Spatial Preservation of Nuclear Chromatin Architecture during Three-Dimensional Fluorescence in Situ Hybridization (3D-FISH)

Solovei, Irina; Cavallo, Antonio; Schermelleh, Lothar; Jaunin, Françoise; Scasselati, Catia; Cmarko, Dušan; Cremer, Christoph; Fakan, Stanislav; Cremer, Thomas

doi:10.1006/excr.2002.5513

Cited by 249 publications

(202 citation statements)

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“…19) (see Table 1 for their features), were tested on different cell types, as indicated on the x axis by capital letters. 'A', 'B', and 'C' are peripheral lymphocytes from three different individuals; 'D', and 'E' are two different lymphoblastoid cell lines; 'F' and 'G' are the neuroblastoma SK-N-BE cell line prepared using the standard method of preparation, and the method to preserve large-scale chromatin structure (Solovei et al 2002), respectively. The radial location of each probe in the different cell types was determined by considering the median value (and the relative confidence interval, C.I.)…”

Section: Discussionmentioning

confidence: 99%

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The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

et al. 2008

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Section: Discussionmentioning

confidence: 99%

“…Moreover, the SK-N-BE cells were also prepared under conditions to preserve the 3D large-scale chromatin structure in the nuclei (Solovei et al 2002). This latter procedure involves the use of freshly prepared 4% buffered paraformaldehyde to fix the cells.…”

Section: Preparation Of Chromosomes and Nucleimentioning

confidence: 99%

The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

et al. 2008

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“…It is important that after fixation of cells on glass slides, they retain, as near as possible, their in vivo morphology (1)(2)(3). A protocol of cell fixation based on paraformaldehyde has been developed to perform FISH in the three-dimensionally (3D) preserved structure of nuclei in cells in which the relative position of chromatin and genes are quite well conserved (4). This technique, named 3D-FISH, has opened the way to study the organization of the chromatin in interphase nuclei.…”

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Smart 3D‐fish: Automation of distance analysis in nuclei of interphase cells by image processing

et al. 2005

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Background: Detection of fluorescent probes by fluorescence in situ hybridization in cells with preserved threedimensional nuclear structures (3D-FISH) is useful for studying the organization of chromatin and localization of genes in interphase nuclei. Fast and reliable measurements of the relative positioning of fluorescent spots specific to subchromosomal regions and genes would improve understanding of cell structure and function. Methods: 3D-FISH protocol, confocal microscopy, and digital image analysis were used. Results: New software (Smart 3D-FISH) has been developed to automate the process of spot segmentation and distance measurements in images from 3D-FISH experiments. It can handle any number of fluorescent spots and incorporate images of 4 0 ,6-diamino-2-phenylindole counterstained nuclei to measure the relative positioning of

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“…In a number of cases, the H19 locus showed simultaneous interactions with several other chromosomes, suggesting a highly ordered CT neighborhood not confirmed to date by 3D-FISH experiments. The combination of 3C and circular chromosome conformation capture assays with single-cell assays, such as 3D-FISH and advanced light microscopic approaches, provides a perfect match for studies of nuclear architecture (11,43) provided that the danger of data biased because of the inherent methodological problems of both approaches are carefully avoided (44)(45)(46).…”

Section: Discussionmentioning

confidence: 99%

“…Human skin fibroblasts were grown directly on coverslips in DMEM supplemented with 10% FCS. All cells were fixed and prepared for 3D-FISH according to standard protocols (45,46). Briefly, cells were attached to coverslips by polylysin, fixed with 4% formaldehyde in 0.3ϫ PBS (lymphocytes and lymphoblastoid cells) or in 1ϫ PBS (fibroblasts), permeabilized with 0.5% Triton-X100, incubated in 20% glycerol, repeatedly frozen in liquid nitrogen, incubated in 0.1 M HCl for 5 min, and stored in 50% formamide/2ϫ SSC before use.…”

Section: Methodsmentioning

confidence: 99%

Maintenance of imprinting and nuclear architecture in cycling cells

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et al. 2007

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

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(14) provided a first case-in-point. They performed FISH experiments in normal, phytohemagglutinin (PHA)-stimulated human lymphocytes with a probe covering a segment of the Angelman syndrome/Prader-Willi syndrome (AS/PWS) locus on HSA 15q11-13 and measured 3D interdistances between the two signals in samples of 50 nuclei at different interphase stages. Approximately 38% of nuclei at G 1 , Ϸ18% at early S phase, Ϸ58% at late S phase, and Ϸ18% at G 2 revealed distances Յ2 m, which were considered as a proximity criterion for a possible functional interaction between the two AS/PWS loci. Notably, the significant increase of associations at late S phase was not found in cells from PWS and AS patients. Furthermore, LaSalle and Lalande (14) mentioned that they obtained similar results for the Beckwith-Wiedemann syndrome (BWS) locus, a second imprinted locus on HSA 11p15.5. Based on these findings, LaSalle and Lalande formulated the kissing hypothesis, which states that a transient spatial association between the oppositely imprinted regions during late S phase is a necessary condition to maintain the imprinted status of cycling cells from one cell cycle to the next. This hypothesis was widely cited and further supported by a 2D FISH study from Riesselmann and Haaf (15), who measured 2D interhomolog distances between oppositely imprinted loci on MMU 7 as a fraction of the nuclear diameter in flattened mouse fibroblast nuclei obtained in cytospin preparations. Somatic pairing was assumed when hybridization signals apparently overlapped or touched each other. By this criterion, significantly higher values were found for BrdU-positive nuclei (13%; 27/208 nuclei) compared with BrdUnegative nuclei (9.2%; 21 of 228 nuclei). No increase of pairing frequencies during S phase was found for control regions not subject to imprinting. In contrast, Nogami et al. (16), who studied the frequency of associations between the two AS loci with probes for the imprinted SNRPN alleles in 3D preserved interphase nuclei of human myeloid leukemia HL60 cells, did not find evidence to further support the hypothesis.Importantly, a large variability of 3D positions was noted for pairs of HSA 11 and HSA 15 chromosome territories (CTs) both within nuclei and between nuclei of different cells. Given this variability, a transient kissing event during late S phase between loci widely separated at other stages of interphase requires specific, long-range, directed chromatin movements. The present study was initiated with the intent to clarify whether such movements lead to the transient juxtaposition of entire homologous CTs or, alternatively, whether homologous CTs stay in place, whereas giant loops harboring genes poised for a kissing event expand from territory surfaces and meet each other in between (1,6,8,17). We visualized the two AS/PWS regions in PHA-stimulated lymphocytes by 3D-FISH with a BAC contig covering most of the AS/PWS locus and measured the 3D distances between the two regions in G 1 , early, mid, and late S phase, G 2 , as well as in...

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Spatial Preservation of Nuclear Chromatin Architecture during Three-Dimensional Fluorescence in Situ Hybridization (3D-FISH)

Cited by 249 publications

References 30 publications

The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

Smart 3D‐fish: Automation of distance analysis in nuclei of interphase cells by image processing

Maintenance of imprinting and nuclear architecture in cycling cells

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