is independent of genomic distance along the
chromosome while for lengths below 1Mb there is a strong dependence. I
propose a polymer model that can explain this levelling-off by means of
looping probabilities, i.e., interaction between genes. A detailed
investigation of this model as well as a model for the 30nm chromatin
fibre that I will present shows that loops on all scales are necessary
to explain the experimental data. Thus we can link the interaction
between genes in the gene regulatory and interaction network to the
physical structure as well as the gene expression opening new inroads
to our understanding.
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Julien Mozziconacci
(The Computer
Laboratory, Cambridge University) |
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Graphical
Modeling of the Beta-Globin Transcription Factory |
We gathered
DNA-transcription-related
structural data available in order to reconstitute a plausible 3D
representation of a eukaryotic transcription factory. We hypothesized
that this factory is located on the Beta-Globin locus control region
(LCR). Published biological data were used to guide us in our modeling.
The resulting 3D model of the whole human Beta-Globin gene cluster
leads us to propose a scenario for gene expression switching during
development.
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John van Noort
(Institute of
Physics, Leiden University) |
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Pulling
on Single Chromatin Fibers |
The compaction of eukaryotic DNA into
chromatin has
been implicated in the regulation of all cellular processes whose
substrate is DNA. To understand this regulation, it is essential to
reveal the structure and mechanism by which chromatin fibers fold and
unfold. I will discuss how we used magnetic tweezers to probe the
mechanical properties of chromatin fibers consisting of a single,
well-defined array of 25 nucleosomes and compare the results with data
obtained when pulling on a single nucleosome. It appears that
neighboring nucleosomes stabilize DNA folding into a nucleosome. When
an array of nucleosomes is folded into a 30 nm fiber, representing the
first level of chromatin condensation, the fiber stretched like a
Hookian spring at forces up to 4 pN. Together with a
nucleosome-nucleosome stacking energy of 14 kT, four times larger than
previously reported, this points to a solenoid as the underlying
topology of the 30 nm fiber. Surprisingly, linker histones do not
affect the length or stiffness of the fibers, but stabilize fiber
folding up to forces of 7 pN. Fibers with a nucleosome repeat length of
167 bp instead of 197 bp are significantly stiffer, consistent with a
two-start helical arrangement. The extensive thermal breathing of the
chromatin fiber that is a consequence of the observed high compliance
provides a structural basis for understanding the balance between
chromatin condensation and transparency for DNA transactions.
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Karsten Rippe
(Research
Group Genome Organization Function, BioQuant Center
German Cancer Research Center (DKFZ)) |
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Controlling
DNA accessibility via the local nucleosome geometry and
internucleosomal interactions |
The folding of the nucleosome
chain
into a chromatin fiber is an important aspect in the regulation of DNA
accessibility. Based on model structures with atomic resolution a new
coarse-grained model for the nucleosome geometry was implemented. The
dependence of the chromatin
fiber conformation on the spatial orientation of nucleosomes and the
path and length of the linker DNA were systematically explored by Monte
Carlo simulations. Two fiber types were analyzed in detail that
represent nucleosome chains without and with linker histones,
respectively: two-start helices with crossed-linker DNA (CL
conformation) and interdigitated one-start helices (ID conformation)
with different nucleosome tilt angles. The CL conformation was derived
from a tetranucleosome crystal structure that was extended into a
fiber. At thermal equilibrium the fiber shape persisted but relaxed
into a structure with a somewhat lower linear mass density of 3.1
0.1
nucleosomes/11 nm fiber while a much higher mass density of up to 7.9
0.2 nucleosomes/11 nm fiber was obtained for the ID fibers. A model is
proposed in which the transition between a CL and ID fiber is mediated
by relatively small changes of the local nucleosome geometry.
Furthermore, simulations of chromatin fiber stretching experiments will
be presented to investigate the effect of the fiber geometry, the
nucleosome repeat length (NRL), and the interaction strength between
neighboring nucleosomes.
References:
Stehr, R., Kepper, N., Rippe, K. and Wedemann, G. (2008). The effect of
the internucleosomal interaction potential on the folding of the
chromatin fiber. Biophys. J. 95, 3677-3691.
Kepper, N., Foethke, D., Stehr, R., Wedemann, G. and Rippe, K. (2008).
Nucleosome geometry and internucleosomal interactions control the
chromatin fiber conformation, Biophys. J. 95, 3692-3705.
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Andrew Routh
(MRC
Laboratory of Molecular Biology, Cambridge) |
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Chromatin
higher-order structure |
During the past decade it has
emerged that the packaging of eukaryotic DNA by histones into chromatin
is a key regulator of transcription, replication, recombination, and
repair. An altered pattern of epigenetic modifications, such as
post-translational modification of histone proteins, is central to many
common human diseases including cancer. Very little is understood about
the mechanisms by which these modifications regulate chromatin
condensation. Such an understanding is dependent on knowledge of the
structure and dynamics of chromatin. I will describe our studies on the
higher orders structure of chromatin with two primary aims:
1) Biophysical characterization of the effects of the linker histone on
the compaction and stability of chromatin higher order structure.
2) Determination of the structure of the "30nm" chromatin fibre by
cryo-electron tomography and single particle electron microscopy.
References:
(1) Routh A, Sandin S, Rhodes D., Nucleosome repeat length and linker
histone stoichiometry determine
chromatin fiber structure. Proc Natl Acad Sci USA. 2008 Jul
1;105(26):8872-7.
(2) Robinson PJ, An W, Routh A, Martino F, Chapman L, Roeder RG, Rhodes
D., 30 nm chromatin fibre decompaction requires both H4-K16 acetylation
and
linker histone eviction. J Mol Biol. 2008 Sep 12;381(4):816-25.
(3) Robinson PJ, Rhodes D., Structure of the "30 nm" chromatin fibre: a
key role for the linker
histone. Curr Opin Struct Biol. 2006 Jun;16(3):336-43.
(4) Robinson PJ, Fairall L, Huynh VA, Rhodes D., EM measurements define
the dimensions of the "30-nm" chromatin fiber:
evidence for a compact, interdigitated structure. Proc Natl Acad Sci
USA. 2006 Apr 25;103(17):6506-11.
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Helmut Schiessel
(Lorentz
Institute for Theoretical Physics, Leiden University) |
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Structure
of chromatin on larger scales |
l discuss the structure of
chromatin on two
length scales: that of the chromatin fiber and that of a whole
interphase chromosome. For chromatin fibers I argue that possible
structures can be predicted from the dense packing of the wedge-shaped
nucleosomes -- in a similar fashion as the DNA double helices can be
predicted from the stacking of the propeller-twisted basepairs. This
leads to the prediction of a discrete set of possible fiber diameters,
three of which have already been observed experimentally. For the
structure of a whole interphase chromosome I argue that current data
from FISH experiments are compatible with poor solvent polymer
statistics over the whole range of genomic distances (ideal chain
statistics for short distances, plateau for large distances).
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Tamar Schlick
(Dept. of
Chemistry and Courant Inst. of Mathematical Sciences, New York
University) |
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Mesoscale
modeling of chromatin organization |
Recent work on mesoscale
modeling of chromatin will be descried, with emphasis on structures in
the presence of linker histones and divalent ions. A surprising new
polymorphic fiber structure arises.
Keywords: chromatin structure, zigzag, solenoid, linker histones,
divalent ions, modeling and simulation
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Paul Wiggins
(Whitehead
Institute for Biomedical Research, Cambridge MA, USA) |
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Chromatin
Organization in E.coli |
Prokaryotic organisms must
strike a balance between DNA accessibility and condensation:
facilitating the genetic processes of transcription, replication, and
DNA repair while simultaneously enabling the structural and physical
processes of chromosome condensation and segregation in rapidly
dividing cells. Recent studies have revealed that prokaryotic
chromosomes are intricately structured but the mechanism for this
physical organization is not yet understood. In this study, we describe
the construction of a large number of E.coli strains carrying three
spectrally distinct, fluorescently labeled genetic loci. The origin and
terminus of replication as well as a strain-specific locus are labeled
and tracked simultaneously in live cells. The positions of the labeled
loci are determined with respect to the cell body and used to compute
both the distribution of locations of single loci within the cell (thus
revealing "chromosome territories") as well as the correlations between
fluctuations in the positioning of different loci. This data is
analyzed in the context of a simple polymer model of the E.coli
chromosome and it provides quantitative support for a nucleoid-centered
mechanism of chromosome organization.
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Kenichi Yoshikawa
(Department
of Physics, Kyoto University) |
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Hierarchical
Dynamics of Chromatin: Physical Aspects |
We will discuss the structural
dynamics of chromatin, together with the conformational characteristics
of giant DNA molecule.
1) Large discrete transition of giant DNA molecule:
A polymer chain exhibits plural number of length-scales, such as
contour length L, persistence length l, and width d. When L
l d, we call the chain as
semi-flexible. In usual genomic DNA molecules, L ~ cm, whereas double
stranded DNA structure is characterized as l = ca. 50 nm, and d = ca.
2nm. It is obvious that genomic DNA molecules are classified as
semi-flexible chain. A semi-flexible chain exhibits unique properties.
i) A single chain undergoes large discrete transition between elongated
coil and folded compact states. ii) The transition is classified as a
first-order phase transition under the criterion of Landau. iii) The
compact state exhibits poly-morphology, rich variety of steric
structures are found such as toroid, rod, spool-like, pearling.
2) Nucleosome structure:
We explore the origin of the chiral selection, left-handed wrapping,
from the viewpoint of the intrinsic elasticity of DNA. We adapt the
approximation of worm-like chain as a model of DNA. We adapt a model by
taking into account of the coupling of twisting with stretching and
also bending. We will present dynamical evidence that the asymmetric
coupling between bending and twisting of DNA gives rise to the
selection of the direction of wrapping by using the Langevin dynamics
at the coarse-grained level. We have also performed systematic study to
make a model of chromatin by used of artificial cationic nano-partices
with different diameters. It has become clear that DNA wrapped around a
nano-particle in a regular manner only when its diameter exhibits
proper size.
3) Reconstituted Chromatin:
We will report the experimental study on the reconstruction of
chromatin from giant DNA molecules, by focusing the individual role of
core histone, linker histone, and topoII. It is found that chromatin
exhibits discrete transition between dispersed and condensed states. It
is also shown that chromatin fibers reconstituted in vitro from core
histones and linker histone H1 became thinner (30 nm to 20 nm in width)
upon acetylation. In the AFM images, the gyration radius of the
nucleosomal fiber became larger after acetylation, distribution in
nucleosomes.
References:
(1) K. Yoshikawa, M. Takahashi, V. V. Vasilevskaya, and A. R.
Khokhlov,Large discrete transition in a single DNA molecule
appears continuous
in the ensemble, Phys. Rev. Lett. 76, 3029-3031(1996).
(2) K. Yoshikawa, and Y. Yoshikawa,Compaction and
condensation of
DNA, in "Pharmaceutical Perspectives of Nucleic Acid-Based
Therapeutics", eds., R. I. Mahato et. al. (2002), pp. 137-163.
(3) T. Yanao, and K. Yoshikawa,Elastic origin of chiral
selection in
DNA wrapping, Phys. Rev., E 77, 021904 (2008).
(4) A. A. Zinchenko, K. Yoshikawa, and D. Baigl,Compaction of
Single-Chain DNA by Histone-Inspired Nanoparticles, Phys. Rev. Lett.,
95, 2281011 (2005).
(5) A. A. Zinchenko, T. Sakaue, S. Araki, K. Yoshikawa, and D. Baigl,
D.,Single-Chain Compaction of Long Duplex DNA by Cationic
Nanoparticles: Modes of Interaction and Comparison with Chromatin,
Phys. Chem. B, 111, 3019 (2007).
(6) K. Yoshikawa,Field Hypothesis on the Self-regulation of
Gene
Expression, J. Bio. Phys., 28, 701 (2002).
(7) Y. Takenaka, H. Nagahara, H. Kitahata, and K.
Yoshikawa,Large-scale on-off switching of genetic activity
mediated by the
folding-unfolding transition in a giant DNA molecule: An hypothesis,
Phys. Rev., E 77, 031905 (2008).
(8) K. Hizume, S. Araki, K. Yoshikwa, and T.
Takeyasu,Topoisomerase
II, a scaffold component, promotes chromatin-compaction in vitro in a
linker-histone H1-dependent manner, Nucleic Acids Research 35, 2787
(2007).
Key Words: On/off switching of the higher-order structure, giant DNA,
nucleosome, reconstituted chromatin, folding transition
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Jordanka Zlatanova
(Department
of Molecular Biology, Wyoming University) |
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Nucleosome
dynamics: single-molecule studies |
We have recently argued that
the nucleosome can no longer be viewed as a single static entity:
rather, it comprises a family of particles differing in their
structural and dynamic properties, leading to different functionalities
(The Nucleosome Family: Dynamic and Growing, J. Zlatanova, T.C. Bishop,
J.-M. Victor, V. Jackson K. van Holde, Structure, in press).
The classical nucleosome that contains one H3/H4 tetramer and two
H2A/H2B dimers exhibits steady-state variability of the length of the
DNA wrapped around the histone octamer, accommodating anywhere from
~100 to 170 bp of DNA. In addition, the particle possesses intrinsic
conformational dynamics, i.e. nucleosomal DNA can partially uncoil from
the histone core in motions that we term "opening" and "breathing". We
will present our results on nucleosomal opening obtained by single-pair
FRET, and will discuss the possible physiological functions of such
motions.
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| Contributed Talks
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Mithun Biswas
(IWR,
University of Heidelberg) |
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Loop
formation in nucleosome with a linear elasticity model |
DNA loop mediated nucleosome
repositioning is supposed to be a dominant mechanism for regulating
access to genomic DNA. The theory of loop formation with DNA modeled as
a worm like chain (WLC) has been discussed by Kuli? et. al[1]. However,
it is expected that DNA, in the length scale of tens of base pairs, is
softer than the DNA modelled as WLC with a persistence length of ~50
nm. Here I have chosen a local elasticity model for DNA proposed by
Wiggins et. al.[2]
(called sub-elastic chain) which offers a softer bending potential for
large deflections and compared the results with WLC model. It is found
that the minimum energy of loop formation obtained from the sub-elastic
chain model is less than that calculated from WLC for short DNA
segments but becomes higher for long DNA segments. I also discuss
nucleosome repositioning dynamics mediated by small DNA loops.
References:
1.Kuli? et. al. 2003, Nucleosome repositioning via loop
formation.Biophys J., 84(5):3197-211.
2.Wiggins et. al. 2006, High flexibility of DNA on short length scales
probed by atomic force microscopy. Nat Nanotechnol., 1(2):100-1.
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Philipp Diesinger
(Institut fr
Theoretische Physik, University of Heidelberg) |
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The
Effects of Nucleosome and Linker Histone Depletion on Properties of
Chromatin: A Monte Carlo Model |
A Monte Carlo model for genome
folding at the 30nm scale with focus on depletion effects will be
presented. Depletion of linker histones and nucleosomes affects
massively the flexibility and the extension of chromatin fibers.
Increasing the amount of nucleosome skips can either lead to a collapse
or to swelling of chromatin fibers. These oppositional effects will be
discussed. Moreover, the role of random chromatin contacts is an
important issue for the interpretation of 3C data sets. Therefore,
their strength was investigated in the framework of the Monte Carlo
model and will be discussed in this talk. Finally the random contacts
will be compared with interaction data from actual Capturing Chromatin
Conformation experiments.
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Yair Field
(Weizmann
Institute of Science, Israel) |
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Distinct
Modes of Regulation by Chromatin Encoded through Nucleosome Positioning
Signals |
The detailed positions of
nucleosomes profoundly impact gene regulation and are partly encoded by
the genomic DNA sequence. However, less is known about the functional
consequences of this encoding. We first address this question using a
genome-wide map of nucleosomes in the yeast S. cerevisiae that we
sequenced in their entirety. Utilizing the high resolution of our map,
we refine our understanding of how nucleosome organizations are encoded
by the DNA sequence, and demonstrate that the genomic sequence is
highly predictive of the in vivo nucleosome organization, even across
new nucleosome-bound sequences that we isolated from fly and human. We
find that Poly(dA:dT) tracts are an important component of these
nucleosome positioning signals, and that their nucleosome-disfavoring
action results in large nucleosome-depletion over them and over their
flanking regions, and enhances the accessibility of transcription
factors to their cognate sites. These results suggest that the yeast
genome may utilize these nucleosome positioning signals to regulate
gene expression with different transcriptional noise and activation
kinetics, and DNA replication with different origin efficiency. These
distinct functions may be achieved by encoding both relatively closed
(nucleosome-covered) chromatin organizations over some factor binding
sites, where factors must compete with nucleosomes for DNA access, and
relatively open (nucleosome-depleted) organizations over other factor
sites, where factors bind without competition. In further work we have
investigated the DNA-encoded nucleosome organization of promoters in
the two related yeast species S. cerevisiae and C. albicans. For that
we have measured in-vivo nucleosome positions in both species, and
further have measured the in-vitro nucleosome positions of purified
histone octamers assembled on purified genomic DNA from both species.
The latter is thus the direct measurement of the DNA sequence
contribution to the nucleosome positioning and is independent of
transcription and replication states, and of the action and binding of
chromatin remodelers and transcription factors. We first show that most
changes in the nucleosome organization of promoters between these
species (measured in-vivo) are attributed to changes in the DNA
sequence (measured in-vitro and predicted by our model). We then show a
global relationship between transcriptional programs of genes (based on
microarray expression profiles of genes along different conditions and
cellular states) and the DNA-encoded nucleosome organizations of their
promoters that is remarkably conserved across these yeast species, even
in the presence of expression divergence. Growth related genes that are
by 'default' on, tend to have the open DNA-encoded nucleosome
organization for their promoters, which presumably facilitates for them
a default accessible promoter state. Inducible genes (condition or
cellular state specific genes) that are by default off tend to have the
closed DNA-encoded nucleosome organization, which presumably
facilitates for them a default inaccessible promoter state. In summary,
in these work we report on progress in understanding the way in which
nucleosome organization is encoded in the DNA, and in identifying
functional consequences of the DNA-encoded nucleosome organization in
both replication and transcription regulation.
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Evgeny Gladilin
(DKFZ
Heidelberg) |
|
Contactless
investigation of nuclear mechanics using a 3D image- and model-based
framework |
Mechanical properties of the
cell nucleus are of general interest for epigenetics and medicine. Many
severe diseases that are manifested and diagnosed on the basis of
pathologically altered nuclear shape are directly linked to abnormal
mechanical properties of the nuclear matter. Canonic material constants
such as stiffness and compressibility play an important role in
characterization of mechanical properties of cellular matter.
Experimental determination of material properties of intracellular
structures, such as the cell nucleus, is a nontrivial task. Most
micromanipulation techniques of experimental cell mechanics are based
on application of controlled forces onto the cellular boundary.
Consequently, these methods provide information about the overall cell
properties (e.g. stiffness of the whole cell) or those of its part
which is directly accessible by the measurement (e.g. cell membrane).
In vivo probing intracellular structures, which are not directly
accessible for a measurement, is not possible with conventional
micromanipulation methods. Meanwhile, modern microscopic imaging
techniques, such as confocal laser scanning microscopy (CLSM), yield
highly resolved 3D images of intracellular structures. Time series of
3D CLSM images depicting successive cell deformation under the impact
of external forces provide valuable insights into mechanical behavior
of intracellular matter on the local scale and can serve for estimation
of material parameters of constitutive models. In this work, we present
an approach to contactless investigation of nuclear mechanics by means
of 3D image- and model-based analysis of drug-induced cell deformation.
In particular, we focus on (i) comparative analysis of 3D structural
response of the nuclear matter with respect to external forces in
normal and Lamin mutant cells, as well as (ii) determination of the
scarcely-investigated nuclear compressibility.
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Thomas Hfer
(DKFZ and
BioQuant Center, Heidelberg) |
|
Stochasticity
and specificity in DNA repair: a dynamic model for chromatin-associated
regulatory processes |
The processes that control the
functioning of our genome transcription, replication, chromatin
organization and repair are carried out by multi-component molecular
machines. Little is known on how such macromolecular complexes form and
act in living cells. We have quantitated and modelled the in-vivo
dynamics of a mammalian DNA repair process, nucleotide excision repair
(NER), that removes UV-induced lesions. Although NER requires tens of
minutes up to a few hours, all its individual components were observed
to exchange much more rapidly (within tens of seconds) between
DNA-bound and free states. To rationalize this behaviour, we have
constructed and parameterized a comprehensive kinetic model. It
distinguishes reversible protein binding events to DNA lesions, most of
them occurring stochastically, and energy-driven unidirectional
reactions of DNA unwinding, damage excision and repair synthesis. The
fitting of the model to a large set of fluorescence-imaging data yields
a biophysically realistic set of kinetic parameters (on- and off-rate
constants and enzymatic rate constants). I will discuss the
implications of this model for the interpretation of the experimental
data and the testing of its predictions. At first sight, stochastic
protein-complex assembly may seem an uneconomical solution to put
together the NER machinery. However, our analysis suggests that the
kinetic design of NER realizes a trade-off between conflicting demands
of high specificity in recognizing DNA lesions and rapid repair.
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Jrg Langowski
(Deutsches
Krebsforschungszentrum (DKFZ), Heidelberg) |
|
Coarse-grained
modelling of nucleosome dynamics |
A coarse-grained simulation
model for the nucleosome is developed, where protein residues and DNA
nucleotides are represented as single beads, interacting through
harmonic (for neighboring) or Morse (for nonbonded) potentials.
Force-?eld parameters were estimated by Boltzmann inversion of the
corresponding radial distribution functions obtained from all-atom
molecular dynamics (MD) simulation. This self-consistent multiscale
approach yields a coarse-grained model that is capable of reproducing
equilibrium structural properties calculated from all-atom MD
simulations. It speeds up nucleosome simulations by a factor of 10^3
and can therefore be used to examine biologically relevant dynamical
nucleosome phenomena on the microsecond timescale and beyond. Examples
given will be the partial dissociation of the linker DNA and the effect
of histone tail acetylation.
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Yaakov (Koby) Levy
(Weizmann
Institute of Science, Israel) |
|
Protein
sliding along DNA: Dynamics and structural characterization |
Efficient search of DNA by
proteins is fundamental to the control of cellular regulatory
processes. It is currently believed that protein sliding, hopping, and
transfer between adjacent DNA segments, during which the protein
non-specifically interacts with the DNA, are central to the speed of
their specific recognition. In this study, we focused on the structural
and dynamic features of proteins when they scan the DNA. Using a simple
computational model that represents the protein-DNA interactions by
electrostatic forces, we identified that the protein makes use of the
identical binding interface for both nonspecific and specific DNA
interactions. Accordingly, in its 1D diffusion along the DNA, the
protein is bound at the major groove and performs a helical motion,
which is stochastic and driven by thermal diffusion. The microscopic
structural insight into sliding from our model, which is governed by
electrostatic forces, corroborates previous experimental studies which
suggested that the active site of some regulatory proteins do
continually face the interior of the DNA groove while sliding along the
sugar-phosphate rails. The diffusion coefficient of the spiral motion
along the major groove of the DNA is not affected by salt
concentration, but the efficiency of the search can be significantly
enhanced by increasing salt concentration due to a larger number of
hopping events. We found that the most efficient search comprises ~20%
sliding along the DNA and ~80% hopping and 3D diffusion. The presented
model that captures various experimental features of facilitated
diffusion has the potency to address other questions regarding the
nature of DNA search such as the sliding characteristics of oligomeric
and multi-domain DNA-binding proteins that are ubiquitous in the cell.
Givaty O and Levy Y, J. Mol. Biol. (In press)
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Lars Nordenskild
(Nanyang
Technological University, School of Biological Sciences, Singapore) |
|
Counterion
Induced Electrostatic Condensation and Self-Assembly of Nucleosome Core
Particles (NCP) and Chromatin Arrays |
Lars Nordenskild*,
Nikolay Korolev*, Alexander P. Lyubartsev#, Abdollah Allahverdi*, Ye
Yang*, Chenning Lu*, Ying Liu* and Nikolay Berezhnoy*
*School of Biological Sciences, Nanyang
Technological University, 60 Nanyang Drive, Singapore 637551,
#Division of Physical Chemistry, Arrhenius Laboratory, Stockholm
University, 106 91 Stockholm, Sweden
Analytical ultracentrifugation,
dynamic light scattering and
precipitation assay measurements of cation induced compaction of
recombinant 12-mer chromatin arrays are presented. Furthermore,
counterion induced aggregation of such arrays as well as of recombinant
mononucleosomes (177 bp) and 147 bp nucleosome core particles (NCP)
were investigated. Effects of the N-terminal tails have also been
studied. The potency of inducing compaction or aggregation, in all
three systems follow the order: spermine4+ Co(NH3)63+
spermidine3+ Mg2+ Ca2+ Na+ K+. This trend is the
same as that observed for the condensation behaviour (compaction as
well as aggregation) of DNA, which indicates similarity in the
underlying electrostatic mechanism causing condensation in DNA and in
chromatin.
MD (Langevin) computer simulations in continuum description of
coarse-grained chromatin and NCP models have been performed. The 12-mer
chromatin array is described in a model with a central spherical
particle with DNA in the form of connected charged beads wrapped around
this central histone octamer. Protruding out from the core are 8
positively charged flexible histone tails. Additional DNA beads
modelling the linker DNA, connects such core particles to form an
array. Explicit mobile counterions of charge and size mimicking Cl-,
K+, Mg2+, and Co(NH3)63+ are included to describe the effects of
counterion valence and results are compared with calculations using
salt represented by a DH screening potential. The latter description is
found to be inadequate for multivalent ions. The simulation results
with explicit ions agree with experimental trends and are in accordance
with polyelectrolyte theory and a mechanism of condensation due to salt
screening, attractive ion-ion correlations and histone tail bridging.
|
|
Georgi V. Pachov
(EML
Research, Heidelberg ) |
|
DNA
flexibility does not affect linker histone protein binding mode |
In the cell nucleus, DNA wraps
around histone proteins, forming nucleosome particles, and packs into a
highly negatively charged structure, the chromatin fiber. The linker
histone is a protein that binds to the nucleosome and determines how
the nucleosomes are linked to each other. To simulate the
nucleosome-linker histone interactions, we applied a Brownian Dynamics
(BD) technique together with normal mode analysis (NMA). NMA of the
nucleosome revealed the most prominent modes of motion of its two
linker DNAs. The results were used to generate conformations of the
linker DNAs which were used in BD simulations of the rigid-body docking
of a linker histone and its mutants to the nucleosome. From the
simulations, two distinct binding sites and one non-binding site on the
linker histone were identified. The residues found to be most important
for binding in the simulations with the linker histone mutants are
consistent with experimental data. Moreover, a unique binding mode of
the linker histone to the nucleosome was found for a wide range of
conformations of the linker DNAs. As well as providing insights into
the determinants of linker histone-nucleosome binding, the results are
valuable for higher order modelling of the chromatin fiber.
|
|
Ren Stehr
(University
of Applied Sciences Stralsund, Germany) |
|
Dynamic
numerical phase diagrams of chromatin |
Ren Stehr 1, Robert Schpflin
1, Ramona Ettig 2, Nick Kepper 2, Karsten Rippe 2 and Gero Wedemann 1
1 University of Applied Sciences Stralsund, System Engineering and
Information Management, Zur Schwedenschanze 15, 18435 Stralsund,
Germany
2 Deutsches Krebsforschungszentrum and BioQuant, Research Group Genome
Organization Function, Im Neuenheimer Feld 280, 69120
Heidelberg,
Germany.
In several studies, the
three-dimensional structure of chromatin has
been investigated by two-dimensional phase diagrams for the frequently
used two-angle model that describes the chromatin fiber conformation by
a torsion angle between nuclesomes and a DNA entry-exit angle at the
nucleosome. In these diagrams, only the fiber geometry is considered
and sterical possible or impossible conformations can be identified.
Here we introduce a new form of numerical phase diagrams. Basing upon
our previously developed 6-angle computer model of chromatin we
extended the pure geometric phase space by including the energy
associated with a given chromatin conformation to evaluate the
probability of its occurrence. This analysis was applied for different
chromatin models as recently derived from native and reconstituted
chromatin. The resulting energy phase diagrams exhibited crucial
differences even between different sterically possible conformations
indicating more and less favourable structures. Furthermore, we
extended the phase diagrams by applying Monte Carlo simulations and
observed changes of the phase diagrams due to transitions to lower
energy states and entropic effects. These dynamic numerical phase
diagrams demonstrate that geometrical impossible conformations can
transform into stable states due to DNA bending and torsion. Based on
this framework, new models for the chromatin fiber and mechanisms for
the control of the chromatin structure are proposed.
|
|
Vladimir Teif
(German
Cancer Research Center, Heidelberg) |
|
Predicting
nucleosome positions on the DNA: combining intrinsic affinities and
remodeler activities |
Vladimir B. Teif 1,2 and
Karsten Rippe 1
1 Research Group Genome Organization Function, Deutsches
Krebsforschungszentrum and BioQuant, Im Neuenheimer Feld 280, 69120
Heidelberg, Germany,
2 Institute of Bioorganic Chemistry, Belarus National Academy of
Sciences, Kuprevich 5/2, 220141, Minsk, Belarus.
The positions of nucleosomes
on the DNA are determined by the intrinsic affinities of the histone
octamer to a given DNA sequence and by the ATP-dependent activity of
nucleosome translocation complexes referred to as chromatin remodelers.
Here, we report a statistical-mechanical approach to take into account
both contributions. Three main remodeler activities were revealed in
the calculations: (i) the enrichment of nucleosomes at certain DNA
sites, (ii) the removal of nucleosomes from preferred binding sites,
and (iii) the establishment of a regular spacing between strongly
positioned nucleosomes. These predicted actions of amplifier, remover
and spacer activities were used to reveal mechanisms for nucleosome
redistributions observed in genome-wide experimental data for the
resting and activated human T-cells. It appeared that in vivo
nucleosome distributions are much less periodic then one would expect
from the DNA sequence preferences. Upon T-cell activation, the
remodeler activity increases, leading to the eviction and shifts of
individual nucleosomes by 10 to 80 bp, the shift length being a
multiple of 10. This remodeler action makes the nucleosome positioning
pattern in the activated cells slightly more periodic in comparison
with the resting ones. Our study of the nucleosome dynamics using
developed models suggests that in addition to nucleosome dislocation,
nucleosome eviction is an important factor to speed up nucleosome
repositioning during the cell cycle and differentiation.
|
|
Jean-Marc Victor
(CNRS, Paris) |
|
Magnetic
tweezers turn nucleosomes inside-out: in vitro modeling and in vivo
predictions |
Single chromatin fiber
manipulations allow to probe the mechanical properties and
conformational flexibility of a nucleosome. The most dramatic
conformational transition observed to date is unquestionably the one
observed with magnetic tweezers when extensive level of positive stress
is placed on the fiber. A careful analysis of the extension-rotation
curves reveals that the nucleosome undergoes a chiral transition into a
metastable right-handed structure called "reversome". In this talk I
will show how to model this transition with rigid body dynamics and 3D
animation tools. I will also show how to get an all atom structure of
the reversome. I will also discuss the in vivo relevance of the
reversome. This structure is expected to form under conditions in which
high levels of transcription-induced positive stress are present. Such
conditions are commonplace in vivo because Pol II exerts a positive
torque sufficient to trigger the transition. I will show that a
"reversome wave" might progress downstream an elongating Pol II at a
rate ~300 bp/s, destabilizing nucleosomes at a distance and thus
facilitating the progression of Pol II through the chromatin fiber
|
|
| Posters
|
|
|
Sumiko Araki
(Kyoto
University, Japan) |
|
Effect
of DNA architecture on the formation of nucleosome |
Nucleosome is a fundamental
unit in the folding process of eukaryote: DNA wraps around protein
named core histone 1.7 times and this complex is called nucleosome. The
structure of chromatin plays important roles in not only compaction of
DNA but also regulation of genetic function. On the other hand, DNA is
often found in circular form. In circular DNA, we recognize two
possible contributions to the stochastic mechanics of the formation of
nucleosomes: internal twisting rigidity and ring architecture. While
circular DNA is sometimes discussed in association with twisting
rigidity, little is known for the ring architecture itself on the
complexation of DNA with proteins. In this study, we focused on the
effect of ring architecture on the formation of nucleosomes and found
that the efficiency of nucleosome formation on ring DNA chain was
higher than that on linear chain.
|
|
Maria Barbi
(Laboratoire
de Physique Theorique de la Matiere Condensee, Paris, France) |
|
Modelling
the hysteretic behaviour of chromatin fibers under magnetic tweezers:
the reversome transition |
Magnetic tweezers
micromanipulations of chromatin fibers reveal unexpected mechanical
properties of these assemblies. After having studied the highly
resilient behaviour of fibers at low number of turns, we now address
the question of modelling the hysteretic behaviour observed at higher
torsion, which involves dramatic structural changes of the nucleosomes.
The experimental results are interpreted indeed by assuming that the
nucleosomes undergo a chiral transition into a metastable right-handed
structure called reversome. A combined approach involving 3D modelling,
geometrical analysis, mechanics, statistical physics and kinetic
processes allows to reproduce the observed behaviour and fit the
experimental curves.
|
|
Manfred Bohn
(Institute of
Theoretical Physics, Heidelberg University) |
|
A
heterogeneous Random Loop Model for Chromatin Folding |
Genome function in higher
eukaryotes involves major changes in the spatial organization of the
chromatin fiber. Nevertheless, our understanding of chromatin folding
is remarkably limited. Polymer models have been used to describe
chromatin folding. However, none of the proposed models gives a
satisfactory explanation of experimental data. In particularly, they
ignore that each chromosome occupies a confined space, i.e. the
chromosome territory. Here, we present a polymer model that is able to
describe key properties of chromatin over length scales ranging from
0.5 to 75 Mb. This random loop (RL) model assumes a random walk folding
of the polymer backbone and defines a probability P for two monomers to
interact, creating loops of a broad size range. On the 0.5 to 3 Mb
length scale chromatin compaction differs in different subchromosomal
domains. This aspect of chromatin structure is incorporated in the RL
model by introducing heterogeneity along the fiber contour length due
to different local looping probabilities. The RL model creates a
quantitative and predictive framework for the identification of nuclear
components that are responsible for chromatin-chromatin interactions
and determine the three-dimensional organization of the chromatin
fiber.
|
|
Marc Emanuel
(Lorentz
Institute for Theoretical Physics, Leiden University ) |
|
The
physics of large scale organization of chromatin |
We show that measurements of
the distribution of chromatin in the nuclei of eukaryotes available
today do not justify any detailed polymer model of chromatin, but can
be described as a confined semidilute solution of chromatin fiber. We
discuss the benefits of decoupling the mechanism behind the
condensation from the organization itself as a first order description.
|
|
Ramona Ettig
(DKFZ
Bioquant, Heidelberg ) |
|
Investigating
nucleosome mobility by Molecular Dynamics simulations |
Ramona Ettig 1, Rene Stehr 2,
Nick Kepper 1, Gero Wedemann 2 and Karsten Rippe 1
1 Deutsches Krebsforschungszentrum and BioQuant, Research Group Genome
Organization Function, Im Neuenheimer Feld 280, 69120
Heidelberg, Germany.
2 University of Applied Science Stralsund, System Engineering and
Information Management, Zur Schwedenschanze 15, 18435 Stralsund,
Germany.
The central building block of chromatin is the nucleosome. It comprises
an octamer core of two copies each of histones H2A, H2B, H3 and H4
around which the DNA is wrapped in 1.67 turns. The linker DNA between
nucleosomes is more accessible for DNA-binding proteins as compared to
the tightly wound intranucleosomal DNA. Accordingly, the positions of
the nucleosomes at promoter and enhancer regions have been shown to
directly affect gene regulation. Translocation of a nucleosome with
respect to the DNA sequence is therefore an important mechanism to
regulate interactions of protein factors with the genome. The access to
the DNA occluded by the nucleosome can be either due to spontaneous
thermal fluctuations (including a transient detachment of the DNA from
the histones) or the result of the activity of ATP-dependent remodeling
complexes. Chromatin remodeler appear to use a loop recapture model to
partial unwrap a small segment of 10 50 bp intranucleosomal DNA and
subsequently propagate this loop around the histone core [1]. Here, we
investigate possible mechanisms for forming a DNA loop and
translocating it with respect to the histone core. Molecular Dynamics
(MD) simulations in explicit water were performed for 100 ns for a
nucleosome with an inserted DNA loop of 40 bp. It was examined how the
interaction sites between the histone core and the nucleosomal DNA
adjacent to the loop break and (re)associate. The results allow first
estimates for the time required for translocations of a DNA loop around
the histone octamer protein core.
1. Strohner, R., Wachsmuth, M., Dachauer, K., Mazurkiewicz, J.,
Hochsttter, J., Rippe, K. Lngst, G. (2005). A 'loop
recapture' mechanism for ACF-dependent nucleosome remodeling. Nat.
Struct. Mol. Biol. 12, 683-690.
|
|
Nick Kepper
(Kirchhoff
Institut for Physics, Bioquant and DKFZ, Heidelberg) |
|
Force
spectroscopy of chromatin fibers: Extracting energetic and structural
information from Monte Carlo simulations |
Nick Kepper*, Ramona Ettig*,
Rene Stehr, Gero Wedemann and Karsten Rippe*
*
Deutsches Krebsforschungszentrum and BioQuant, Research Group
GenomeOrganization Function, Im Neuenheimer Feld 280, 69120
Heidelberg, Germany
University of Applied Science Stralsund, System Engineering and
Information Management, Zur Schwedenschanze 15, 18435 Stralsund,
Germany
Folding of the nucleosome chain into a chromatin fiber is an important
factor for the regulation of DNA accessibility. The interactions
between nucleosomes crucially affect the organization in the chromatin
fiber. However, details on the energetic parameters of this process are
still scarce. Force spectroscopy experiments of single chromatin fibers
are ideally suited to give an insight in the nucleosome organization in
the chromatin fiber. Here, we introduce a Monte Carlo program for the
simulation of chromatin fiber stretching experiments to investigate the
effect of the fiber geometry, the nucleosome repeat length (NRL), and
the interaction strength between neighboring nucleosomes. Since the
contribution of the different energetic terms like DNA bending,
torsion, electrostatics and nucleosome-nucleosome interactions can be
directly derived from the simulations their contribution to various
regimes of the force-distance curves was evaluated. From comparing the
simulation with experimental data sets the effective nucleosome
interaction energy for fibers without linker histones was estimated.
Furthermore, an additional potential was included in the simulations to
account for the unwrapping of the DNA from the histone octamer protein
core in the force regime from 5-15 pN.
|
|
Zbyszek Otwinowski
(UT Southwestern Medical Center at Dallas, USA) |
|
Topology of Eukaryotic Chromatin |
Chromatin structure undergoes many changes
during the cell cycle and in response to regulatory events. However,
very little is known about how nucleosomes are arranged into
higher-order structures in vivo, even though the efficiency and
precision of cell division imply high levels of structural
organization. I propose abandoning the current paradigm of chromatin
organization based on thermodynamics of the lowest energy state and
replace it with the idea of a topologically restrained, high-energy
structure. I propose that DNA is subject to a recursive topological
restraint, and is anchored by hemicatenates that are part of the
chromosomal scaffold. Long-distance cis-regulation of transcription is
a natural consequence of recursive topological restraint. This new
theory of chromatin structure has a multitude of consequences for key
aspects of cellular biology.
|
|
Robert Schpflin
(University
of Applied Siences Stralsund, Germany) |
|
Dynamic
numerical phase diagrams of chromatin |
Rene Stehr 1, Robert Schpflin
1, Ramona Ettig 2, Nick Kepper 2, Karsten Rippe 2 and Gero Wedemann 1
1 University of Applied Sciences Stralsund, System Engineering and
Information Management, Zur Schwedenschanze 15, 18435 Stralsund,
Germany
2 Deutsches Krebsforschungszentrum and BioQuant, Research Group Genome
Organization Function, Im Neuenheimer Feld 280, 69120
Heidelberg, Germany.
In several studies, the three-dimensional structure of chromatin has
been investigated by two-dimensional phase diagrams for the frequently
used two-angle model that describes the chromatin fiber conformation by
a torsion angle between nuclesomes and a DNA entry-exit angle at the
nucleosome. In these diagrams, only the fiber geometry is considered
and sterical possible or impossible conformations can be identified.
Here we introduce a new form of numerical phase diagrams. Basing upon
our previously developed 6-angle computer model of chromatin we
extended the pure geometric phase space by including the energy
associated with a given chromatin conformation to evaluate the
probability of its occurrence. This analysis was applied for different
chromatin models as recently derived from native and reconstituted
chromatin. The resulting energy phase diagrams exhibited crucial
differences even between different sterically possible conformations
indicating more and less favourable structures. Furthermore, we
extended the phase diagrams by applying Monte Carlo simulations and
observed changes of the phase diagrams due to transitions to lower
energy states and entropic effects. These dynamic numerical phase
diagrams demonstrate that geometrical impossible conformations can
transform into stable states due to DNA bending and torsion. Based on
this framework, new models for the chromatin fiber and mechanisms for
the control of the chromatin structure are proposed.
|
|
Ramasubramanian Sundaramoorthy
(University
of Dundee, Scotland) |
|
|
The Eukaryotic genomes are
organised into a
condensed structure termed chromatin. Chromatin consists of repeating
units called nucleosomes, made up of highly conserved four histone
proteins. Nucleosomes exhibit a high dynamism, understanding of which
may provide vital clues on fundamental biological processes. Although,
high resolution crystal structures of nucleosome have been solved, it
provides limited information on the dynamic nature since it renders
only a static picture of the molecule. We intended to study and
understand the dynamism of nucleosome using electron paramagnetic
resonance (EPR) spectroscopy. Using EPR spectroscopy the geometry of
the molecule can be obtained from the distance related magnetic dipolar
interaction energy between the unpaired electron spins. Recent advances
in EPR spectroscopic methods, especially usage of S-band high EPR
frequency (180-GHz electron Larmor frequency) PELDOR (pulsed
electron-electron double resonance) type experiment allow us to measure
the long distance range (8nm) between the two spin labels introduced in
strategic locations by site directed mutagenesis. By carefully
measuring the distance between the multiple pairs of spin labels
introduced at directed locations on nucleosome we can apply the
approach to study a range of chromatin related structures.
|
|
Annika Wedemeier
(German
Cancer Research Center, Heidelberg) |
|
Modeling
the diffusional transport in the interphase cell nucleus |
In recent years great progress
has been made in the view of the living cell as a regulatory network in
time. However, a quantitative description of the transport of
biomolecules in the dense macromolecular network of chromatin fibers in
the interphase cell nucleus is still missing. Furthermore, it is not
yet clear to what extent macromolecular mobility is affected by
structural components of the nucleus. This work contributes to the
understanding of this process by developing a theoretical description
of network diffusion in the interphase cell nucleus. To model the
situation in the cell nucleus a lattice approach is used minimizing
computational time and effort. Our model leads to a quantitative
understanding of transport behaviour which is directly related to
chromatin morphology. Changes of these characteristics are known to
occur upon apoptosis or malignant transformations. The crowded
environment of chromatin fibers in the nucleus is simulated by a
simplified version of the bond fluctuation method originally desrcibed
by Carmesin et al (Macromolecules 1988,21, p.2819) in combination with
a Metropolis Monte Carlo procedure. This yields well equilibrated
polymer chains satisfying static properties such as end-to-end
distance. It is investigated how the diffusion coefficient of particles
of a given size depends on the 3D geometry of the network of chromatin
fibers and their density in the nucleus. We show that the diffusion
cofficient is proportional to the volume fraction of the freely
accessible space. Additionally, we investigate to what extent
structural properties of the fibers, such as persistence length and
contour length, influence the diffusion coefficient. We observe that
neither the contour length nor the persistence length of the fibers
affects the diffusional transport of small particles.
|
|
Paul Wiggins
(Whitehead
Institute for Biomedical Research, Cambridge MA, USA) |
|
Chromatin
Organization in E.coli |
Prokaryotic organisms must
strike a balance between DNA accessibility and condensation:
facilitating the genetic processes of transcription, replication, and
DNA repair while simultaneously enabling the structural and physical
processes of chromosome condensation and segregation in rapidly
dividing cells. Recent studies have revealed that prokaryotic
chromosomes are intricately structured but the mechanism for this
physical organization is not yet understood. In this study, we describe
the construction of a large number of E.coli strains carrying three
spectrally distinct, fluorescently labeled genetic loci. The origin and
terminus of replication as well as a strain-specific locus are labeled
and tracked simultaneously in live cells. The positions of the labeled
loci are determined with respect to the cell body and used to compute
both the distribution of locations of single loci within the cell (thus
revealing "chromosome territories") as well as the correlations between
fluctuations in the positioning of different loci. This data is
analyzed in the context of a simple polymer model of the E.coli
chromosome and it provides quantitative support for a nucleoid-centered
mechanism of chromosome organization.
|
|
Tomasz Wocjan
(DKFZ,
Heidelberg) |
|
Brownian
dynamics simulation of DNA unrolling from the nucleosome |
Tomasz Wocjan, Konstantin
Klenin, Jrg Langowski
In eukaryotic cells DNA is compacted into chromatin. The basic packing
unit, the nucleosome, consists of a histone octamer around which ~ 147
bp of DNA are wrapped in 1 3/4 turns. The mechanism by which this
structure can be opened, giving access to DNA-processing enzymes is of
fundamental biological importance. Here we develop a model for the
attachement of DNA on the histone core and simulate the unwinding of
DNA from the nucleosome core particle by mechanical forces, a process
which has been analyzed experimentally [1,2]. We use a Brownian
dynamics simulation [3] with a coarse-grained model in which the linear
DNA is represented by a chain of linear segments interacting via
potentials for torsion, bending and stretching. A renormalized
Debye-Hckel potential includes electrostatic interaction between the
chain segments, while hydrodynamics is treated using the Rotne-Prager
tensor. The interaction between the negatively charged DNA and the
positively charged surface of the histone octamer with cylindrical
geometry is modeled by a short-ranged potential. The dynamics of the
nucleosome is studied by varying the force loading rate applied to the
DNA. This allows to analyze the transition from the conformation with
the DNA helically coiled around the histone core to the state of fully
extended DNA and to obtain information about the kinetics and energy
barriers of the transition.
[1] B.D. Brower-Toland, C.L. Smith, R.C. Yeh, J.T. Lis, C.L. Peterson,
M.D. Wang, PNAS 99, 4 (2002)
[2] L.H. Pope, M.L. Bennink, K.A. van Leijenhorst-Groener, D.Nikova,
J.Greve, J.F. Marko, Biophys. J. 88, 3572-3583 (2005)
[3] K.Klenin, H.Merlitz, J.Langowski, Biophys. J. 74, 780-788 (1998)
|
|
Mai Zahran
(IWR,
Heidelberg University) |
|
Sequence
specific DNA recognition |
EcoRV is a restriction enzyme
of type II produced by Eschrichia Coli. The function of EcoRV is to
destroy invading foreign DNA by cleaving it at a GATATC sequence. Upon
binding to DNA, EcoRV induces a kink of 50 degrees at the central base
pair (TA) of the recognition site. It is an important defense mechanism
against viral attacks, because E. Coli own's DNA is protected from
cleavage by methylation at the first adenine of the recognition
sequence. The EcoRV system provides a so far unique example among type
II restriction endonuclease of major protein-induced conformational
change of the DNA. We are interested in understanding the role of the
DNA bending on its interaction with EcoRV.
|
|
| Other Participants
|
|
| Gesa von Bornstaedt
(DKFZ,
Heidelberg) |
|
| Anneke Brmmer
(DKFZ,
Research Group Modeling of Biological Systems, Heidelberg) |
|
| Vlad Cojocaru
(EML
Research, Heidelberg) |
|
| Fabian Erdel
(DKFZ
BioQuant, Heidelberg) |
|
| Anna Feldman-Salit
(EML Research
Heidelberg) |
|
| Miriam Fritsche
(Institut for
Theoretical Physics, Heidelberg University) |
|
| Maria Hondele
(EMBL
Heidelberg, Gene Expression) |
|
| Erica Hong
(Harvard
Medical School, USA) |
|
| Benot Knecht
(Institut for
Theoretical Physics, Heidelberg University) |
|
| Michael Martinez
(EML
Research, Heidelberg) |
|
| Paolo Mereghetti
(EML
Research, Heidelberg) |
|
| Manohar Pilli
(EML
Research, Heidelberg) |
|
| Nima Hamedani Radja
(Lorentz
Institute, Leiden University, The Netherlands) |
|
| Eberhard Schmitt
(Kirchhoff-Institut
fr Physik, Heidelberg) |
|
| Matthias Stein
(EML
Research, Heidelberg) |
|
| Karine Voltz
(IWR, University of Heidelberg) |
|
| Rebecca Wade
(EML
Research, Heidelberg) |
|
|
