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The Universality of Nucleosome Positioning: From Yeast to Human
Monday, February 13, 2017 Solution-exchanging fragmented fluorophore and the SunTag system greatly extended the allowed observation time. We report successful tracking of an arbitrary genomic locus by targeting a sequence of only several repeats using our new design. This technique will enable the general application of CRISPR-based imaging to genome-wide loci. 1073-Pos Board B141 Nucleosome Opening Kinetics and the Influence of Histone Modifications Studied by Single Molecule FRET Alexander Gansen1, Suren Felekyan2, Kathrin Lehmann1, Ralf K€uhnemuth2, Ruihan Zhang1, Katalin To´th1, Claus A.M. Seidel2, Jo¨rg Langowski1. 1 Biophysics of Macromolecules, German Cancer Research Center, Heidelberg, Germany, 2Molecular Physical Chemistry, Heinrich-HeineUniversit€at, D€ usseldorf, Germany. Nucleosomes compact the genome and regulate access by wrapping and unwrapping DNA. Here we characterized NaCl-induced opening of mononucleosomes by single-molecule FRET, using donor/acceptor labels on the DNA and various histones. By species-selective fluorescence lifetime and photon distribution analysis we identified new nucleosome opening intermediates and developed a kinetic model [1]. Opening proceeds through a weakening of the H2A-H2B dimer/(H3-H4)2 tetramer interface on a 0.1 ms time scale, then by a slower two-step release of the dimers coupled to DNA unwrapping, extending from several ms to minutes. Nucleosome opening and detachment of histone dimers proceed asymmetrically depending on the DNA sequence. Mutations at the H2A/H3 interface (H2A R81A, R88A, R81A/R88A, R81E/ R88E) facilitate the initial opening, confirming the importance of the dimer:tetramer interface for nucleosome stability. This is also supported by molecular dynamics (MD) simulations that show enhanced DNA fluctuations for the mutants. Partially opened states such as described here might be a convenient nucleation point for DNA-recognizing proteins. For characterizing the role of histone tails in nucleosome interaction and stability, we performed MD simulations in explicit and implicit solvent on the H4 tail interacting with the acidic patch of a neighboring nucleosome. The simulations show that H4K16 acetylation decreases the interactions between H4 tail and the acidic patch of the neighboring nucleosome, a process that is central to the regulation of chromatin compaction [2,3]. References [1] A. Gansen, A. Valeri, F. Hauger, S. Felekyan, S. Kalinin, K. To´th, J. Langowski, and C. A. M. Seidel. Nucleosome disassembly intermediates characterized by single-molecule FRET. Proc Natl Acad Sci U S A, 106(36):15308-13, Sep 2009. [2] J. Erler, R. Zhang, L. Petridis, X. Cheng, J. C. Smith, and J. Langowski. The role of histone tails in the nucleosome: A computational study. Biophys. J., 107(12):2911-2922, 2014. [3] R. Zhang, J. Erler, J. Langowski. How does histone acetylation regulate chromatin accessibility? A computational study of the role of H4K16 in inter-nucleosome interaction. Biophys. J., under revision. 1074-Pos Board B142 Effect of Chromatin Architecture on Dynamic Correlations and Rheological Responses of Interphase Chromosomes Min Hyeok Kim, Lei Liu, Changbong Hyeon. Korea Institute for Advanced Study, Seoul, Korea, Republic of. The spatial organization of interphase chromatin is tightly linked to its biological functions. The intra-chromosomal contact frequencies revealed by Hi-C indicated that there is a fundamental variation in the chromatin organization from species to species and along the developmental stage. In this study, we used two different Hi-C contact maps for Human embryonic stem cell (ESC) and fibroblast (FB) to model distinct spatial organization of chromatin into a Gaussian network model and calculated dynamic functions and rheological response. Our calculation suggests that dynamics of loci in FB is associated with longer range cross-correlation than that of ESC. Furthermore, despite the higher contact frequency, FB chromosome retains smaller loss modulus due to the compartmentalized architecture. We discuss these finding in context of current knowledge of the gene expression and regulation during different stages of cell differentiation. 1075-Pos Board B143 Spatial Organization of Chromatin Domains and Compartments in Single Chromosomes Siyuan Wang1, Jun-Han Su1, Brian J. Beliveau2, Bogdan Bintu1, Jeffrey R. Moffitt1, Chao-ting Wu2, Xiaowei Zhuang1. 1 Harvard University, Cambridge, MA, USA, 2Harvard Medical School, Boston, MA, USA. The spatial organization of chromatin critically affects genome function. Recent chromosome-conformation-capture studies have revealed topologically associ-
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ating domains (TADs) as a conserved feature of chromatin organization, but how TADs are spatially organized in individual chromosomes remains unknown. Here, we developed an imaging method for mapping the spatial positions of numerous genomic regions along individual chromosomes and traced the positions of TADs in human interphase autosomes and X chromosomes. We observed that chromosome folding deviates from the ideal fractal-globule model at large length scales and that TADs are largely organized into two compartments spatially arranged in a polarized manner in individual chromosomes. Active and inactive X chromosomes adopt different folding and compartmentalization configurations. These results suggest that the spatial organization of chromatin domains can change in response to regulation. 1076-Pos Board B144 The Universality of Nucleosome Positioning: From Yeast to Human Razvan V. Chereji, David J. Clark. NICHD, National Institutes of Health, Bethesda, MD, USA. Nucleosome organization is important for the understanding of promoter accessibility and transcription regulation. In the past decade, in vivo nucleosome mapping suggested a stereotypical and conserved nucleosome organization, consisting of nucleosome-depleted regions (NDRs) around the transcription start sites (TSSs), flanked by regular arrays of nucleosomes on gene bodies. We study the factors that determine the nucleosome positioning, and their universality across different organisms, and we develop biophysical models that incorporate the common rules of nucleosome organization. We show that in vitro reconstituted nucleosomes occupy different locations from those observed in vivo. Thus, the DNA sequence has a limited role in nucleosome positioning. Using ChIP-seq and MNase-seq techniques, we show that non-histone proteins bind to gene promoters and compete with the histones, creating NDRs. We go on to apply rigorous biophysical models to explain the in vivo nucleosome phasing relative to the TSS, incorporating the ATP-dependent chromatin remodelers. In collaboration with other labs, we compare chromatin organization in yeast, fly, mouse and human. We find that most of yeast genes have a nucleosome depleted and DNase I accessible promoter. However, surprisingly, using MNase-seq, DNase-seq and RNA-seq data, we find that in higher organisms genes fall into two distinct, tissue-specific classes: the promoters of inactive genes are tightly packed with nucleosomes, not phased relative to the TSS and stabilized by H1 linker histones. By contrast, the active genes have DNase I accessible promoters and phased arrays of nucleosomes along the gene bodies. Using data available from the ENCODE project, we verify that most of the characterized epigenetic marks also have this bimodal distribution, correlated with the nucleosome organization of the gene promoters. References: Chereji RV et al - Phys. Rev. E 83, 050903 (2011) Chereji RV, Morozov AV - J. Stat. Phys. 144, 379 (2011) Chereji RV, Morozov AV - Proc. Natl. Acad. Sci. U.S.A. 111, 5236 (2014) Ganguli D, Chereji RV et al - Genome Res. 24, 1637 (2014) Cole HA et al - Nucleic Acids Res. 42, 12512 (2014) Elfving N, Chereji RV et al - Nucleic Acids Res. 42, 5468 (2014) Chereji RV, Morozov AV - Brief. Funct. Genomics 14, 50 (2015) Chereji RV, Kan T-W et al - Nucleic Acids Res. 44, 1036 (2016) Ocampo J, Chereji RV et al - Nucleic Acids Res. 44, 4625 (2016) Qiu H, Chereji RV et al - Genome Res. 26, 211 (2016) 1077-Pos Board B145 Protein Diffusion Around Bacterial Nucleoid Asli Yildirim1, Tadashi Ando2, Yuji Sugita3, Michael Feig4. 1 Chemistry, Michigan State University, East Lansing, MI, USA, 2Department of Applied Electronics, Tokyo University of Science, Tokyo, Japan, 3RIKEN, Wako, Japan, 4Biochemistry, Michigan State University, East Lansing, MI, USA. The effect of macromolecular crowding on protein diffusion has been extensively studied in order to understand the protein dynamics in cellular environments. Previous studies have generally focused on proteins as crowding agents as they are known to occupy 20-30% of the cell volume. The volume fraction of the nucleoid in bacterial cells is also significant, around 10-20%, but the effect of chromosomal DNA as a crowding agent on protein diffusion has drawn relatively limited attention. Using recently build high-resolution models of bacterial nucleoids, Brownian and Stokesian dynamics simulations of coarse-grained model systems containing chromosomal DNA and proteins were carried out to investigate the effect of nucleoid crowding on protein diffusion. The coarse-grained model for the nucleoid is an experimentally-derived 3D model of the Caulobacter crescentus chromosome at base-pair resolution built by combining topological information of chromosomal DNA with distance restraints obtained from high-throughput Chromosome Conformation
217a
ating domains (TADs) as a conserved feature of chromatin organization, but how TADs are spatially organized in individual chromosomes remains unknown. Here, we developed an imaging method for mapping the spatial positions of numerous genomic regions along individual chromosomes and traced the positions of TADs in human interphase autosomes and X chromosomes. We observed that chromosome folding deviates from the ideal fractal-globule model at large length scales and that TADs are largely organized into two compartments spatially arranged in a polarized manner in individual chromosomes. Active and inactive X chromosomes adopt different folding and compartmentalization configurations. These results suggest that the spatial organization of chromatin domains can change in response to regulation. 1076-Pos Board B144 The Universality of Nucleosome Positioning: From Yeast to Human Razvan V. Chereji, David J. Clark. NICHD, National Institutes of Health, Bethesda, MD, USA. Nucleosome organization is important for the understanding of promoter accessibility and transcription regulation. In the past decade, in vivo nucleosome mapping suggested a stereotypical and conserved nucleosome organization, consisting of nucleosome-depleted regions (NDRs) around the transcription start sites (TSSs), flanked by regular arrays of nucleosomes on gene bodies. We study the factors that determine the nucleosome positioning, and their universality across different organisms, and we develop biophysical models that incorporate the common rules of nucleosome organization. We show that in vitro reconstituted nucleosomes occupy different locations from those observed in vivo. Thus, the DNA sequence has a limited role in nucleosome positioning. Using ChIP-seq and MNase-seq techniques, we show that non-histone proteins bind to gene promoters and compete with the histones, creating NDRs. We go on to apply rigorous biophysical models to explain the in vivo nucleosome phasing relative to the TSS, incorporating the ATP-dependent chromatin remodelers. In collaboration with other labs, we compare chromatin organization in yeast, fly, mouse and human. We find that most of yeast genes have a nucleosome depleted and DNase I accessible promoter. However, surprisingly, using MNase-seq, DNase-seq and RNA-seq data, we find that in higher organisms genes fall into two distinct, tissue-specific classes: the promoters of inactive genes are tightly packed with nucleosomes, not phased relative to the TSS and stabilized by H1 linker histones. By contrast, the active genes have DNase I accessible promoters and phased arrays of nucleosomes along the gene bodies. Using data available from the ENCODE project, we verify that most of the characterized epigenetic marks also have this bimodal distribution, correlated with the nucleosome organization of the gene promoters. References: Chereji RV et al - Phys. Rev. E 83, 050903 (2011) Chereji RV, Morozov AV - J. Stat. Phys. 144, 379 (2011) Chereji RV, Morozov AV - Proc. Natl. Acad. Sci. U.S.A. 111, 5236 (2014) Ganguli D, Chereji RV et al - Genome Res. 24, 1637 (2014) Cole HA et al - Nucleic Acids Res. 42, 12512 (2014) Elfving N, Chereji RV et al - Nucleic Acids Res. 42, 5468 (2014) Chereji RV, Morozov AV - Brief. Funct. Genomics 14, 50 (2015) Chereji RV, Kan T-W et al - Nucleic Acids Res. 44, 1036 (2016) Ocampo J, Chereji RV et al - Nucleic Acids Res. 44, 4625 (2016) Qiu H, Chereji RV et al - Genome Res. 26, 211 (2016) 1077-Pos Board B145 Protein Diffusion Around Bacterial Nucleoid Asli Yildirim1, Tadashi Ando2, Yuji Sugita3, Michael Feig4. 1 Chemistry, Michigan State University, East Lansing, MI, USA, 2Department of Applied Electronics, Tokyo University of Science, Tokyo, Japan, 3RIKEN, Wako, Japan, 4Biochemistry, Michigan State University, East Lansing, MI, USA. The effect of macromolecular crowding on protein diffusion has been extensively studied in order to understand the protein dynamics in cellular environments. Previous studies have generally focused on proteins as crowding agents as they are known to occupy 20-30% of the cell volume. The volume fraction of the nucleoid in bacterial cells is also significant, around 10-20%, but the effect of chromosomal DNA as a crowding agent on protein diffusion has drawn relatively limited attention. Using recently build high-resolution models of bacterial nucleoids, Brownian and Stokesian dynamics simulations of coarse-grained model systems containing chromosomal DNA and proteins were carried out to investigate the effect of nucleoid crowding on protein diffusion. The coarse-grained model for the nucleoid is an experimentally-derived 3D model of the Caulobacter crescentus chromosome at base-pair resolution built by combining topological information of chromosomal DNA with distance restraints obtained from high-throughput Chromosome Conformation