A method for simultaneously delineating multiple targets in 3D-FISH using limited channels, lasers, and fluorochromes

Many studies have suggested a link between the spatial organization of genomes and fundamental biological processes such as genome reprogramming, gene expression, and differentiation. Multicolor fluorescence in situ hybridization on three-dimensionally preserved nuclei (3D-FISH), in combination with confocal microscopy, has become an effective technique for analyzing 3D genome structure and spatial patterns of defined nucleus targets including entire chromosome territories and single gene loci. This technique usually requires the simultaneous visualization of numerous targets labeled with different colored fluorochromes. Thus, the number of channels and lasers must be sufficient for the commonly used labeling scheme of 3D-FISH, “one probe-one target”. However, these channels and lasers are usually restricted by a given microscope system. This paper presents a method for simultaneously delineating multiple targets in 3D-FISH using limited channels, lasers, and fluorochromes. In contrast to other labeling schemes, this method is convenient and simple for multicolor 3D-FISH studies, which may result in widespread adoption of the technique. Lastly, as an application of the method, the nucleus locations of chromosome territory 18/21 and centromere 18/21/13 in normal human lymphocytes were analyzed, which might present evidence of a radial higher order chromatin arrangement.     

Smart 3D-FISH: automation of distance analysis in nuclei of interphase cells by image processing.

BACKGROUND: Detection of fluorescent probes by fluorescence in situ hybridization in cells with preserved three-dimensional 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',6-diamino-2-phenylindole counterstained nuclei to measure the relative positioning of spot loci in the nucleus and inter-spot distance. Results from a pilot experiment using Smart 3D-FISH on ENL, MLL, and AF4 genes in two lymphoblastic cell lines were satisfactory and consistent with data published in the literature. CONCLUSION: Smart 3D-FISH should greatly facilitate image processing and analysis of 3D-FISH images by providing a useful tool to overcome the laborious task of image segmentation based on user-defined parameters and decrease subjectivity in data analysis. It is available as a set of plugins for ImageJ software.     

Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence

Using in situ hybridization techniques, a fixation step must precede denaturation to prevent disintegration of the chromosomes. The analysis of nuclei fixed by paraformaldehyde, preserving the native structure (three-dimensional or 3D fixation and analysis) has become possible with the development of confocal microscopy; however, the analysis of those fixed by methanol and acetic acid, dehydrating the nuclei (two-dimensional or 2D fixation and analysis), remains a very valuable tool for practical use in diagnostics and also in many cases for research. We compared both types of fixation and analyses using different cell lines and different DNA probes. Fixation of cells by methanol and acetic acid leads to the enlargement of contact of nuclei with the slide surface, resulting in a substantial increase of nuclear diameter, flattening of the nucleus, and consequently to a distortion of gene topology. Our results indicate that chromatin structures located in the outer parts of the nuclear volume (e.g., heterochromatin of some centromeres) are relatively shifted to the membrane of these nuclei, keeping the absolute centromere-membrane distance constant. On the other hand, euchromatin located in the inner parts of the nuclear volume is not shifted outside proportionally to the increase of molecular dimensions; consequently, the relative distances for the center of nucleus to gene are smaller after methanol-acetic acid fixation. The limitations of the analysis of dehydrated preparations for practical use and in research are discussed.     

Spatio-temporal organization of DNA replication in murine embryonic stem, primary, and immortalized cells

The extent to which chromosomal domains are reorganized within the nucleus during differentiation is central to our understanding of how cells become committed to specific developmental lineages. Spatio-temporal patterns of DNA replication are a reflection of this organization. Here, we demonstrate that the temporal order and relative duration of these replication patterns during S-phase are similar in murine pluripotent embryonic stem (ES) cells, primary adult myoblasts, and an immortalized fibroblast line. The observed patterns were independent of fixation and denaturation techniques. Importantly, the same patterns were detected when fluorescent nucleotides were introduced into living cells, demonstrating their physiological relevance. These data suggest that heritable gene silencing during commitment to specific cell lineages is not mediated by global changes in the sub-nuclear organization and replication timing of chromosome domains.     

The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture

Three-dimensional structured illumination microscopy (3D-SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ~100 nm range. We present first results and assess the potential using 3D-SIM in combination with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution. We address the topography of DAPI-stained DNA in nuclei before and after 3D-FISH, nuclear pores and the lamina, chromosome territories, chromatin domains, and individual gene loci. We also look at the replication patterns of chromocenters and the topographical relationship of Xist-RNA within the inactive X-territory. These examples demonstrate that an appropriately adapted 3D-FISH/3D-SIM approach preserves key characteristics of the nuclear ultrastructure and that the gain in information obtained by 3D-SIM yields new insights into the functional nuclear organization.     

CRISPR-based genomic loci labeling revealed ordered spatial organization of chromatin in living diploid human cells

The eukaryotic genome is compacted in the form of chromatin within the nucleus. Whether the spatial distribution of the genome is ordered or not has been a longstanding question. Answering this question would enable us to understand nuclear organization and cellular processes more deeply. Here, we applied a modified CRISPR/dCas9 system to label the randomly selected genomic loci in diploid living cells, which were visualized by high-resolution wide-field imaging. To analyze the spatial positions of three pairs of genomic loci, three sets of parameters were progressively measured: i) the linear distance between alleles; ii) the radial distribution of the genomic loci; and iii) the linear distances between three pairs of genomic loci on nonhomologous chromosomes. By accurate labeling, geometric measuring and statistical analysis, we demonstrated that the distribution of these genomic loci in the 3D space of the nucleus is relatively stable in both late G1 and early S phases. Collectively, our data provided visual evidence in live cells, which implies the orderly spatial organization of chromatin in the nucleus. The combination of orderliness and flexibility ensures the methodical and efficient operation of complex life systems. How the nucleus adopts this ordered 3D structure in living cells is thought-provoking.     

Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes

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.     

Chromatin dynamics during DSB repair

We show that double strand breaks (DSBs) induced in chromatin of low as well as high density by exposure of human cells to gamma-rays are repaired in low-density chromatin. Extensive chromatin decondensation manifested in the vicinity of DSBs by decreased intensity of chromatin labelling, increased H4K5 acetylation, and decreased H3K9 dimethylation was observed already 15 min after irradiation. Only slight movement of sporadic DSB loci for short distances was noticed in living cells associated with chromatin decondensation around DSBs. This frequently resulted in their protrusion into the low-density chromatin domains. In these regions, the clustering (contact or fusion) of DSB foci was seen in vivo, and in situ after cell fixation. The majority of these clustered foci were repaired within 240 min, but some of them persisted in the nucleus for several days after irradiation, indicating damage that is not easily repaired. We propose that the repair of DSB in clustered foci might lead to misjoining of ends and, consequently, to exchange aberrations. On the other hand, the foci that persist for several days without being repaired could lead instead to cell death.     

Supercoiling of 30-nm chromatin fibers around a nuclear axis in human retinal pigment cell nuclei: Electron microscope evidence for a chromatin macrostructure

Nucleosomes and higher-order chromatin structures have been identified in the range of resolution of 10–30 nm. No information is available on higher configurations that involve chromatin arrangements at the nuclear level. Thin sections of human retinal pigment cell nuclei reveal an orderly array of 30-nm chromatin fibers on the inner nuclear surface. The observations can be three-dimensionally explained by a coiled or a serially circular arrangement of fibers around a central axis of the nucleus. The axial chromatin orientation shows no apparent relationship to the epithelial cell polarity nor to the irregular shape of the nuclei. The described configuration, which is compatible with a chromatin structure of third order, represents the first example of chromatin architecture coherently organized at the nuclear level.     

Evolutionarily conserved,cell type and species-specific higher order chromatin arrangements in interphase nuclei of primates

Several studies demonstrated a gene-density-correlated radial organization of chromosome territories (CTs) in spherically shaped nuclei of human lymphocytes or lymphoblastoid cells, while CT arrangements in flat-ellipsoidal nuclei of human fibroblasts are affected by both gene density and chromosome size. In the present study, we performed fluorescence in situ hybridization (FISH) experiments to three-dimensionally preserved nuclei (3D-FISH) from human and nonhuman primate cultured lymphoblastoid cells and fibroblasts. We investigated apes, Old, and New World monkeys showing either evolutionarily conserved karyotypes, multiple translocations, fusions, or serial fissions. Our goal was to test whether cell type specific differences of higher order chromatin arrangements are evolutionarily conserved in different primate lineages. Whole genome painting experiments and further detailed analyses of individual chromosomes indicate a gene-density-correlated higher order organization of chromatin in lymphoblastoid cell nuclei of all studied primate species, despite evolutionary chromosome reshuffling. In contrast, in primate fibroblast nuclei evolutionary translocations, fissions and fusions resulted in positional shifts of orthologous chromosome segments, thus arguing against a functional role of chromosome size-dependent spatial chromatin arrangements and for geometrical constraints in flat-ellipsoidal fibroblast nuclei. Notably, in both cell types, regions of rearranged chromosomes with distinct differences in gene density showed polarized arrangements with the more gene-dense segment oriented towards the nuclear interior. Our results indicate that nonrandom breakage and rejoining of preferentially gene-dense chromosomes or chromosome segments may have occurred during evolution. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Replication labeling patterns and chromosome territories typical of mammalian nuclei are conserved in the early metazoan<Emphasis Type="Italic"> Hydra</Emphasis>

To investigate the evolutionary conservation of higher order nuclear architecture previously described for mammalian cells we have analyzed the nuclear architecture of the simple polyp Hydra. These diploblastic organisms have large nuclei (8–10 m) containing about 3×10⁹ bp of DNA organized in 15 chromosome pairs. They belong to the earliest metazoan phylum and are separated from mammals by at least 600 million years. Single and double pulse labeling with halogenated nucleotides (bromodeoxyuridine, iododeoxyuridine and chlorodeoxyuridine) revealed striking similarities to the known sequence of replication labeling patterns in mammalian nuclei. These patterns reflect a persistent nuclear arrangement of early, mid-, and late replicating chromatin foci that could be identified during all stages of interphase over at least 5–10 cell generations. Segregation of labeled chromatids led after several cell divisions to nuclei with single or a few labeled chromosome territories. In such nuclei distinct clusters of labeled chromatin foci were separated by extended nuclear areas with non-labeled chromatin, which is typical of a territorial arrangement of interphase chromosomes. Our results indicate the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals and suggest the existence of conserved mechanism(s) controlling this architecture.Abbreviations CT Chromosome territory - BrdU Bromodeoxyuridine - IdU Iododeoxyuridine - CldU Chlorodeoxyuridine Communicated by E.A. Nigg     

High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains

We analyzed the incorporation of bromodeoxyuridine (BrdUrd) into DNA in exponentially growing murine erythroleukemia cells (FLC-745), using fluorescent anti-BrdUrd antibodies with light microscopy and flow cytometry. The fine localization of the DNA replicating sites was investigated at the ultrastructural level by using a second antibody conjugated with colloidal gold. The latter approach, which does not require acidic denaturation of the DNA, enables preservation of good morphology and obtains a better resolution power than that of electron microscopic autoradiography, the percentage of labeled cells obtained with the two techniques being comparable. After short BrdUrd pulses, characteristic distribution of the labeling can be identified in the heterochromatin, in interchromatin domains, or at the boundary between the dispersed and the condensed chromatin. Similar patterns are also observable in the nuclear structures which condense after acid denaturation, suggesting that DNA replication takes place at fixed sites associated with the nuclear matrix.     

不同固定方法对植物细胞扫描电镜观察用冷冻断裂样品的影响

用冷冻断裂法在扫描电镜下研究了洋葱(Allium cepa)根端分生组织细胞内部的三维结构。采用了两种固定方法。冷冻断裂前只用1％锇酸固定的材料容易在细胞质和核之间断开,而用卡诺固定液(无水乙醇:冰醋酸3:1)前固定,然后再用1％锇酸固定的材料容易使细胞核断裂。前一固定方法适于研究细胞质的内部结构(细胞骨架的纤维、线粒体、内质网等及其三维分布关系):后一固定方法适于研究核内结构(染色质、核仁、核基质纤维)的三维形象,特别是核仁纤维中心染色质的三维结构。     

Effects of Different Fixatives on Freeze-Fractured Samples of Plant Cells for SEM Observations

Three-dimensional structures of meristematic cells of Allium cepa were studied using freeze-fracture method under the scanning electron microscope. Two fixation procedures were used. The cells were often fractured between eytoplasm and nucleus when the materials were fixed in 1% OsO4 alone before freeze fracture, whereas the nuclei, were frequently fractured if the materials were fixed first in Carnoy's, solution (ethanol: acetic acid=3:l) and then in 1% OsO4 before freeze fracture. The former fixation procedure is suitable for the study of the interior structures of cytoplasm such as cytoskeleton fibres, mitochondria, endoplasmic reticulum and their three-dimensional topography. The latter fixation method is suitable for the study of interior structures of nucleus such as chromatin, nucleoli, nuclear matrix filaments and their 3-dimensional architectures, especially the 3-dimensional structures of chromatin in fibrillar centre of the nucleolus.     

Distinct chromatin organization in the germ line founder cell of the Caenorhabditis elegans embryo

Cells belonging to the germ lineage segregate physically and molecularly from their somatic neighbors during embryogenesis. While germ line‐specific chromatin modifications have been identified at later stages in the Caenorhabditis elegans nematode, none have been found in the single P₄ germ line founder cell that arises at the beginning of gastrulation. Using light and electron microscopy, we now report that the chromatin organization in the germ line founder cell of the early C. elegans embryo is distinct from that in the neighboring somatic cells. This unique organization is characterized by a greater chromatin compaction and an expansion of the interchromatin compartment. The ultrastructure of individual chromatin domains does not differ between germ line and somatic cells, pointing to a specific organization mainly at the level of the whole nucleus. We show that this higher order reorganization of chromatin is not a consequence of the P₄ nucleus being smaller than somatic nuclei or having initiated mitosis. Imaging of living embryos expressing fluorescent markers for both chromatin and P granules revealed that the appearance of a distinct chromatin organization in the P₄ cell occurs approximately 10 min after its birth and coincides with the aggregation of P granules around the nucleus, suggesting a possible link between these two events. The higher order reorganization of chromatin that is reported here occurs during the establishment of definitive germ cell identity. The changes we have observed could therefore be a prerequisite for the programming of chromatin totipotency.     

Intranuclear Distribution of Galectin-3 in Mouse 3T3 Fibroblasts: Comparative Analyses by Immunofluorescence and Immunoelectron Microscopy

The intracellular distribution of carbohydrate binding protein 35 (CBP35), recently named galectin-3, was studied in mouse 3T3 fibroblasts, using immunofluorescence at the light microscope level and immunogold labeling at the ultrastructural level. In general, serum-stimulated, proliferating cells showed higher levels of labeling than quiescent cultures of the same cells. In the proliferating cells, the labeling intensity was higher in the nucleus than in the cytoplasm. Treatment of permeabilized cells or thin sections with ribonuclease A decreased the immunolabeling intensity, whereas parallel control treatments with deoxyribonuclease I failed to yield the same effect. While there appears to be general agreement between the immunofluorescence and the ultrastructural results regarding the level of CBP35 and its association with nuclear ribonucleoprotein complexes, there was one striking difference in terms of labeling of specific subnuclear structures. Immunofluorescence results indicate diffuse distribution of CBP35 within the nucleus, but the label appears to be excluded from certain "black holes," which most probably correspond to nucleoli. On the other hand, immunogold particles were observed in electron microscopy, mainly in interchromatin spaces, except for interchromatin granule clusters, at the border of condensed chromatin, on the dense fibrillar component, and at the periphery of the fibrillar centers of nucleoli.     

Innate structure of DNA foci restricts the mixing of DNA from different chromosome territories

The distribution of chromatin within the mammalian nucleus is constrained by its organization into chromosome territories (CTs). However, recent studies have suggested that promiscuous intra- and inter-chromosomal interactions play fundamental roles in regulating chromatin function and so might define the spatial integrity of CTs. In order to test the extent of DNA mixing between CTs, DNA foci of individual CTs were labeled in living cells following incorporation of Alexa-488 and Cy-3 conjugated replication precursor analogues during consecutive cell cycles. Uniquely labeled chromatin domains, resolved following random mitotic segregation, were visualized as discrete structures with defined borders. At the level of resolution analysed, evidence for mixing of chromatin from adjacent domains was only apparent within the surface volumes where neighboring CTs touched. However, while less than 1% of the nuclear volume represented domains of inter-chromosomal mixing, the dynamic plasticity of DNA foci within individual CTs allows continual transformation of CT structure so that different domains of chromatin mixing evolve over time. Notably, chromatin mixing at the boundaries of adjacent CTs had little impact on the innate structural properties of DNA foci. However, when TSA was used to alter the extent of histone acetylation changes in chromatin correlated with increased chromatin mixing. We propose that DNA foci maintain a structural integrity that restricts widespread mixing of DNA and discuss how the potential to dynamically remodel genome organization might alter during cell differentiation.