Participants 




Invited Talks
Andy Andrews (Department of Biochemistry and Molecular Biology, Colorado State University)

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.



Thomas C. Bishop (Center for Computational Science, Tulane University )

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.



Peter R. Cook (The Sir William Dunn School of Pathology, Oxford University)

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.



Dieter W. Heermann (Institute for Theoretical Physics, Heidelberg University)

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.



Julien Mozziconacci (The Computer Laboratory, Cambridge University)

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.



John van Noort (Institute of Physics, Leiden University)

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.



Karsten Rippe (Research Group Genome Organization Function, BioQuant Center German Cancer Research Center (DKFZ))

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.




Andrew Routh (MRC Laboratory of Molecular Biology, Cambridge)

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.



Helmut Schiessel (Lorentz Institute for Theoretical Physics, Leiden University)

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).



Tamar Schlick (Dept. of Chemistry and Courant Inst. of Mathematical Sciences, New York University)

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



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.



Kenichi Yoshikawa (Department of Physics, Kyoto University)

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



Jordanka Zlatanova (Department of Molecular Biology, Wyoming University)

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.


Contributed Talks

Mithun Biswas (IWR, University of Heidelberg)

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.



Philipp Diesinger (Institut fr Theoretische Physik, University of Heidelberg)

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.



Yair Field (Weizmann Institute of Science, Israel)

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.



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.



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.



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.



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)



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)


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