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  • IN-VIVO  (10)
  • 1
    Keywords: IN-VIVO ; MAMMALIAN-CELLS ; LIVING CELLS ; EMBRYONIC STEM-CELLS ; LYSINE-9 METHYLATION ; HP1 PROTEINS ; DNA-METHYLATION ; HISTONE H3 ; CHROMATIN-REMODELING COMPLEXES ; NUCLEOSOME MODIFICATION
    Abstract: The cell establishes heritable patterns of active and silenced chromatin via interacting factors that set, remove, and read epigenetic marks. To understand how the underlying networks operate, we have dissected transcriptional silencing in pericentric heterochromatin (PCH) of mouse fibroblasts. We assembled a quantitative map for the abundance and interactions of 16 factors related to PCH in living cells and found that stably bound complexes of the histone methyltransferase SUV39H1/2 demarcate the PCH state. From the experimental data, we developed a predictive mathematical model that explains how chromatin-bound SUV39H1/2 complexes act as nucleation sites and propagate a spatially confined PCH domain with elevated histone H3 lysine 9 trimethylation levels via chromatin dynamics. This "nucleation and looping" mechanism is particularly robust toward transient perturbations and stably maintains the PCH state. These features make it an attractive model for establishing functional epigenetic domains throughout the genome based on the localized immobilization of chromatin-modifying enzymes.
    Type of Publication: Journal article published
    PubMed ID: 25134515
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  • 2
    Keywords: APOPTOSIS ; CANCER ; CELLS ; EXPRESSION ; CELL ; Germany ; IN-VIVO ; VIVO ; PROTEIN ; cell line ; DIFFERENTIATION ; DNA ; DOMAIN ; image analysis ; FLOW ; cell cycle ; CELL-CYCLE ; CYCLE ; SPECTROSCOPY ; IDENTIFICATION ; PROGRESSION ; CHROMATIN ; INDUCED APOPTOSIS ; CYCLE PROGRESSION ; CELL-LINE ; LINE ; acetylation ; REGION ; REGIONS ; MAMMALIAN-CELLS ; LENGTH ; REORGANIZATION ; STRUCTURAL-CHANGES ; HISTONE DEACETYLASE ; FLOW-CYTOMETRY ; INTERPHASE ; CHROMATIN STRUCTURE ; S-PHASE ; DOMAINS ; TRICHOSTATIN-A ; CORRELATION SPECTROSCOPY ; ARREST ; ACETYLTRANSFERASE ; SCALE ; DEPENDENCE ; fractal dimension ; DEACETYLASE INHIBITORS ; GENE-CONTROL ; HYPERACETYLATION ; image correlation ; NUCLEOSOME CORE ; TSA
    Abstract: The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to 〉1 mum was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied
    Type of Publication: Journal article published
    PubMed ID: 15292402
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  • 3
    Keywords: IN-VIVO ; fluorescence correlation spectroscopy ; nuclear architecture ; ANOMALOUS DIFFUSION ; GEOMETRY ; CHROMATIN ACCESSIBILITY ; MEMBRANE DYNAMICS ; TIME-DEPENDENT DIFFUSION ; DYNAMIC ORGANIZATION ; POROUS-MEDIA
    Abstract: In living cells, most proteins diffuse over distances of micrometres within seconds. Protein translocation is constrained due to the cellular organization into subcompartments that impose diffusion barriers and guide enzymatic activities to their targets. Here, we introduce an approach to retrieve structural features from the scale-dependent mobility of green fluorescent protein monomer and multimers in human cells. We measure protein transport simultaneously between hundreds of positions by multi-scale fluorescence cross-correlation spectroscopy using a line-illuminating confocal microscope. From these data we derive a quantitative model of the intracellular architecture that resembles a random obstacle network for diffusing proteins. This topology partitions the cellular content and increases the dwell time of proteins in their local environment. The accessibility of obstacle surfaces depends on protein size. Our method links multi-scale mobility measurements with a quantitative description of intracellular structure that can be applied to evaluate how drug-induced perturbations affect protein transport and interactions.
    Type of Publication: Journal article published
    PubMed ID: 25058002
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  • 4
    Keywords: IN-VIVO ; ATOMIC-FORCE MICROSCOPY ; LIGAND-BINDING ; GENE-REGULATION ; COOPERATIVE BINDING ; TARGET SITES ; TRANSCRIPTION-FACTOR-BINDING ; ONE-DIMENSIONAL LATTICES ; NUCLEOSOME POSITIONS ; LINEAR LATTICE
    Abstract: Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibria measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical-mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.
    Type of Publication: Journal article published
    PubMed ID: 21386588
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  • 5
    Keywords: IN-VIVO ; PROTEIN ; transcription ; DNA ; DYNAMICS ; ASSOCIATION ; MAMMALIAN-CELLS ; LIVING CELLS ; HETEROCHROMATIN ; H4-K16 ACETYLATION
    Abstract: The eukaryotic nucleus harbors the DNA genome, which associates with histones and other chromosomal proteins into a complex referred to as chromatin. It provides an additional layer of so-called epigenetic information via histone modifications and DNA methylation on top of the DNA sequence that determines the cell's active gene expression program. The nucleus is devoid of internal organelles separated by membranes. Thus, free diffusive transport of proteins and RNA can occur throughout the space accessible for a given macromolecule. At the same time, chromatin is partitioned into different specialized structures such as nucleoli, chromosome territories, and heterochromatin domains that serve distinct functions. Here, we address the question of how the activity of chromatin-modifying enzymes is confined to chromatin subcompartments. We discuss mechanisms for establishing activity gradients of diffusive chromatin-modifying enzymes that could give rise to distinct chromatin domains within the cell nucleus. Interestingly, such gradients might directly result from immobilization of the enzymes on the flexible chromatin chain. Thus, locus-specific tethering of these enzymes to chromatin could have the potential to establish, maintain, or modulate epigenetic patterns of characteristic domain size.
    Type of Publication: Journal article published
    PubMed ID: 24033539
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  • 6
    Keywords: CELLS ; CELL ; Germany ; IN-VIVO ; MICROSCOPY ; VIVO ; imaging ; INFORMATION ; SYSTEM ; SYSTEMS ; COMPLEX ; BIOLOGY ; MOLECULAR-BIOLOGY ; SIGNAL ; FIELD ; genetics ; LOCALIZATION ; NETHERLANDS ; FLUORESCENCE ; heredity ; ARCHITECTURE ; molecular biology ; molecular ; RE ; FLUORESCENCE MICROSCOPY ; analysis ; NUCLEAR ; 3D ; SIGNATURE ; 2 OBJECTIVE LENSES ; 3D WIDEFIELD MICROSCOPY ; AXIAL RESOLUTION ; TEMPERATURE ; EXCITATION ; ERRORS ; three-dimensional ; live cell imaging ; SECONDS ; STORM ; DATA-ACQUISITION ; 3-DIMENSIONAL SUPERRESOLUTION ; DISTANCE MEASUREMENTS ; FPALM ; localization microscopy ; nanosizing ; NANOSTRUCTURE ; OPTICAL RECONSTRUCTION MICROSCOPY ; PALM ; PALMIRA ; SALM ; SIZE DETERMINATION ; SMI microscopy ; SPDM ; Vertico-SMI microscope
    Abstract: Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved its applicability to a wide range of biological questions. For the SMI live cell imaging this system was enhanced in terms of the development of a completely new upright configuration. This so called Vertico-SMI transfers the advantages of SMI nanoscaling to vital biological systems, and is shown to work consistently at different temperatures using both oil- and water-immersion objective lenses. Furthermore, we increased the speed of data acquisition to minimize errors in the detection signal resulting from cellular or object movement. By performing accurate characterization, the present Vertico-SMI now offers a fully-fledged microscope enabling a complete three-dimensional (3D) SMI data stack to be acquired in less than 2 seconds. We have performed live cell measurements of a tet-operator repeat insert in U2OS cells, which provided the first in vivo signatures of subnuclear complexes. Furthermore, we have successfully implemented an optional optical configuration allowing the generation of high-resolution localization microscopy images of a nuclear pore complex distribution
    Type of Publication: Journal article published
    PubMed ID: 18461478
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  • 7
    Keywords: IN-VIVO ; INFORMATION ; GENE-EXPRESSION ; DNA ; BINDING ; SEQUENCE ; ORGANIZATION ; DROSOPHILA EMBRYO ; NUMBERS ; LATTICE
    Abstract: A recent study of transcription regulation in Drosophila embryonic development revealed a complex non-monotonic dependence of gene expression on the distance between binding sites of repressor and activator proteins at the corresponding enhancer cis-regulatory modules (Fakhouri et al 2010 Mol. Syst. Biol. 6 341). The repressor efficiency was high at small separations, low around 30 bp, reached a maximum at 50-60 bp, and decreased at larger distances to the activator binding sites. Here, we propose a straightforward explanation for the distance dependence of repressor activity by considering the effect of the presence of a nucleosome. Using a method that considers partial unwrapping of nucleosomal DNA from the histone octamer core, we calculated the dependence of activator binding on the repressor-activator distance and found a quantitative agreement with the distance dependence reported for the Drosophila enhancer element. In addition, the proposed model offers explanations for other distance-dependent effects at eukaryotic enhancers
    Type of Publication: Journal article published
    PubMed ID: 21666293
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  • 8
    Keywords: EXPRESSION ; IN-VIVO ; GENOME ; CHROMATIN ; LIVING CELLS ; PERICENTRIC HETEROCHROMATIN ; ORGANIZATION ; BODIES ; LONG NONCODING RNA ; TRANSCRIPTION FACTORIES ; INACTIVE X-CHROMOSOME ; XIST RNA
    Abstract: The dynamic organization of the cell nucleus into subcompartments with distinct biological activities represents an important determinant of cell function. Recent studies point to a crucial role of RNA as an architectural factor for shaping the genome and its nuclear environment. Here, we outline general principles by which RNA organizes functionally different nuclear subcompartments in mammalian cells. RNA is a structural component of mobile DNA-free nuclear bodies like paraspeckles or Cajal bodies, and is involved in establishing specific chromatin domains. The latter group comprises largely different structures that require RNA for the formation of active or repressive chromatin compartments with respect to gene expression as well as separating boundaries between these
    Type of Publication: Journal article published
    PubMed ID: 22281031
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  • 9
    Keywords: IN-VIVO ; GENE ; EXPRESSION ANALYSIS ; REGULATORY ELEMENTS ; ORGANIZATION ; HIGH-RESOLUTION ; CHROMATIN-STRUCTURE ; HISTONE ; SEQUENCE PREFERENCES ; TRANSCRIPTIONAL NETWORK
    Abstract: We determined genome-wide nucleosome occupancies in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell-type- and protein-specific binding preferences of transcription factors to sites with either low (Myc, Klf4 and Zfx) or high (Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome-depleted regions around transcription start and transcription termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to CpG content or histone methylation marks. Throughout the genome, nucleosome occupancy was correlated with certain histone methylation or acetylation modifications. In addition, the average nucleosome repeat length increased during differentiation by 5-7 base pairs, with local variations for specific regions. Our results reveal regulatory mechanisms of cell differentiation that involve nucleosome repositioning.
    Type of Publication: Journal article published
    PubMed ID: 23085715
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  • 10
    Keywords: IN-VIVO ; ESCHERICHIA-COLI ; MUTATION ; TRYPANOSOMA-BRUCEI ; IMMUNODEFICIENCY-VIRUS TYPE-1 ; STRAND-BREAK REPAIR ; ANTIGENIC VARIATION ; DNA BREAKS ; CHROMATIN ORGANIZATION ; MUTAGENIC REPAIR
    Abstract: The study of evolution has entered a revolutionary new era, where quantitative and predictive methods are transforming the traditionally qualitative and retrospective approaches of the past. Genomic sequencing and modern computational techniques are permitting quantitative comparisons between variation in the natural world and predictions rooted in neo-Darwinian theory, revealing the shortcomings of current evolutionary theory, particularly with regard to large-scale phenomena like macroevolution. Current research spanning and uniting diverse fields and exploring the physical and chemical nature of organisms across temporal, spatial, and organizational scales is replacing the model of evolution as a passive filter selecting for random changes at the nucleotide level with a paradigm in which evolution is a dynamic process both constrained and driven by the informational architecture of organisms across scales, from DNA and chromatin regulation to interactions within and between species and the environment.
    Type of Publication: Journal article published
    PubMed ID: 23691975
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