Figure 1 - uploaded by Miguel R Branco
Content may be subject to copyright.
Chromosome Territories Intermingle in the Nucleus of Human Cells Chromosomes were painted in cryosections (approximately 150 nm thick) of human lymphocytes and visualized by fluorescence microscopy (A–F) or indirectly immunolabeled with gold particles before imaging by electron microscopy (G–I). (A) Example of a nuclear section showing intermingling between Chromosomes 5 and 7. (B–E) Intermingling is best seen in gray-scale images after the mask for one chromosome (white line) is overlaid on the image of the other chromosome. The intersection between masks for both chromosomes is shown in the merged images (yellow line), representing areas of intermingling. Fluorescence intensity line scans of relevant areas in images (B–E) can be in Figure S1. (F and G) Five- and 10-nm gold particles labeling Chromosomes 1 and 2 (G; pseudo-colored green and red, respectively) are intermingled within the intersections identified on the light microscope in the same nuclear section (F). Gold particles labeling different CTs can be found in close proximity within areas of intermingling (G, arrows in inset). (H and I) Stereoviews of a region of colocalization (*) between Chromosome 1 (5 nm gold) and 2 (10 nm gold) were obtained by collecting images tilted À 6 8 (H) and þ 6 8 (I) relative to the z -axis. 3D visualization shows that differently-sized particles lie adjacent in the same z planes. (J and K) Histone H2B was indirectly immunolabeled with 5-nm gold particles and imaged on the EM before (J) and after (K) mock FISH; gold particles are spatially preserved in both heterochromatic (arrow 1) and euchromatic (arrow 2) regions. Bars: (A and F) 1 l m; (B–E) 0.5 l m; (G–K) 50 nm. DOI: 10.1371/journal.pbio.0040138.g001
Source publication
After mitosis, mammalian chromosomes partially decondense to occupy distinct territories in the cell nucleus. Current models propose that territories are separated by an interchromatin domain, rich in soluble nuclear machinery, where only rare interchromosomal interactions can occur via extended chromatin loops. In contrast, recent evidence for chr...
Contexts in source publication
Context 1
... test for chromosome intermingling we cohybridized pairs of whole chromosome paints to sections of phytohe- magglutinin-activated human lymphocytes (Figure 1). Binary masks were obtained for each CT and their intersections used to identify areas of colocalization ( Figure 1B-1E). ...
Context 2
... test for chromosome intermingling we cohybridized pairs of whole chromosome paints to sections of phytohe- magglutinin-activated human lymphocytes (Figure 1). Binary masks were obtained for each CT and their intersections used to identify areas of colocalization ( Figure 1B-1E). Fluores- cence intensity profiles confirm that these areas contain DNA from two chromosomes ( Figure S1). ...
Context 3
... masks were obtained for each CT and their intersections used to identify areas of colocalization ( Figure 1B-1E). Fluores- cence 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). ...
Context 4
... 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). ...
Context 5
... 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. ...
Context 6
... 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. ...
Context 7
... 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 (approx- imately 150 nm) of the section. ...
Context 8
... 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 (approx- imately 150 nm) of the section. ...
Context 9
... 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). ...
Context 10
... 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). ...
Context 11
... intersection between masks for both chromosomes is shown in the merged images (yellow line), representing areas of intermingling. Fluorescence intensity line scans of relevant areas in images (B-E) can be in Figure S1. (F and G) Five-and 10-nm gold particles labeling Chromosomes 1 and 2 (G; pseudo-colored green and red, respectively) are intermingled within the intersections identified on the light microscope in the same nuclear section (F). ...
Similar publications
Selected childhood and adult neoplasm exemplify fundamental differences in their propensity for genomic change. DNA replication is essential for the formation of neuroepithelial tumors, probably because the genome can be remodeled. Nonetheless, several differentiated and stable childhood neoplasms retain their nuclear controls for differentiation....
The organization of chromosomes is important for various biological processes and is involved in the formation of rearrangements often observed in cancer. In mammals, chromosomes are organized in territories that are radially positioned in the nucleus. However, it remains unclear whether chromosomes are organized relative to each other. Here, we ex...
Citations
... Cremer and Branco have demonstrated that acrocentric chromosomes, including chromosomes 15, 21, and 22, are frequently positioned near each other. This spatial proximity within the nucleus can facilitate interchromosomal interactions, which may be relevant to both functional interactions and regulatory mechanisms of chromosomes, including replication time and ICE [44,45]. ...
Chromosomal rearrangements can interfere with the disjunction and segregation of other chromosome pairs not involved in the rearrangements, promoting the occurrence of numerical abnormalities in resulting gametes and predisposition to trisomy in offspring. This phenomenon of interference is known as the interchromosomal effect (ICE). Here we report a prenatal case potentially generated by ICE. The first-trimester screening ultrasound of the pregnant woman was normal, but the NIPT indicated a high risk for three copies of chromosome 21, thus suspecting trisomy 21 (T21). After a comprehensive clinical evaluation and genetic counseling, the couple decided to undergo amniocentesis. The prenatal karyotype confirmed T21 but also showed a balanced translocation between the long arm of chromosome 15 (q22) and the long arm of chromosome 22. The parents’ karyotypes also showed that the mother had the 15;22 translocation. We reviewed T21 screening methods, and we performed a literature review on ICE, a generally overlooked phenomenon. We observed that ours is the first report of a prenatal case potentially due to ICE derived from the mother. The recurrence risk of aneuploidy in the offspring of translocated individuals is likely slightly increased, but it is not possible to estimate to what extent. In addition to supporting observations, there are still open questions such as, how frequent is ICE? How much is the aneuploidy risk altered by ICE?
... The final and highest order of chromatin are chromosome territories. Each chromosome is found in a discrete region of nucleus, and the only instances of overlapping between chromosomes is at the boundaries of their territories [39,40]. Generally, these territories are spherical, as in many mammalian and Arabidopsis cells [41,42]. ...
Chromatin is the complex of DNA and associated proteins found in the nuclei of living organisms. How it is organized is a major research field as it has implications for replication, repair, and gene expression. This review summarizes the current state of the chromatin organization field, with a special focus on chromatin motor complexes cohesin and condensin. Containing the highly conserved SMC proteins, these complexes are responsible for organizing chromatin during cell division. Additionally, research has demonstrated that condensin and cohesin also have important functions during interphase to shape the organization of chromatin and regulate expression of genes. Using the model organism C. elegans, the authors review the current knowledge of how these complexes perform such diverse roles and what open questions still exist in the field.
... Beyond mulKplexed protein imaging, a potenKally even more powerful aspect of cryosecKons is that the same secKons can be subject to both immunostaining and fluorescence in situ hybridizaKon 39,69 , enabling analyses of the interplay between targeted proteins and specific sequences of RNA and/or DNA. Here, we performed proof-of-principle tokPAINT imaging of α-tubulin in cryosecKons that had addiKonally . ...
DNA-PAINT combined with total Internal Reflection Fluorescence (TIRF) microscopy enables the highest localization precisions, down to single nanometers in thin biological samples, due to TIRF’s unique method for optical sectioning and attaining high contrast. However, most cellular targets elude the accessible TIRF range close to the cover glass and thus require alternative imaging conditions, affecting resolution and image quality. Here, we address this limitation by applying ultrathin physical cryosectioning in combination with DNA-PAINT. With “tomographic & kinetically-enhanced” DNA-PAINT (tokPAINT), we demonstrate the imaging of nuclear proteins with sub-3 nanometer localization precision, advancing the quantitative study of nuclear organization within fixed cells and mouse tissues at the level of single antibodies. We believe that ultrathin sectioning combined with the versatility and multiplexing capabilities of DNA-PAINT will be a powerful addition to the toolbox of quantitative DNA-based super-resolution microscopy in intracellular structural analyses of proteins, RNA and DNA in situ .
... Reconstruction of 3D chromatin conformations is a promising approach to improve our understanding of processes in the cell. While coarse information about the existence of chromosome territories has already been obtained with different methods (Cremer et al., 1993;Cremer and Cremer, 2001;Bolzer et al., 2005;Branco and Pombo, 2006), chromosome conformation capture methods like Hi-C (Lieberman-Aiden et al., 2009) provide more detailed and accurate information about the organization of chromatin. These results can, e.g., be used to improve the understanding of the cell cycle (Naumova et al., 2013), gene regulation (Cremer and Cremer, 2001) and differentiation of cell types (Dixon et al., 2015). ...
Detailed understanding of the 3D structure of chromatin is a key ingredient to investigate a variety of processes inside the cell. Since direct methods to experimentally ascertain these structures lack the desired spatial fidelity, computational inference methods based on single cell Hi-C data have gained significant interest. Here, we develop a progressive simulation protocol to iteratively improve the resolution of predicted interphase structures by maximum-likelihood association of ambiguous Hi-C contacts using lower-resolution predictions. Compared to state-of-the-art methods, our procedure is not limited to haploid cell data and allows us to reach a resolution of up to 5,000 base pairs per bead. High resolution chromatin models grant access to a multitude of structural phenomena. Exemplarily, we verify the formation of chromosome territories and holes near aggregated chromocenters as well as the inversion of the CpG content for rod photoreceptor cells.
... SVs may not always be isolated mutational events; when an SV occurs, it may be one of many interrelated disruptive events happening in the genome. For example, CRs such as fusions and translocations may disrupt nuclear organization, predisposing the formation of additional rearrangements (Branco and Pombo 2006; E.L. Berdan et al. et al. 2021). Similarly, SVs formed by the insertion, deletion, or duplication of TEs can emerge together during a burst of activity in specific TE families (Wells and Feschotte 2020). ...
Research on the genomic architecture of speciation has increasingly revealed the importance of structural variants (SVs) that affect the presence, abundance, position, and/or direction of a nucleotide sequence. SVs include large chromosomal rearrangements such as fusion/fissions and inversions and translocations, as well as smaller variants such as duplications, insertions, and deletions (CNVs). Although we have ample evidence that SVs play a key role in speciation, the underlying mechanisms differ depending on the type and length of the SV, as well as the ecological, demographic, and historical context. We review predictions and empirical evidence for classic processes such as underdominance due to meiotic aberrations and the coupling effect of recombination suppression before exploring how recent sequencing methodologies illuminate the prevalence and diversity of SVs. We discuss specific properties of SVs and their impact throughout the genome, highlighting that multiple processes are at play, and possibly interacting, in the relationship between SVs and speciation.
... They are located in the cell nucleus. Each chromosome contains many genes, which are specific regions of DNA that code for proteins or functional RNA molecules [7]. ...
The decoding of the human genome has been a landmark achievement in the field of genomics, generating vast amounts of DNA sequencing data that necessitate sophisticated analysis techniques. In recent years, machine learning has emerged as a powerful tool in unravelling the complexities of genomic data and expediting research discoveries. This article explores the integration of machine learning techniques in DNA sequencing analysis, elucidating their applications in genome assembly, variant calling, personalized medicine, and drug discovery. Additionally, it addresses the ethical considerations surrounding the use of genomic data. By harnessing the potential of machine learning, researchers are unlocking new insights into human genetics and paving the way for transformative advancements in healthcare and scientific understanding.
... Earlier studies also reported a correlation between inter-chromosome interactions and gene translocation in Drosophila and human cells. 25,58 To quantify the amount of overlap between our polymer-grafted colloids, we calculate the number of steric contacts between polymer chains of neighboring colloids by assigning a contact distance of r bb = 1.5 between the beads of any two colloidal particles (Fig. 4). This distance corresponds to the occurrence of a steric contact between any two beads (cf. ...
Interphase chromosomes are known to organize non-randomly in the micron-sized eukaryotic cell nucleus and occupy certain fraction of nuclear volume, often without mixing. Using extensive coarse-grained simulations, we model such chromosome structures as colloidal particles whose surfaces are grafted by cyclic polymers. This model system is known as Rosetta. The cyclic polymers, with varying polymerization degrees, mimic chromatin loops present in interphase chromosomes, while the rigid core models the chromocenter section of the chromosome. Our simulations show that the colloidal chromosome model provides a well-separated particle distribution without specific attraction between the chain monomers. As the polymerization degree of the grafted cyclic chains decreases while maintaining the total chromosomal length (e.g., the more potent activity of condensin-family proteins), the average chromosomal volume becomes smaller, inter-chromosomal contacts decrease, and chromocenters organize in a quasi-crystalline order reminiscent of a glassy state. This order weakens for polymer chains with a characteristic size on the order of the confinement radius. Notably, linear-polymer grafted particles also provide the same chromocenter organization scheme. However, unlike linear chains, cyclic chains result in less contact between the polymer layers of neighboring chromosome particles, demonstrating the effect of DNA breaks in altering genome-wide contacts. Our simulations show that polymer-grafted colloidal systems could help decipher 3D genome architecture along with the fractal globular and loop-extrusion models.
... Transcribable locus-rich areas are likely to be found at the boundaries of chromatin territories (Fig. 2) (Shah et al. 2018;Schoenfelder et al. 2010). Different chromosomal territories can interact with one another, especially near their borders (Branco and Pombo 2006). At the megabase level, genetic areas with comparable chromatin properties likely communicate with one another (Lieberman-Aiden et al. 2009). ...
In
eukaryotes, the genome
does not emerge in a specific shape but rather as a hierarchial bundle within the nucleus. This multifaceted genome organization consists of multiresolution cellular structures, such as chromosome territories, compartments, and topologically associating domains, which are frequently defined by architecture, design proteins including CTCF and cohesin, and chromatin loops. This review briefly discusses the advances in understanding the basic rules of control, chromatin folding, and functional areas in early embryogenesis. With the use of chromosome capture techniques, the latest advancements in technologies for visualizing chromatin interactions come close to revealing 3D genome formation frameworks with incredible detail throughout all genomic levels, including at single-cell resolution. The possibility of detecting variations in chromatin architecture might open up new opportunities for disease diagnosis and prevention, infertility treatments, therapeutic approaches, desired exploration, and many other application scenarios.
... In interphase, chromosomes decondense within the nucleus, but instead of mixing, they become organized in distinct regions (territories) of the nucleus [3]. While the chromosomes are organized into distinct territories, overlapping of those territories is also observed in recent studies, suggesting that the different chromosomes are partially mixed at the borders of territories [4][5][6][7][8][9]. In Drosophila and human lymphocytes, about 40% of the chromosome territories shows some intermixing [5,10]. ...
... While the chromosomes are organized into distinct territories, overlapping of those territories is also observed in recent studies, suggesting that the different chromosomes are partially mixed at the borders of territories [4][5][6][7][8][9]. In Drosophila and human lymphocytes, about 40% of the chromosome territories shows some intermixing [5,10]. Even if the mixing is far from complete, it can have implications for correlations in gene expression in the regions of the boundaries of the chromosomes that do partially mix within realistic times [11]. ...
... These calculations suggest that the dynamics of chromosome mixing is a slow process and that most cells will likely divide before chromosomes are completely mixed. Of course, partial mixing does occur, as discussed above and in [5,29]. Different orders of magnitudes of the mixing time correspond to the different colors (from violet to red) in the heat map. ...
Chromosomes are arranged in distinct territories within the nucleus of animal cells. Recent experiments have shown that these territories overlap at their edges, suggesting partial mixing during interphase. Experiments that knock-down of condensin II proteins during interphase indicate increased chromosome mixing, which demonstrates control of the mixing. In this study, we use a generic polymer simulation to quantify the dynamics of chromosome mixing over time. We introduce the chromosome mixing index, which quantifies the mixing of distinct chromosomes in the nucleus. We find that the chromosome mixing index in a small confinement volume (as a model of the nucleus), increases as a power-law of the time, with the scaling exponent varying non-monotonically with self-interaction and volume fraction. By comparing the chromosome mixing index with both monomer subdiffusion due to (non-topological) intermingling of chromosomes as well as even slower reptation, we show that for relatively large volume fractions, the scaling exponent of the chromosome mixing index is related to Rouse dynamics for relatively weak chromosome attractions and to reptation for strong attractions. In addition, we extend our model to more realistically account for the situation of the Drosophila chromosome by including the heterogeneity of the polymers and their lengths to account for microphase separation of euchromatin and heterochromatin and their interactions with the nuclear lamina. We find that the interaction with the lamina further impedes chromosome mixing.
... We posed the idea that these NGEFTs somehow serve as templates to aberrantly drive the repair process. [45][46][47][48] However, up to that time, no experimental tools were available to set-up experiments to validate this hypothesis. ...
Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all “cancer genes” were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: (1) proximity of genes able to produce prematurely terminated transcripts, which lead to the production of (2) trans-spliced fusion RNAs, and finally, the induction of (3) DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced. The implications of these findings will be discussed.
