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  • 1
  • 2
    Keywords: Germany ; CT ; DENSITY ; TOOL ; GENE ; DIFFERENTIATION ; DOMAIN ; CONTRAST ; fibroblasts ; NUCLEI ; CHROMATIN ; IN-SITU HYBRIDIZATION ; LYMPHOCYTES ; FISH ; HUMAN GENOME ; MAMMALIAN-CELLS ; SPATIAL-ORGANIZATION ; TERRITORIES ; CHROMOSOME TERRITORIES ; ARCHITECTURE ; INTERPHASE NUCLEUS ; FUNCTIONAL IMPLICATIONS ; CELL-NUCLEI ; ORDER CHROMATIN ARRANGEMENTS
    Abstract: Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling ( early S-phase) human diploid fibroblasts ( 46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes - independently of their gene density - were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding
    Type of Publication: Journal article published
    PubMed ID: 15839726
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  • 3
    Keywords: CELL ; Germany ; human ; MODEL ; SUPPORT ; VOLUME ; GENE ; GENE-EXPRESSION ; DIFFERENTIATION ; MECHANISM ; mechanisms ; BIOLOGY ; CHROMATIN ; DIFFERENCE ; IN-SITU HYBRIDIZATION ; REGION ; REGIONS ; LOCALIZATION ; INTERPHASE CHROMOSOMES ; FAILURE ; CHROMOSOME TERRITORIES ; ORGANIZATION ; ARCHITECTURE ; fibroblast ; LIGHT ; GENOME ORGANIZATION ; FLUORESCENCE MICROSCOPY ; MODULATED ILLUMINATION MICROSCOPY ; ENGLAND ; comparison ; quantitative ; LIMITS ; 3D chromatin architecture ; confocal laser scanning microscopy (CLSM) ; fluorescence in situ hybridization (FISH) ; HISTORICAL-PERSPECTIVE ; NM AXIAL RESOLUTION ; POINT-SPREAD FUNCTION ; Prader-Willi syndrome (PWS) ; SCALE CHROMATIN ORGANIZATION ; spectral precision distance microscopy (SPDM)
    Abstract: Despite the major advancements during the last decade with respect to both knowledge of higher order chromatin organization in the cell nucleus and the elucidation of epigenetic mechanisms of gene control, the true three-dimensional (3D) chromatin structure of endogenous active and inactive gene loci is not known. The present study was initiated as an attempt to close this gap. As a model case, we compared the chromatin architecture between the genetically active and inactive domains of the imprinted Prader-Willi syndrome (PWS) locus in human fibroblast and lymphoblastoid cell nuclei by 3D fluorescence in situ hybridization and quantitative confocal laser scanning microscopy. The volumes and 3D compactions of identified maternal and paternal PWS domains were determined in stacks of light optical serial sections using a novel threshold-independent approach. Our failure to detect volume and compaction differences indicates that possible differences are below the limits of light optical resolution. To overcome this limitation, spectral precision distance microscopy, a method of localization microscopy at the nanometer scale, was used to measure 3D distances between differentially labeled probes located both within the PWS region and in its neighborhood. This approach allows the detection of intranuclear differences between 3D distances down to about 70-90 nm, but again did not reveal clearly detectable differences between active and inactive PWS domains. Despite this failure, a comparison of the experimental 3D distance measurements with computer simulations of chromatin folding strongly supports a non-random higher order chromatin configuration of the PWS locus and argues against 3D configurations based on giant chromatin loops. Our results indicate that the search for differences between endogenous active and inactive PWS domains must be continued at still smaller scales than hitherto possible with conventional light microscopic procedures. The possibilities to achieve this goal are discussed
    Type of Publication: Journal article published
    PubMed ID: 18039333
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  • 4
    Keywords: CELLS ; EXPRESSION ; proliferation ; MATURATION ; MEMBRANE ; NUCLEAR-ENVELOPE ; CHROMATIN-STRUCTURE ; emerin ; INTEGRITY ; B RECEPTOR
    Abstract: Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR -and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.
    Type of Publication: Journal article published
    PubMed ID: 23374351
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  • 5
    Keywords: EXPRESSION ; CELL ; Germany ; MICROSCOPY ; IMAGES ; transcription ; DIFFERENTIATION ; COMPLEX ; COMPLEXES ; BIOLOGY ; MOLECULAR-BIOLOGY ; chromosome ; CHROMATIN ; FISH ; HUMAN GENOME ; REGION ; REGISTRATION ; REGIONS ; NETHERLANDS ; NUCLEAR-ORGANIZATION ; IMAGE REGISTRATION ; CHROMOSOME TERRITORIES ; ORDER ; molecular biology ; molecular ; FEATURES ; RE ; analysis ; 3D ; CELL-NUCLEI ; GENOMIC REGION ; SET ; NOV ; SHAPE ; CELL BIOLOGY ; Cell nuclei ; 3D chromatin structure ; 3D multicolor FISH microscopy ; Geometrical morphometrics ; LOCAL GENE DENSITY ; MORPHOMETRICS ; Non-rigid image registration ; Shape distribution ; Shape normalization ; Statistical shape theory
    Abstract: The 3D folding structure formed by different genomic regions of a chromosome is still poorly understood. So far, only relatively simple geometric features, like distances and angles between different genomic regions, have been evaluated. This work is concerned with more complex geometric properties, i.e., the complete shape formed by genomic regions. Our work is based on statistical shape theory and we use different approaches to analyze the considered structures, e.g., shape uniformity test, 3D point-based registration, Fisher distribution, and 3D non-rigid image registration for shape normalization. We have applied these approaches to analyze 3D microscopy images of the X-chromosome where four consecutive genomic regions (BACs) have been simultaneously labeled by multicolor FISH. We have acquired two sets of four consecutive genomic regions with an overlap of three regions. From the experimental results, it turned out that for all data sets the complete structure is non-random. In addition, we found that the shapes of active and inactive X-chromosomal genomic regions are statistically independent. Moreover, we reconstructed the average 3D structure of chromatin in a small genomic region (below 4 Mb) based on five BACs resulting from two overlapping four BAC regions. We found that geometric normalization with respect to the nucleus shape based on non-rigid image registration has a significant influence on the location of the genomic regions. (c) 2008 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 18789978
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  • 6
    Keywords: Germany ; human ; CELL ; CLONES ; BIOLOGY ; chromosome ; ARRANGEMENT ; CHROMOSOME TERRITORIES
    Type of Publication: Meeting abstract published
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  • 7
    Keywords: Germany ; MODEL ; MODELS ; CT ; DISTINCT ; TIME ; DOMAIN ; CHROMATIN ; REGION ; NUCLEUS ; MAMMALIAN-CELLS ; DNA-REPLICATION ; INTERPHASE CHROMOSOMES ; LIVING CELLS ; CHROMOSOME TERRITORIES ; DOMAINS ; nuclear architecture ; ELECTRON-MICROSCOPY ; HELA-CELLS ; LEVEL ; CHANNELS ; chromatin condensation ; CHROMATIN FIBER ; interchromatin compartment ; PERICHROMATIN FIBRILS ; REPLICON CLUSTERS ; VIEW ; X-INACTIVATION
    Abstract: In spite of strong evidence that the nucleus is a highly organized organelle, a consensus on basic principles of the global nuclear architecture has not so far been achieved. The chromosome territory-interchromatin compartment (CT-IC) model postulates an IC which expands between chromatin domains both in the interior and the periphery of CT. Other models, however, dispute the existence of the IC and claim that numerous chromatin loops expand between and within CTs. The present study was undertaken to resolve these conflicting views. (1) We demonstrate that most chromatin exists in the form of higher-order chromatin domains with a compaction level at least 10 times above the level of extended 30 nm chromatin fibers. A similar compaction level was obtained in a detailed analysis of a particularly gene-dense chromosome region on HSA 11, which often expanded from its CT as a finger-like chromatin protrusion. (2) We further applied an approach which allows the experimental manipulation of both chromatin condensation and the width of IC channels in a fully reversible manner. These experiments, together with electron microscopic observations, demonstrate the existence of the IC as a dynamic, structurally distinct nuclear compartment, which is functionally linked with the chromatin compartment
    Type of Publication: Journal article published
    PubMed ID: 17115328
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  • 8
    ISSN: 1573-6849
    Keywords: Columba ; lampbrush chromosomes ; fluorescencein situ hybridization ; repeated DNA sequences ; RNA transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A highly repetitive, centromerically localized DNA sequence (PR1) has been isolated from the genomic DNA of two species of pigeon (Columba livia andC. palumbus). PR1 is approximately 900 bp long. It includes a sequence that is similar to the CENP-B box of mammals. It represents about 5% of the genome inC. livia and 2% inC. palumbus. In both species, tandem arrays ofPR1 form part of larger repeating units. The organization of PR1 repeats and the larger repeating units is strikingly different in the two species. The large repeating units inC. livia include long (at least 14 units) tandem arrays of PR1 interspersed with relatively short intervening sequences. The large repeats ofC. palumbus have much shorter (4 units or fewer) PR1 arrays interspersed with longer sections of non-PR1 DNA. PR1 is transcribed on short lampbrush loops in the centromeric regions of all lampbrush bivalents ofC. palumbus. InC. livia, it is not transcribed at any of the major pericentromeric sites at which it is known to be present, although it is transcribed at one minor centromeric site on chromosome 2. It is proposed that transcription of the noncoding PR1 sequence on lampbrush chromosomes of pigeons relates to its genomic organization. The proposal is discussed with regard to the ‘read-through’ hypothesis for transcription on lampbrush loops.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1573-6849
    Keywords: birds ; FISH ; lampbrush chromosome ; telomere ; transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The arrangement of loops and chromomeres at the ends of lampbrush chromosomes in four species of bird is described with reference to chromomeres, loops and transcription units. Unlike the situation described in lampbrush chromosomes of amphibians, the lampbrush chromosomes of birds end in a terminal chromomere with conspicuous loops emerging from it. The fine-scale morphology of the ribonuclear protein matrix of these terminal loops is different from that of the majority of loops elsewhere on the chromosomes. In many cases the loops associated with the terminal chromomere are open ended, emerging from the chromomere but not returning to it at the other end. The distal ends of terminal openended loops therefore represent the true ends of the chromatids that make up a lampbrush half-bivalent. The pattern of binding of three telomeric DNA sequence probes to the terminal regions of bird lampbrush chromosomes, under conditions of DNA/DNA and DNA/RNA transcriptin situ hybridization has been investigated by fluorescencein situ hybridization. All three probes gave the same results. With DNA/DNA and DNA/RNA transcript hybridization, three classes of structure were labelled: the terminal chromomere, a small number of interstitial chromomeres and the terminal transcription unit on telomere loops. Labelling of telomere loops, but not of terminal or interstitial chromomeres, was eliminated by ribonuclease treatment beforein situ hybridization. The labelled regions of telomere loops were spaced away from the labelled terminal chromomere by an unlabelled sub telomeric transcription unit. After DNA/DNAin situ hybridization, no labelled loops were seen. DNA/RNA transcriptin situ hybridization with single-stranded hexamers of each strand of telomeric DNA showed that the terminal transcription unit on telomere loops represents transcription exclusively from the C-rich strand of the repeat outwards towards the end of the chromosome. It is concluded that transcription specifically of the C-rich strand of strictly terminal clusters of telomere repeats is an obligatory event on the lampbrush chromosomes of birds and is unlikely to represent indiscriminate readthrough from proximally located gene elements.
    Type of Medium: Electronic Resource
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