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Dimensional Organization of Chromosome Territories In View metadata, citation and similar papers at core.ac.uk brought to you by CORE Three - Dimensional Organization of Chromosome Territories in provided by Erasmus University Digital Repository the Human Interphase Cell Nucleus Human 3D Genome Study Group Tobias A. Knoch, Christian Münkel and Jörg Langowski 1) 2) 3) Joachim Rauch, Harald Bornfleth and Christoph Cremer with Irina Solovei and Thomas Cremer 1) Division Biophysics of Macromolecules, German Cancer Research Center, Im Neuenheimer Feld 280 D - 69120 Heidelberg, Federal Republic of Germany German Human 2) Instiute for Applied Physics, Albert Ueberle Str. 3-5, 3) Institute for Anthropology and Human Genetics, 69120 Heidelberg, FRG http://www.DKFZ-Heidelberg.de/Macromol/Welcome.html Richard Wagner Str. 10, 8033Munich, FRG Genome Project PURPOSE SIMULATIONS The eukaryotic cell is a prime example of a functioning nano With Monte Carlo and Brownian Dynamics methods Random Walk / Giant Loop model Multi-Loop-Subcompartment model machinery. The synthesis of proteins, maintenance of structure and we simulated various models (see sketch left) of human (RW/GL) (MLS) interphase chromosome 15 assuming a flexible polymer chain. To save computer power we start with duplication of the eukaryotic cell itself are all fine-tuned biochemical Sachs et al. (1995) Münkel et al. (1997) ~3,500 300nm=31kbp and later we relax with ~21,000 50nm=5,2 kbp long segments. For simulation of a single processes that depend on the precise structural arrangement of the chromosome it is placed in a potential well whose height R bands is related to the excluded volume interaction (EVI). The cellular components. The regulation of genes – their transcription G bands EVI keeps the chain from self crossing. Starting configurations have the approximate form and size of a and replication - has been shown to be connected closely to the loop size: 5Mbp metaphase chromosome (Fig. 1) from which the following decondensation into interphase resembles the natural three-dimensional organization of the genome in the cell nucleus. process. Despite the successful linear sequencing of the human genome its Fig. 1 three-dimensional structure is widely unknown. With the simulation of chromosomes and cell nuclei in comparison with fluorescence in situ hybridization we show here an approach loop size: 126kbp backbone leading to the detailed determination of the three-dimensional (non DNA) organization of the human genome: attachment rosette size: 1-2Mbp Rosettes in the Multi-Loop-Subcompartment model points (according to interphase correspond to the size of chromosomal interphase band Best agreement between simulations and experiment is reached for ideogram bands) domains. For simulation of a whole interphase nucleus 46 a Multi-Loop-Subcompartment model, thus the human genome Linker consists of DNA methaphase chromosomes are placed randomly in a chromatin fiber, 30nm thick nucleus confined by an EVI. The simulations are made shows a higher degree of determinism than previously thought. (no separate backbone needed) on two IBM SP2 parallel computers with 80 and 512 nodes. Fig. 4 Fig. 2 Fig. 3 Simulation of a human interphase cell Ray traced image of the Ray traced image of the Multi-Loop nucleus with all 46 chromosomes with Random-Walk/Giant-Loop model, loop Subcompartment model, loop size 1,200,000 polymer segments after 0.5s size 5Mbp, after ~80.000 Monte-Carlo 126kbp, linker size 126 kbp, after Brownian Dynamics simulation with 10s steps. and 1000 relaxing Brownian Dynamics ~50.000 Monte-Carlo and 1000 The MLS-model leads to the formation of steps. Large loops intermingle freely relaxing Brownian- Dynamics steps. distinct chromosome territories and thus forming no distinct features like in Here rosettes form subcompartments subcompartments. the MLS model. as separated organizational and dynamic entities. Typical textbook illustration of the human cell nucleus: 1) human cell nuclei differ from spherical shape, 2) the DNA is not a closed pipe, 3) nucleosomes are not regularly organized into chromatin, 4) chromatin does not float around randomly in the nucleus. V. Hennings (illustrator) in Molecular and Cellular Biology by Stephen L. Wolfe, 1993. EXPERIMENTS CONCLUSION Best agreement between simulations and experiments is reached for a Measurement of 3D-Distances between Genomic Markers Multi-Loop-Subcompartment model with a loop size of roughly126kbp and a linker length of 1,200nm. Supposed that defined loop bases exist it might be Prader-Willi Region (paternal chromosome) YAC 48 Fluorescence in situ hybridization (FISH) in connection with arm 1Mbp possible to determine the mean positioning of genes relative to each other. Angelmann Region (maternal chromosome) YAC 60 confocal light microscopy is used for the specific marking of centromer 1.5 small chromosomal DNA regions. Despite the low spatial resolution 15q11-13 PLW/A-Region, FISH, RW/GL-model: Fig. 7 of FISH, it is possible to interprete the results (f. e. the 3D distance our data. loop size: 5 Mbp clones and their genomic size Lymphocytes 11q13 FISH, K. Monier, Institut 3 Mbp Fibroblasts 11q13 } Albert Bonniot, Grenoble. between genetic markers as a function of their genomic distance) 1.0 Mbp λ 48.1 λ 48.17 λ λ 48.14 48.7 with our simulations. Yokota et. al., 1995. 0,5 Mbp 0,12 Mbp p 17,3 17,8 13.0 11.0 q Chromosome 15 and the Prader-Labhard-Willi/Angelmann 1 arm 69.2 156,6 17,0 Syndrom region was chosen, because the genomic distance between markers is well known (see sketch left) and because the 225.8 [kbp] PLW/A-syndrom is a candidate for structure mutation (in contrast 173.6 to the common base pair mutation). 0.5 242.8 Collaboration with B. Horsthemke, Institute for Human MLS-model (loop size 126 kbp): linker length: 251kbp genomic distances between probe pairs mean spatial distance [m] Genetics, Essen, FRG. 188kbp 126kbp 62kbp Methods: Human fibroblast cells grown on coverslips to confluent Distance Distribution Fig. 5 0 layers and being assumed to rest now in the same cell cycle phase 0 0.2 0.4 0.6 0.8 1 .08 # of distances: 305 are fixed in isotonic environment with paraformaldehyde. genomic distance between genomic markers [Mbp] Mean: (77623)nm For Hybridization we use digoxigenin labeled DNA probes. The 20 For calculating more general properties of chromosomes the fractal dimension probes are detected with fluorescent dyes. .05 15 of the chromatin fiber was determined from the simulations. The fractal analysis resulted in multifractal behaviour (data not shown here) in good 10 Confocal image series were taken with a Leica TCS NT confocal agreement with predictions drawn from porous network research (Avnir, 1989; smooth fit microscope with an axial displacement of z = 200nm. The images Frequency .03 Mandelbrot, private communications). 5 normal fit are median and background filtered. After manual threshold 5 RW/GL: Excluded Volume 0.1kT Fig. 8 [%] [N] determination from an extended focus view (Fig. 6) for spot finding LoopSize 5Mbp, LinkerLength 3600nm, 20 Loops LoopSize 126kbp, LinkerLength 600nm, 561 Loops we proceed with image reconstruction specially adjusted to the MLS: LoopSize 126kbp, Excluded Volume 0.1kT 4 LinkerLength 600nm microscope. Finally the 3D-spatial distances are determined LinkerLength 1200nm LinkerLength 1800nm 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 between the centers of mass of the spots (Fig. 5). The Euclidean Cut Off LinkerLength 2400nm D = 1.1 MLS: LoopSize 126kbp, Excluded Volume 1.0kT Spatial Distance [nm] experimental distance distributions are then compared to the D = 2.43 3 computed ones. We use a workstation cluster of 10 Silicon D = 1.89 Graphics Indigo and Indigo II for computation. D = 2.48 D = 2.88 2 D = 2.93 D = 2.01 The fibroblast nuclei are found to have their in vivo size (~20m * D = 2.98 10m * 6m) so that we conclude that at least on the micrometer 1 length scale we preserved the nuclear structure. With two colour D = 2.03 FISH it is possible to detect 3D-distances below the optical Log(Curve Length in Number of Measures) SegmentLength 300nm resolution. 0 1.5 2 2.5 3 3.5 4 Log (Measure Length [nm]) Fig. 6 Chromosomes form distinct territories in interphase and genomic The simulation of a whole human cell nucleus with all 46 human markers lay clearly separable within the territories. chromosomes in connection with the simulation of single chromosomes Left: Territory painting by FISH of chromosome 15; by chance the resulted in the formation of distinct chromosome territories as predicted. In two territories neighbour each other. contrast to the RW/GL-model the MLS-model leads to low overlap between Right: Genomic markers YAC48 and YAC60, genomic separation chromosome territories as well as chromosome arms, in agreement with overlap 1Mbp. analysis of confocal image series (data not shown here). Three-Dimensional Organization of Chromosome Territories in the Human Interphase Cell Nucleus Knoch, T. A., Münkel, C. & Langowski, J. Functional Organization of the Cell Nucleus - EMBO Workshop, Prague, Czech Republic, 9th - 12th August 1999. Abstract To study the three-dimensional organization of chromosome territories and the human interphase cell nucleus we developed models which could be compared to experiments. Despite the successful linear sequencing of the human genome its 3D-organization is widely unknown. Using Monte Carlo and Brownian dynamics simulations we managed to model the chromatin fibre as a wormlike-chain polymer. A typical chromosome consists of 20.000 and a nucleus with all 46 chromosomes of 1.200.000 polymer chain segments. The parallel simulations are performed on a SP2512 and a Cray T3E. With fluorescent in situ hybridization and confocal microscopy we determined genomic marker distributions and chromosome arm overlap. Best agreement between simulations and experiments is reached for a Multi-Loop-Subcompartment model (126 kbp loops connected to rosettes connected by a 126 kbp chromatin linker).
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