Chromatin Organization in the Mammalian Nucleus

Gilbert, Nick; Gilchrist, Susan; Bickmore, Wendy A.

doi:10.1016/s0074-7696(04)42007-5

Cited by 133 publications

(108 citation statements)

References 322 publications

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“…Indeed, the results obtained here revealed for the fixed nuclei a very high standard deviation even surpassing the half the IOD arithmetic means (X 5 38.20; S 5 19.80) ( Table 2), which is in agreement with more than one ploidy degree being present (20)(21)(22)(23)(24)(25)(26). The variation in DNA content retained in the hepatocyte nuclear remnants may be due to a structural constraint imposed by part of the chromatin organization possibly because the examined nuclei were engaged into different functional activities (8,9). Supporting this hypothesis is the previously reported frequency of adult hepatocytes forming ECFs by the action of gravity in fixed preparations subjected to a long lysis protocol, which has been found not to exceed 22% in fully nourished mice, and which significantly decreases with starvation (5) and increases with aging (27).…”

Section: Resultssupporting

confidence: 69%

“…Should a variation occurs, it could even mean that the putative structural constraint to DNA flow might be caused by differences in nuclear architecture possibly in association to differences in nuclear matrix interactions and transcriptional activities (5,8,9) in the hepatocyte nuclei. In addition, the presence of especial richness in DNA AT bases in the nuclear remnants of mouse hepatocytes could be associated with chromocenters generated by condensed centromere regions originally rich in repetitive AT-containing DNA (10)(11)(12).…”

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Nucleus image properties assessed by video image analysis in mouse hepatocytes under a short lysis for extended chromatin fiber formation

2006

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Background: How much DNA remains in mouse hepatocyte nuclei after extended chromatin fiber (ECF) formation or whether this content varies within the nuclear population is not known. This information could be relevant to understanding chromatin extensibility as related to chromatin organization, possibly associated with variable nuclear activities in hepatocytes. Methods: A protocol for ECF formation under the gravity action, image analysis of Feulgen-stained unfixed mouse hepatocyte remnants, and DAPI fluorescence were used. Results: Areas, shape, Feulgen-DNA amounts, and chromatin texture were affected in unfixed, lysed nuclei. The Feulgen-DNA values in nuclear remnants represented 37% of the content in fixed, nonlysed nuclei in terms of median values; the coefficient of variation of Feulgen-DNA values in the nuclear remnants was much higher than those in controls. Enhancement in DAPI fluorescence was

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Section: Resultssupporting

confidence: 69%

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confidence: 99%

Nucleus image properties assessed by video image analysis in mouse hepatocytes under a short lysis for extended chromatin fiber formation

2006

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“…Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3,4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5)(6)(7)(8)(9)(10)(11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells.…”

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confidence: 99%

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Beliveau

Joyce

Apostolopoulos

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

385

472

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A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.T he role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome. Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5-11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells. In addition, because FISH is applied to fixed cells, it can reveal the positioning of chromosomes relative to nuclear, cytoplasmic, and even tissue structures. FISH can also be used to visualize RNA, permitting the simultaneous assessment of gene expression, chromosome position, and protein localization.FISH probes are typically derived from cloned genomic regions or flow-sorted chromosomes, which are labeled directly via nick translation or PCR in the presence of fluorophore-conjugated nucleotides or labeled indirectly with nucleotide-conjugated haptens, such as biotin and digoxigenin, and then visualized with secondary detection reagents. Probe DNA is often fragmented into ∼150-to 250-bp pieces to facilitate its penetration into fixed cells (12) and, as many genomic clones contain repetitive sequences that occur abundantly in the genome, hybridization is typically performed in the presence of unlabeled repetitive DNA (13). Another limitation to clone-based probes is that the genomic regions that can be visualized with them are restricted by the availability of clones and the size of ...

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“…For instance, transcriptionally active regions are often co-localised in super-enhancer hubs [8] or transcription factories [14], whereas transcriptionally inactive regions may form large heterochromatic foci [15], mega-base (Mbp) size lamin-associated domains [16] or Barr [17] and Polycomb bodies [18].…”

Section: Introductionmentioning

confidence: 99%

Epigenetic Transitions and Knotted Solitons in Stretched Chromatin

Michieletto

Orlandini

Marenduzzo

2017

Preprint

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The spreading and regulation of epigenetic marks on chromosomes is crucial to establish and maintain cellular identity. Nonetheless, the dynamical mechanism leading to the establishment and maintenance of a given, cell-line specific, epigenetic pattern is still poorly understood. In this work we propose, and investigate in silico, a possible experimental strategy to illuminate the interplay between 3D chromatin structure and epigenetic dynamics. We consider a set-up where a reconstituted chromatin fibre is stretched at its two ends (e.g., by laser tweezers), while epigenetic enzymes (writers) and chromatin-binding proteins (readers) are flooded into the system. We show that, by tuning the stretching force and the binding affinity of the readers for chromatin, the fibre undergoes a sharp transition between a stretched, epigenetically disordered, state and a crumpled, epigenetically coherent, one. We further investigate the case in which a knot is tied along the chromatin fibre, and find that the knotted segment enhances local epigenetic order, giving rise to "epigenetic solitons" which travel and diffuse along chromatin. Our results point to an intriguing coupling between 3D chromatin topology and epigenetic dynamics, which may be investigated via single molecule experiments.

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Chromatin Organization in the Mammalian Nucleus

Cited by 133 publications

References 322 publications

Nucleus image properties assessed by video image analysis in mouse hepatocytes under a short lysis for extended chromatin fiber formation

Nucleus image properties assessed by video image analysis in mouse hepatocytes under a short lysis for extended chromatin fiber formation

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Epigenetic Transitions and Knotted Solitons in Stretched Chromatin

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