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Single-Molecule Analysis of Colocalized Epigenetic Modifications
66a
Sunday, February 28, 2016
Chromosomes segregate into discrete regions of the nucleus during interphase, which are referred to as ‘‘chromosome territories’’; however, the level of independence (or conversely, the level of interaction) between the territories is vigorously debated. Chromosome conformation and capture techniques, such as Hi-C, can provide detailed information on the organization of the genome. From an analysis of Hi-C data, we have found that the chromosomes of both mouse and human embryonic stem (ES) cells tend to interact much less than the chromosomes of differentiated cells. DNA FISH experiments with chromosome paints support the Hi-C data showing that chromosome territories in ES cells tend to be farther apart; in fact, a global decrease in inter-chromosomal interactions correlated with an increase in average nuclear size. Surprisingly, the primary transcriptional hardware, RNAPII, did not show clear organizational changes upon cellular differentiation. Direct stochastic optical reconstruction (dSTORM) microscopy in both ES and differentiated cell nuclei showed that RNAPII maintain a constant density and level of clustering. Our data reveal a structural difference in genome organisation between ES cells and differentiated cells and suggest that the genome undergoes a fundamental reorganization after cellular differentiation. 345-Pos Board B125 Genome-Wide Mapping of Chromatin Secondary Structure using Ionizing Radiation Coupled with Sequencing Viviana I. Risca, Sarah Denny, Alicia Schep, Aaron Straight, William J. Greenleaf. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA. Chromatin packages two meters of human DNA into a five-micron nucleus while allowing regulated access to the genome. At the intermediate length scale of ~1 kilobase, which spans nucleosome-nucleosome interactions, chromatin structure is expected to play a crucial role in regulating transcription, DNA replication and DNA repair. However, our structural understanding of this level of chromatin organization continues to lag behind our rapidly developing understanding of both single nucleosomes and higher-order, long-range interactions. We have developed a novel method, RICC-seq, for probing chromatin structure at this poorly understood length scale. RICC-seq uses hydroxyl radicals generated by ionizing radiation to create sparse and spatially correlated strand breaks that result in short DNA fragments. The lengths and mapping locations of these fragments can be used to place basepair-resolved pairwise distance constraints on sequence loci in 3D space. In order to understand the detailed architecture underlying chromatin compaction and DNA accessibility, we have generated the first genome-wide RICC-seq maps of chromatin compaction in terminally differentiated fibroblasts, comparing chromatin structure between heterochromatic and euchromatic regions. These data reveal differential packaging geometries for heterochromatic regions with significantly more DNA contacts with specific chromatin post translational modifications. We envision that RICC-seq is a generalizable method for high-resolution understanding of condensed nucleic acids with potential applications to targets as diverse as folded RNA to viral genomes within capsids. 346-Pos Board B126 Characterizing Transcription and Splicing Kinetics by 3D Orbital Tracking Nathan A. Redman1, Eric J. Hayden1,2, Matthew L. Ferguson1,3. 1 Biomolecular Sciences Ph.D. Program, Boise State University, Boise, ID, USA, 2Department of Biological Sciences, Boise State University, Boise, ID, USA, 3Department of Physics, Boise State University, Boise, ID, USA. We describe our recent application of two photon laser scanning microscopy to characterize transcription and splicing rates in living cells with subdiffraction limited spatial resolution and second to hour long temporal resolution by the application of 3D orbital tracking[1] and two color in vivo RNA labelling[2]. We find that measurements agree with previous studies utilizing widefield volumetric imaging but increase the temporal resolution 100 fold and the duration of each experiment by 10 fold characterizing protein-RNA binding, RNA synthesis and splicing and long timescale transcriptional bursting in a single measurement. This technique may have future applications for the study of both natural and synthetic gene regulation in eukaryotes. 1. Levi V, Ruan Q, Gratton E. 3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells. Biophys J. 2005;88: 2919-2928. http://dx.doi.org/10.1529/biophysj.104.044230
2. Coulon A, Ferguson ML, de Turris V, Palangat M, Chow CC, Larson DR. Kinetic competition during the transcription cycle results in stochastic RNA processing. Elife. 2014;3. http://dx.doi.org/10.7554/eLife.03939. 347-Pos Board B127 The NC-SAC: Computational Pipeline for Predicting Structures of 3D Chromatin Chains from Experimental Data: Origin of Scaling Properties, Emergence of Chromosome Territories and Discovery of Novel Loci Interactions Associated with Differential Gene Expression Gamze Gu¨rsoy, Yun Xu, Jie Liang. University of Illinois at Chicago, Chicago, IL, USA. The global architecture of cell nucleus and the spatial organization of chromatin play fundamental roles in gene expression and nuclear function. Techniques such as electron microscopy and Chromosome Conformation Capture (3C, 4C, 5C, Hi-C, ChIA-PET) provide a wealth of information on the three-dimensional organization of genome. However, understanding detailed mechanisms important for cellular activities from existing experimental data requires computational modeling of the 3D structures of chromatin. Here we describe a computational pipeline for constructing ensembles of chromatin chains from diverse experimental data. We showed that the scaling properties of human genome arise from polymer properties under the confinement of the cell nucleus. We also demonstrated that the inter-chromosomal interactions as seen in Hi-C experiments, the emergence of chromosome territories, as well as the co-localization of fragile sites of budding yeast can all be fully accounted for by the nuclear confinement and landmark constraints. We further integrated 5C interactions to our polymer models and obtained up to 20,000 detailed 3D structures of the aglobin locus at 3-15 kb resolution. We uncovered global differences in the spatial structures of the a-globin locus at different expression levels. We further predicted novel chromatin interactions, which are validated by independent ChIA-PET studies. Our predicted structures suggest a complex higher-order regulatory machinery behind the differential gene expression, with a novel mechanistic model that can be tested experimentally. Overall, our pipeline can incorporate diverse experimental data and generate 3D models of self-avoiding chromatin chains. It provides a generally applicable new approach for identifying spatial interactions and assessing their roles in regulating gene activities and nuclear functions. 348-Pos Board B128 Single-Molecule Analysis of Colocalized Epigenetic Modifications Jen-Chien Chang1, Takashi Umehara2, Keisuke Fujita3, Yuichi Taniguchi4, Toshio Yanagida3, Akiko Minoda1. 1 Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Japan, 2Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama, Japan, 3 Laboratory for Cell Dynamics Observation, RIKEN Quantitative Biology Center, Osaka, Japan, 4Laboratory for Single Cell Gene Dynamics, RIKEN Quantitative Biology Center, Osaka, Japan. Post-translational modifications of histones, which mediate many nuclear processes, is one of the most well characterized epigenetic mechanisms. In recent years, it has become evident that specific combinations of histone modifications represent various chromatin states, and are associated with different regulatory functions. However, standard methods like chromatin immunoprecipitation probe one histone modification at a time; thus, the combinatorial patterns at each histone or nucleosome are largely unknown. Here, we apply singlemolecule fluorescent imaging in analyzing the coexisting histone modifications. We first demonstrate the method using reconstituted nucleosomes installing tetra-acetylated-lysines at histone 4. Once the assay is validated, it can be applied in the study of the coexisting active and repressive histone marks in different cell types, especially stem cells during differentiation. Other applications include the colocalization of histone variants, DNA methylation, transcription factors, and chromatin-binding proteins. We expect this method to elucidate the functions of chromatin in different states, with the ultimate goal in providing a complete picture of how epigenome is modulated. 349-Pos Board B129 Physical Modeling of Stress Communication between Chromosome Loci Thomas J. Lampo1, Andrew S. Kennard2, Andrew J. Spakowitz1. 1 Chemical Engineering, Stanford University, Stanford, CA, USA, 2 Biophysics, Stanford University, Stanford, CA, USA. The organizational dynamics of chromosomal DNA are critical to many biological processes including genetic regulation, recombination, condensation, and segregation. The physical understanding of such processes would be greatly improved by predictive, quantitative modeling that can be directly compared to experimental measurements. Previous work has examined or modeled either
Sunday, February 28, 2016
Chromosomes segregate into discrete regions of the nucleus during interphase, which are referred to as ‘‘chromosome territories’’; however, the level of independence (or conversely, the level of interaction) between the territories is vigorously debated. Chromosome conformation and capture techniques, such as Hi-C, can provide detailed information on the organization of the genome. From an analysis of Hi-C data, we have found that the chromosomes of both mouse and human embryonic stem (ES) cells tend to interact much less than the chromosomes of differentiated cells. DNA FISH experiments with chromosome paints support the Hi-C data showing that chromosome territories in ES cells tend to be farther apart; in fact, a global decrease in inter-chromosomal interactions correlated with an increase in average nuclear size. Surprisingly, the primary transcriptional hardware, RNAPII, did not show clear organizational changes upon cellular differentiation. Direct stochastic optical reconstruction (dSTORM) microscopy in both ES and differentiated cell nuclei showed that RNAPII maintain a constant density and level of clustering. Our data reveal a structural difference in genome organisation between ES cells and differentiated cells and suggest that the genome undergoes a fundamental reorganization after cellular differentiation. 345-Pos Board B125 Genome-Wide Mapping of Chromatin Secondary Structure using Ionizing Radiation Coupled with Sequencing Viviana I. Risca, Sarah Denny, Alicia Schep, Aaron Straight, William J. Greenleaf. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA. Chromatin packages two meters of human DNA into a five-micron nucleus while allowing regulated access to the genome. At the intermediate length scale of ~1 kilobase, which spans nucleosome-nucleosome interactions, chromatin structure is expected to play a crucial role in regulating transcription, DNA replication and DNA repair. However, our structural understanding of this level of chromatin organization continues to lag behind our rapidly developing understanding of both single nucleosomes and higher-order, long-range interactions. We have developed a novel method, RICC-seq, for probing chromatin structure at this poorly understood length scale. RICC-seq uses hydroxyl radicals generated by ionizing radiation to create sparse and spatially correlated strand breaks that result in short DNA fragments. The lengths and mapping locations of these fragments can be used to place basepair-resolved pairwise distance constraints on sequence loci in 3D space. In order to understand the detailed architecture underlying chromatin compaction and DNA accessibility, we have generated the first genome-wide RICC-seq maps of chromatin compaction in terminally differentiated fibroblasts, comparing chromatin structure between heterochromatic and euchromatic regions. These data reveal differential packaging geometries for heterochromatic regions with significantly more DNA contacts with specific chromatin post translational modifications. We envision that RICC-seq is a generalizable method for high-resolution understanding of condensed nucleic acids with potential applications to targets as diverse as folded RNA to viral genomes within capsids. 346-Pos Board B126 Characterizing Transcription and Splicing Kinetics by 3D Orbital Tracking Nathan A. Redman1, Eric J. Hayden1,2, Matthew L. Ferguson1,3. 1 Biomolecular Sciences Ph.D. Program, Boise State University, Boise, ID, USA, 2Department of Biological Sciences, Boise State University, Boise, ID, USA, 3Department of Physics, Boise State University, Boise, ID, USA. We describe our recent application of two photon laser scanning microscopy to characterize transcription and splicing rates in living cells with subdiffraction limited spatial resolution and second to hour long temporal resolution by the application of 3D orbital tracking[1] and two color in vivo RNA labelling[2]. We find that measurements agree with previous studies utilizing widefield volumetric imaging but increase the temporal resolution 100 fold and the duration of each experiment by 10 fold characterizing protein-RNA binding, RNA synthesis and splicing and long timescale transcriptional bursting in a single measurement. This technique may have future applications for the study of both natural and synthetic gene regulation in eukaryotes. 1. Levi V, Ruan Q, Gratton E. 3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells. Biophys J. 2005;88: 2919-2928. http://dx.doi.org/10.1529/biophysj.104.044230
2. Coulon A, Ferguson ML, de Turris V, Palangat M, Chow CC, Larson DR. Kinetic competition during the transcription cycle results in stochastic RNA processing. Elife. 2014;3. http://dx.doi.org/10.7554/eLife.03939. 347-Pos Board B127 The NC-SAC: Computational Pipeline for Predicting Structures of 3D Chromatin Chains from Experimental Data: Origin of Scaling Properties, Emergence of Chromosome Territories and Discovery of Novel Loci Interactions Associated with Differential Gene Expression Gamze Gu¨rsoy, Yun Xu, Jie Liang. University of Illinois at Chicago, Chicago, IL, USA. The global architecture of cell nucleus and the spatial organization of chromatin play fundamental roles in gene expression and nuclear function. Techniques such as electron microscopy and Chromosome Conformation Capture (3C, 4C, 5C, Hi-C, ChIA-PET) provide a wealth of information on the three-dimensional organization of genome. However, understanding detailed mechanisms important for cellular activities from existing experimental data requires computational modeling of the 3D structures of chromatin. Here we describe a computational pipeline for constructing ensembles of chromatin chains from diverse experimental data. We showed that the scaling properties of human genome arise from polymer properties under the confinement of the cell nucleus. We also demonstrated that the inter-chromosomal interactions as seen in Hi-C experiments, the emergence of chromosome territories, as well as the co-localization of fragile sites of budding yeast can all be fully accounted for by the nuclear confinement and landmark constraints. We further integrated 5C interactions to our polymer models and obtained up to 20,000 detailed 3D structures of the aglobin locus at 3-15 kb resolution. We uncovered global differences in the spatial structures of the a-globin locus at different expression levels. We further predicted novel chromatin interactions, which are validated by independent ChIA-PET studies. Our predicted structures suggest a complex higher-order regulatory machinery behind the differential gene expression, with a novel mechanistic model that can be tested experimentally. Overall, our pipeline can incorporate diverse experimental data and generate 3D models of self-avoiding chromatin chains. It provides a generally applicable new approach for identifying spatial interactions and assessing their roles in regulating gene activities and nuclear functions. 348-Pos Board B128 Single-Molecule Analysis of Colocalized Epigenetic Modifications Jen-Chien Chang1, Takashi Umehara2, Keisuke Fujita3, Yuichi Taniguchi4, Toshio Yanagida3, Akiko Minoda1. 1 Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Japan, 2Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama, Japan, 3 Laboratory for Cell Dynamics Observation, RIKEN Quantitative Biology Center, Osaka, Japan, 4Laboratory for Single Cell Gene Dynamics, RIKEN Quantitative Biology Center, Osaka, Japan. Post-translational modifications of histones, which mediate many nuclear processes, is one of the most well characterized epigenetic mechanisms. In recent years, it has become evident that specific combinations of histone modifications represent various chromatin states, and are associated with different regulatory functions. However, standard methods like chromatin immunoprecipitation probe one histone modification at a time; thus, the combinatorial patterns at each histone or nucleosome are largely unknown. Here, we apply singlemolecule fluorescent imaging in analyzing the coexisting histone modifications. We first demonstrate the method using reconstituted nucleosomes installing tetra-acetylated-lysines at histone 4. Once the assay is validated, it can be applied in the study of the coexisting active and repressive histone marks in different cell types, especially stem cells during differentiation. Other applications include the colocalization of histone variants, DNA methylation, transcription factors, and chromatin-binding proteins. We expect this method to elucidate the functions of chromatin in different states, with the ultimate goal in providing a complete picture of how epigenome is modulated. 349-Pos Board B129 Physical Modeling of Stress Communication between Chromosome Loci Thomas J. Lampo1, Andrew S. Kennard2, Andrew J. Spakowitz1. 1 Chemical Engineering, Stanford University, Stanford, CA, USA, 2 Biophysics, Stanford University, Stanford, CA, USA. The organizational dynamics of chromosomal DNA are critical to many biological processes including genetic regulation, recombination, condensation, and segregation. The physical understanding of such processes would be greatly improved by predictive, quantitative modeling that can be directly compared to experimental measurements. Previous work has examined or modeled either