Chromosome positioning and male infertility: it comes with the territory

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Abstract

<p class="first" id="Par1">The production of functional spermatozoa through spermatogenesis requires a spatially and temporally highly regulated gene expression pattern, which in case of alterations, leads to male infertility. Changes of gene expression by chromosome anomalies, gene variants, and epigenetic alterations have been described as the main genetic causes of male infertility. Recent molecular and cytogenetic approaches have revealed that higher order chromosome positioning is essential for basic genome functions, including gene expression. This review addresses this issue by exposing well-founded evidences which support that alterations on the chromosome topology in spermatogenetic cells leads to defective sperm function and could be considered as an additional genetic cause of male infertility. </p>

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Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes

Andreas Bolzer, Gregor Kreth, Irina Solovei … (2005)

Introduction Somatic cells within an organism possess genomes that are, with only a few minor exceptions, identical. However, various cell types may possess different epigenomes including the variation of DNA methylation and histone modification patterns. Epigenome variability accounts for cell-type-specific gene expression and silencing patterns in multicellular organisms. The impact of higher-order nuclear architecture on these patterns is not yet known [1]. Studies of higher-order chromatin arrangements in numerous cell types from different species form an indispensable part of a comprehensive approach to understanding epigenome evolution and cell-type-specific variability. Numerous research groups have attempted to map the large-scale organization and distribution of chromatin in cycling and postmitotic cell types (for reviews see [2,3,4,5,6,7,8]). Reliable topological maps, however, for the three-dimensional (3D) and 4D (3D plus spatiotemporal) arrangements of the two haploid chromosome complements in a diploid somatic cell nucleus have been lacking so far. Such 3D and 4D maps would provide the necessary foundation for studying the effect of higher-order chromatin distribution on nuclear functions, and are needed for different cell types at various stages of the cell cycle and at various stages of terminal differentiation. In addition to their importance for epigenome research, these maps should also help to understand karyotype evolution [9,10,11,12] and the formation of chromosomal rearrangements in irradiated or cancer cells [13,14,15,16,17]. In a 2D analysis of human fibroblast prometaphase rosettes, Nagele et al. [18,19] measured distances and angular separations for a number of chromosomes. These authors concluded that the maternal and paternal chromosome sets were separate, and that the heterologous chromosomes in each set showed highly nonrandom distributions. Subsequent studies further emphasized a highly ordered chromosome territory (CT) pattern for the nuclei of polarized human bronchial epithelial cells [20] and for nuclei of quiescent (G0) diploid (46, XY) human fibroblasts in culture [21]. Koss [20] reported that angles between the center of the nucleus and homologous pairs of Chromosome 1, 7, and X CTs were nearly identical in about two-thirds of bronchial epithelial cell nuclei to the angles reported by Nagele et al. for the same chromosome pairs in fibroblast prometaphase rosettes [18]. In contrast, Allison and Nestor [22] found a relatively random array of chromosomes on the mitotic ring of prometaphase and anaphase cells in cultured human diploid fibroblasts, diploid cells from human lung tissue, and human lymphocytes. The causes of these discrepancies have so far remained elusive. For nuclei of human lymphocytes, phytohemagglutinin-stimulated lymphoblasts, and lymphoblastoid cell lines, several groups have consistently reported a preferential positioning of gene-rich CTs (e.g., Homo sapiens chromosome [HSA] 19) towards the center of the nucleus, and of gene-poor CTs (e.g., HSAs 18 and Y) towards the nuclear periphery [23,24,25,26]. We recently confirmed this gene-density-correlated radial CT positioning for several other normal and malignant human cell types [26]. Bickmore and colleagues [23,27] also reported gene-density-correlated CT arrangements for cycling human fibroblasts. In contrast, Sun et al. [28] and our group [23,24,25,26] provided support for chromosome-size-correlated radial arrangements in quiescent fibroblasts. Although Sun et al. refer to nuclei studied in the G1-phase of the cell cycle, we believe that most of the cells included in their analysis were in a quiescent state (G0), since fibroblasts were grown on coverslips to 90%–95% confluence. Bridger et al. [27] reported that Chromosome 18 CTs were significantly closer to the nuclear periphery in S-phase fibroblasts than in quiescent fibroblasts. These findings suggest that cycling and noncycling fibroblasts differ in higher-order chromatin organization. We tested this hypothesis further in the present study. To overcome some of the technical limitations of previous studies, and to explore some of their inconsistencies, we employed 3D fluorescence in situ hybridization (FISH) protocols that allowed the differential coloring of all 24 chromosome types (22 autosomes plus X and Y) simultaneously within a population of human male fibroblasts (46, XY) under conditions preserving the 3D nuclear shape and structure to the highest possible degree [29,30]. In addition, we performed a series of two-color 3D FISH experiments in semi-confluent cultures, and determined the radial 3D positions of a subset of CTs (HSAs 1, 17–20, and Y) in quiescent (G0) and cycling (early S-phase) fibroblasts. Our data demonstrate unequivocally that the 3D arrangements of chromosomes in quiescent and cycling human fibroblasts follow probabilistic rules, and suggest that nuclear functions in human fibroblasts do not require a deterministic neighborhood pattern of homologous and heterologous chromosomes. Throughout, when we use the term “probabilistic chromosome order,” we mean an order that cannot be explained simply as a consequence of geometrical constraints that affect the distribution of chromosomes in mitotic rosettes or of CTs in cell nuclei. Constraints may enforce an arrangement of large and small chromosomes or CTs that deviates significantly from the prediction of a random order of points without any functional implications. Our long-term goal is to contribute to the elucidation of the set of rules (most likely a combination of probabilistic and deterministic) that generate cell-type-specific, functionally relevant higher-order chromatin arrangements. Results Differential Coloring of All 24 Chromosome Types in Nuclei of Human Male Diploid Fibroblasts Early-passage human fibroblast cultures (46, XY) were grown to confluence and maintained at this stage for several days before being fixed with buffered 4% paraformaldehyde. Under these conditions, the overwhelming majority (>99%) of cells were postmitotic (G0), as demonstrated by a lack of both pKi67 staining and incorporation of thymidine analogs (data not shown). Two 3D multiplex FISH (M-FISH) protocols were used for the differential coloring of all 24 human chromosome types (22 autosomes plus X and Y). The first approach was based on 3D M-FISH with 24 chromosome paint probes. Probes were differentially labeled using a combinatorial labeling scheme with seven different haptens/fluorochromes [31]. DAPI was used to stain nuclear DNA. Light-optical serial sections were separately recorded for each fluorochrome using digital wide-field epifluorescence microscopy (Figure 1). A second approach, called ReFISH [32], achieved differential staining of all 24 human chromosome types in two sequential FISH experiments with triple-labeled probe subsets. Light-optical serial sectioning of the same nuclei with laser confocal microscopy was performed after both the first and the second hybridization. Both approaches provided stringent accuracy for color classification of all CTs, and yielded the same results. Therefore, we combined data from 31 nuclei studied with the first approach and from 23 nuclei studied with the second approach (54 nuclei in total). Following careful correction for chromatic shifts, and image deconvolution in the case of wide-field microscopy (Figure S1), we performed overlays of the corresponding light-optical sections from all channels with voxel accuracy. CT classification was carried out on these overlays by the computer program goldFISH [33] (Figures 1B and S1C). This program classifies chromosomes by virtue of differences in the combinatorial fluorescent labeling schemes. Figure 1C shows the 3D reconstruction of a nucleus with all CTs viewed from different angles. Although the present experiments were not designed to address the issue of chromatin intermingling from neighboring CTs, it is obvious that goldFISH should have led to numerous misclassifications if there were excessive, widespread intermingling (for further discussion of CT boundaries, see [34]). For each individual CT the classification achieved by goldFISH was confirmed or rejected by careful visual inspection of light-optical sections. Any CT that could not be classified with certainty was omitted from further consideration. We were thus able to identify 2,030 CTs (82%) from a total of 2,484 CTs present in the 54 diploid fibroblast nuclei. As reference points for all distance and angle measurements reported below, we determined the 3D location of the fluorescence intensity gravity centers (IGCs) of individual painted CTs and the IGC of the nucleus (CN). Unless stated otherwise, when we describe below the position of a CT or prometaphase chromosome (PC) and report distance and angle measurements, we are referring to the 3D position of the CT's or PC's IGC. As a control for the reliability of the CT localizations, we subjected nuclei first studied by 24-color 3D FISH to a sequential five-color FISH experiment with individually labeled paint probes for Chromosomes 1 (Cy5), 3 (Cy3), 10 (FITC), 12 (Cy3.5), and 20 (Cy5.5). We were able to retrieve 11 of the 31 originally studied nuclei and to determine whether 3D positions of CTs first classified in the 24-color 3D FISH experiment could be confirmed after the second hybridization. In 96% of the re-hybridized CTs, the 3D position of the IGC differed by less than 1 μm, the range being between 0.01 and 1.3 μm. Size-Correlated Radial CT Positions in Nuclei of Quiescent (G0) Fibroblasts For every identified CT we measured the 3D radial CN–CT distance (from the CN to the CT's IGC). For a graphic overview of the location of each CT in 2D nuclear projections, the 3D positions of all IGCs obtained for a given CT were normalized and drawn into an ellipse representing the nuclear rim (Figure 2). As representative examples, Figure 2A shows nuclear projections of the normalized 3D IGC locations of CTs of HSAs 1, 7, 11, 18, 19, and Y, while Figure 2B shows cumulative 3D CN–CT graphs for the same CTs. Figures S2 and S4 provide the respective data for the entire chromosome complement. Notably, 3D radial CN–CT distance measurements did not reveal a significant difference between the positions of the gene-poor HSA 18 and the gene-rich HSA 19, although distinctly peripheral and interior locations, respectively, have been found for these two chromosomes in the spherical nuclei of lymphocytes and several other cell types (see Introduction). In summary, our data (Figures 2B, S2, S4, and S7 [left panel]) demonstrate that the territories of all small chromosomes—independent of their gene density—were preferentially found close to the center of the nucleus, while the territories of large chromosomes were preferentially located towards the nuclear rim. Figure 3 displays the positive correlation obtained in quiescent human fibroblasts for the mean normalized radial CN–CT distances and the DNA content of the chromosomes. The broad variability of radial CT positions seen in the set of 54 G0 nuclei indicates that radial CT arrangements in quiescent fibroblasts follow probabilistic, not deterministic, rules. To visualize the relative average positions of the IGCs of all heterologous CTs, we generated multidimensional scaling (MDS) plots [35,36] based on the mean of all normalized 3D CT–CT distances (Figure 4). Consistent with the data shown in Figure 3A, we found CTs from small chromosomes preferentially clustering towards the center of the nucleus, while CTs from large chromosomes were preferentially located towards the periphery. The acrocentric chromosomes (13–15, 21, and 22) carry nucleolar organizer regions (NORs) on their short arms, and active NORs are associated with the nucleoli. Since nucleoli are generally located away from the nuclear envelope in the inner nuclear space, we expected that normalized 3D CN–CT distances for all acrocentric chromosomes should be significantly shorter on average than 3D CN–CT distances for the largest chromosomes. Figure 5 confirms this expectation in the sample of 54 3D evaluated nuclei, emphasizing the sensitivity of the IGC approach. We also found a highly significant difference (p 0.05; Mann-Whitney U-test [U-test]). In contrast, the gene-poor Y territory was slightly more shifted towards the nuclear interior than the gene-rich HSA 17 CTs (Figure 6B and 6E). This shift was significant for cycling fibroblasts (p 0.05; U-test), but located significantly closer to the nuclear center than expected in the case of a uniform radial distribution (p 0.05; one-tailed K-S test of goodness of fit). With few exceptions pairwise comparisons of the mean angular separation between a pair of homologous CTs with the respective mean angle distribution in 60 random point distribution model nuclei did not show a significant difference (p > 0.05; two-tailed K-S test). Significant differences (p 0.05; two-tailed K-S test). (426 KB JPG). Click here for additional data file. Figure S11 Significance Levels for Pairwise Comparisons between Heterologous 3D CT–CN–CT Angles in 54 G0 Fibroblast Nuclei Significance levels were determined by the two-tailed K-S test. Green, not significant, p > 0.05; yellow, p 0.05; two-tailed K-S test). (328 KB JPG). Click here for additional data file. Video S1 Model Nucleus: CT Simulation The video shows the simulation of CT expansion in a fibroblast model nucleus according to the SCD model (compare with Figure 1). (567 KB MPG). Click here for additional data file.

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Genome-wide maps of nuclear lamina interactions in single human cells.

Jop Kind, Ludo Pagie, Sandra de Vries … (2015)

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT.

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Intermingling of Chromosome Territories in Interphase Suggests Role in Translocations and Transcription-Dependent Associations

Miguel R. Branco, Ana Pombo (2006)

Introduction Chromatin organization in the cell nucleus influences gene expression, DNA replication, damage, and repair. When the interphase nucleus forms, chromosomes partially decondense but still occupy distinct territories [ 1], which have nonrandom radial positions that are conserved through evolution [ 2– 5]. Current models suggest that chromosome territories (CTs) are separated by an interchromatin domain (ICD), rich in the nuclear machinery for nucleic acid metabolism. According to the ICD model, active genes are found in direct contact with the ICD [ 6], and occasionally fine chromosome fibers extend into this domain, where rare interchromosomal interactions may occur [ 1, 7– 9]. However, a physical separation between CTs is not supported by data on translocation frequencies and chromatin dynamics. Simulations of chromosome translocations based on models of chromosome organization have suggested the existence of a significant degree of intermingling between CTs [ 10– 12]. Furthermore, in vivo studies have shown that although chromatin domains are relatively stable [ 13], individual loci show diffusion dynamics constrained to approximately 0.4 μm [ 14– 16] and can exhibit movements as large as 1.5 μm [ 15]. This argues against a strict localization of chromatin within a CT that would prevent extensive intermingling. Recently, specific associations between loci on different chromosomes have been reported [ 17, 18], which are reminiscent of intrachromosomal clustering that is essential for correct gene expression [ 17, 19– 23]. It remains unclear whether these are just a few rare examples of interchromosomal associations that occur via chromatin fibers that extend from their own CTs or whether a greater potential exists for interactions through more extensive intermingling of chromosomes in interphase. Such interactions, if abundant, would be expected to determine chromosome organization and thereby influence the range of translocations that occur in each cell type. Previous data on chromosome morphology and organization have mainly originated from painting of whole chromosomes by fluorescence in situ hybridization (FISH) in three-dimensional (3D) nuclei. However, 3D-FISH is known to provide low spatial resolution and to compromise chromatin organization at the local level [ 24]. We have developed a novel FISH procedure for ultrathin cryosections (approximately 150 nm thick; cryo-FISH) of well-fixed [ 25], sucrose-embedded cells, that maximizes chromosome-painting efficiency, provides high resolution, and simultaneously preserves chromatin nanostructure. We show here that chromosomes intermingle significantly in interphase nuclei of human cells, arguing against the presence of an interchromosomal domain that separates CTs. The extent with which particular pairs of CTs intermingle correlates with the frequency of chromosome translocations in the same cell type [ 26]. Furthermore, we show that blocking of transcription changes the pattern of intermingling while preserving general chromosome properties, such as compaction and radial position, indicating that transcription-dependent associations between CTs are frequent enough to influence chromosome organization. In line with this view, we find that activation of the MHC class II gene cluster by interferon-gamma (IFN-γ) causes an increased colocalization of this locus with other chromosomes, concomitant with the relocation to a more external position in relation to its own CT [ 27]. Results/Discussion Chromosome Territories Intermingle Previous studies of chromosome organization during interphase have relied on the painting of chromosomes in whole nuclei, in conditions that compromise painting efficiency to preserve three-dimensionality. However, even in the best conditions, the nanostructure of chromatin at the level of single chromatin domains is lost [ 24]. To overcome this limitation, we developed a FISH procedure (cryo-FISH) using ultrathin cryosections of cells fixed under stringent conditions [ 25]. To test for chromosome intermingling we cohybridized pairs of whole chromosome paints to sections of phytohemagglutinin-activated human lymphocytes ( Figure 1). Binary masks were obtained for each CT and their intersections used to identify areas of colocalization ( Figure 1B– 1E). Fluorescence intensity profiles confirm that these areas contain DNA from two chromosomes ( Figure S1). Intermingling was detected for all chromosome pairs analyzed in these primary cells, but also in other human cell types (resting lymphocytes, HeLa cells, and primary fibroblasts; unpublished data). Due to the low resolution of the light microscope (LM; at best 200 nm in the x and y axes), we tested by electron microscopy (EM) whether DNA from different chromosomes is found in close proximity within areas of intermingling ( Figure 1G). After FISH, sections were first imaged on the LM to locate areas of intermingling ( Figure 1F), before indirectly immunolabeling the fluorochromes in the paints (FITC and rhodamine) with 5- and 10-nm gold particles, respectively ( Figure 1G). CTs labeled by immunogold particles strongly correlate with the corresponding LM image. Areas of intermingling identified by LM were found to contain colocalized gold particles labeling different chromosomes ( Figure 1G, inset, arrows; more than ten sections with intermingled CTs analyzed), showing that they are sufficiently close to interact at the molecular level. Stereoviews of regions of intermingling show that gold particles of different sizes are found at the same z-planes ( Figure 1H and 1I; eight regions of intermingling in four nuclear profiles analyzed), such that the intersection between CTs cannot be simply explained by distant territories that overlap within the thickness (approximately 150 nm) of the section. We next tested whether intermingling could result from artefactual chromatin disruption due to the harsh cryo-ISH procedure, in spite of the stringent fixation used. We compared the distribution of histone H2B, DNA, and sites of transcription labeled with Br-UTP, before and after ISH, and found that intermingling or the close proximity of gold particles labeling different chromosomes could not be explained by loss of fine chromatin structure during the procedure ( Figures 1J, 1K, and S2). Strikingly, the position of gold particles labeling histone H2B remains constant before and after FISH ( Figure 1J and 1K). To determine whether cryo-ISH had simply revealed the rare interactions between looped chromatin or showed more extensive intermingling of CTs, we measured how much of one CT intermingles with all others. We labeled sections with a Chromosome 3 paint, together with a probe that hybridizes with all other chromosomes ( Figure S3), and found that 41% of the volume of Chromosome 3 intermingles with the remaining genome. Although this argues against the existence of an interchromatin space that separates CTs, it remained possible that intermingling involved only loops of less condensed chromatin [ 1, 7, 8]. Therefore, we asked whether chromatin concentration within areas of intermingling is lower to that within a CT. We compared the fluorescence intensity of general DNA dyes (DAPI or TOTO-3 after RNase treatment) in intermingled regions of a CT with nonintermingled regions, or with the whole nucleoplasm, and found no significant differences (ratios of 1.12 ± 0.20 and 1.02 ± 0.52, respectively; n = 32). This shows that similar average DNA concentrations are present in intermingled and nonintermingled regions, indicating that chromatin has similar average properties in both areas. In fact, we also observed mixing of chromatin fibers within a CT (“intramingling”) between both arms of a chromosome (approximately 10% of Chromosome 3 volume; see also [ 28]). Therefore, regions of higher accessibility to transcription and pre-mRNA processing factors do not preferentially locate between CTs but are more uniformly distributed throughout the nucleoplasm, as shown previously [ 29, 30]. Different Extents of Chromosome Intermingling between CTs Correlate with Translocation Frequencies Chromosome intermingling has been suggested by modeling of translocation frequencies [ 10– 12], but not previously visualized, except for rare interactions [ 9]. A prediction of such models is that the extent of intermingling between each pair of chromosomes should be reflected in their translocation potential. We therefore measured the intermingling volumes for 24 pairs of chromosomes in activated human lymphocytes using a simple stereological principle (see Materials and Methods). Chromosome pairs were selected to reflect a wide range of translocation frequencies as measured in the same cell type by Arsuaga et al. [ 26] ( Table S1). The fraction of one chromosome (both homologs) that intermingles with any of the other 22 chromosomes is, on average, 2.1 ± 1.1%. This would correspond to 46% of each chromosome being intermingled with the rest of the genome (2.1% × 22 chromosomes), which is in agreement with the experimental value of 41% obtained for Chromosome 3. To obtain absolute values that are independent of CT volume, we expressed intermingling as a percentage of the nuclear volume ( Figure 2A). These values are representative of the average across the cell population, thus taking into account the frequency of CT association and the extent of intermingling when they are associated. Intermingling volumes between individual chromosome pairs vary by 20-fold ( Figure 2A), and show statistically significant differences ( p 3 h), rinsed in PBS, fixed in 0.5% glutaraldehyde in PBS (10 min), washed in distilled water, and incubated in 2% methylcellulose (10 min). Excess liquid was blotted and grids were left to dry. For the biotin-labeled paint, FITC-conjugated streptavidin (1/500; Sigma) or AlexaFluor350 conjugated Neutravidin (1/100; Molecular Probes) were used. The biotin-labeled BAC probe for the MHC II locus was detected using rhodamine-conjugated neutravidin (1/500; Molecular Probes), followed by a biotin-conjugated goat anti-avidin antibody (1/500; Vector) and rhodamine-conjugated neutravidin. PML was detected with anti-PML rabbit IgG clone H238 (1/10; Santa Cruz Biotechnology), followed by an AlexaFluor 488–conjugated goat anti-mouse antibody (1/1,000; Molecular Probes). Histone H2B was detected with a rabbit anti-histone H2B polyclonal antibody (1/100; Chemicon), followed by a goat anti-rabbit antibody conjugated with 5-nm gold particles (1/50; British BioCell). Serine2-phosphorylated PolII was indirectly immunolabeled with H5 (1/1,000; Covance, Berkeley, California, United States). After immunolabeling and washing (3×) in PBS, antibodies were fixed (1 h) with 8% paraformaldehyde in 250 mM HEPES (pH 7.6), before mock-ISH or chromosome painting. Microscopy For confocal laser scanning microscopy, images were collected sequentially on a Leica TCS SP2 (×100 PL APO 1.40 oil objective) equipped with argon (488 nm) and HeNe (543 nm; 633 nm) lasers or a Leica TCS SP1 (×100 PL APO 1.35 oil objective) equipped with UV (351/364 nm), argon (488 nm), krypton (568 nm), and HeNe (633 nm) lasers. For wide-field LM, images were collected sequentially on a Delta-Vision Spectris system (Applied Precision, Issaquah, Washington, United States) equipped with an Olympus IX70 wide-field microscope (×100 UPlanFl 1.3 oil objective), a charge-coupled device camera, and the following filters: DAPI, FITC, RD-TR-PE, CY-5, CFP, YFP. No bleed-through was detected in these conditions. The use of ultrathin cryosections allows for the use of wide-field microscopy with no reduction in axial (z) resolution and only a small reduction in lateral resolution [ 46]. For EM, images were collected on a JEOL 1011 transmission electron microscope (JEOL UK, Welwyn Garden City, Herts, United Kingdom) equipped with a cooled slow-scan KeenView charge-coupled device camera (1,392 × 1,024 pixels; Soft Imaging System, Münster, Germany). Image analysis and measurements For LM experiments, images (TIFF files) were automatically merged using a MatLab script (kindly provided by Tiago Branco, University College London, London, United Kingdom), saved as new TIFF files, and manually thresholded in Adobe Photoshop (Adobe Systems, Edinburgh, United Kingdom) to define masks for nuclei or CTs. Threshold values were chosen empirically so that the entire CT was selected but no widespread nuclear background was included. Independent drawing of masks by four different people on 10 images were compared to test the reliability of this empirical method. Variability in CT volume was found to be 15%, and in intermingling volumes was 30%, in the same order of magnitude as the variability obtained across independent experiments. The values of the areas of these masks and the intersection between the masks for both CTs were extracted using another MatLab script (Tiago Branco). CT and intermingling volumes were calculated according to stereological methods [ 47] after collecting random images of sections irrespective of their area and whether they contained CT signals (i.e., sections analyzed represented the whole nucleus). CT or intermingling areas were averaged across all sections and divided by the average of the nuclear areas. This ratio (R) is equivalent to the ratio of the respective average volumes, as shown here: where A ROI is the average CT or intermingling area, A NUC is the average nuclear area, V ROI and V NUC are the corresponding average volumes, and t is the section thickness. Using average section volumes for R gives the same result as using average whole nuclei volumes if enough random sections from different cells are included in the calculation. To obtain several values for R within one hybridization experiment (to allow statistical analysis), images were randomly grouped and R was calculated for each group. Standard deviations remained constant with increasing number of groups until a group size was reached at which R did not contain enough information and the standard deviation increased abruptly. The highest number of groups before this increase was used. Group size varied between different chromosome pairs, averaging 55 sections per group, and up to four groups were used in an experiment (a total of 57 to 211 sections were analyzed in individual experiments). Standard deviations obtained by this method were consistent with standard deviations between independent hybridization experiments. The R values were used for statistical tests, and considered to have a normal distribution, as normality plots for the analysis of residuals were positive. Two-sample comparisons were performed by two-tailed t-test and multisample comparisons by ANOVA. Regression analyses using an F-test were performed to test the significance of variable correlations. For the analysis of the MHC II locus data, we used Fisher's exact test for 2 × 2 contingency tables and chi-squared test for larger tables. Supporting Information Figure S1 Fluorescence Intensity Profiles of the Images in Figure 1B– 1E (469 KB JPG) Click here for additional data file. Figure S2 Additional Control Experiments Showing Preservation of Nuclear Structure during cryo-FISH (954 KB JPG) Click here for additional data file. Figure S3 Quantification of the Total Intermingling of Chromosome 3 with the Remaining Genome (214 KB JPG) Click here for additional data file. Figure S4 Graphical Analyses of the Distribution of Active RNA Polymerase II Sites within CTs and Areas of Intermingling (13 KB PDF) Click here for additional data file. Figure S5 Volumes for All CTs in Human Female Lymphocytes and Correlation with DNA Content (12 KB PDF) Click here for additional data file. Protocol S1 Quantification of Nuclear Volume That Contains Intermingled CTs (24 KB DOC) Click here for additional data file. Table S1 Interchange Yields in Phytohemagglutinin-Activated Human Lymphocytes (47 KB PDF) Click here for additional data file.