ABSTRACT
SUMMARY: Genomic sequences are widely used to infer the evolutionary history of a given group of individuals. Many methods have been developed for sequence clustering and tree building. In the early days of genome sequencing, these were often limited to hundreds of sequences but due to the surge of high throughput sequencing, it is now common to have millions of sampled sequences at hand. We introduce MNHN-Tree-Tools, a high performance set of algorithms that builds multi-scale, nested clusters of sequences found in a FASTA file. MNHN-Tree-Tools does not rely on multiple sequence alignment and can thus be used on large datasets to infer a sequence tree. Herein, we outline two applications: a human alpha-satellite repeats classification and a tree of life derivation from 16S/18S rDNA sequences. AVAILABILITY AND IMPLEMENTATION: Open source with a Zlib License via the Git protocol: https://gitlab.in2p3.fr/mnhn-tools/mnhn-tree-tools. MANUAL: A detailed users guide and tutorial: https://gitlab.in2p3.fr/mnhn-tools/mnhn-tree-tools-manual/-/raw/master/manual.pdf. WEBSITE AND FAQ: http://treetools.haschka.net. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Algorithms , Genomics , Humans , Phylogeny , Sequence Alignment , Cluster AnalysisABSTRACT
BACKGROUND: Alpha satellite is the major repeated DNA element of primate centromeres. Evolution of these tandemly repeated sequences has led to the existence of numerous families of monomers exhibiting specific organizational patterns. The limited amount of information available in non-human primates is a restriction to the understanding of the evolutionary dynamics of alpha satellite DNA. RESULTS: We carried out the targeted high-throughput sequencing of alpha satellite monomers and dimers from the Cercopithecus solatus genome, an Old World monkey from the Cercopithecini tribe. Computational approaches were used to infer the existence of sequence families and to study how these families are organized with respect to each other. While previous studies had suggested that alpha satellites in Old World monkeys were poorly diversified, our analysis provides evidence for the existence of at least four distinct families of sequences within the studied species and of higher order organizational patterns. Fluorescence in situ hybridization using oligonucleotide probes that are able to target each family in a specific way showed that the different families had distinct distributions on chromosomes and were not homogeneously distributed between chromosomes. CONCLUSIONS: Our new approach provides an unprecedented and comprehensive view of the diversity and organization of alpha satellites in a species outside the hominoid group. We consider these data with respect to previously known alpha satellite families and to potential mechanisms for satellite DNA evolution. Applying this approach to other species will open new perspectives regarding the integration of satellite DNA into comparative genomic and cytogenetic studies.
Subject(s)
Cercopithecus/genetics , DNA, Satellite , Genetic Variation , Genome , Animals , Centromere , Chromosomes, Mammalian , Consensus Sequence , Datasets as Topic , Genomics/methods , High-Throughput Nucleotide Sequencing , In Situ Hybridization, Fluorescence , Male , PhylogenyABSTRACT
DNA imaging in living cells usually requires transgenic approaches that modify the genome. Synthetic pyrrole-imidazole polyamides that bind specifically to the minor groove of double-stranded DNA (dsDNA) represent an attractive approach for in-cell imaging that does not necessitate changes to the genome. Nine hairpin polyamides that target mouse major satellite DNA were synthesized. Their interactions with synthetic target dsDNA fragments were studied by thermal denaturation, gel-shift electrophoresis, circular dichroism, and fluorescence spectroscopy. The polyamides had different affinities for the target DNA, and fluorescent labeling of the polyamides affected their affinity for their targets. We validated the specificity of the probes in fixed cells and provide evidence that two of the probes detect target sequences in mouse living cell lines. This study demonstrates for the first time that synthetic compounds can be used for the visualization of the nuclear substructures formed by repeated DNA sequences in living cells.
Subject(s)
DNA/chemistry , Fluorescent Dyes/chemistry , Nylons/chemistry , Repetitive Sequences, Nucleic Acid , Animals , Base Sequence , Mice , Microscopy, Fluorescence , NIH 3T3 Cells , Optical ImagingABSTRACT
The cyclin-dependent kinase CDK11(p58) is specifically expressed at G2/M phase. CDK11(p58) depletion leads to different cell cycle defects such as mitotic arrest, failure in centriole duplication and centrosome maturation, and premature sister chromatid separation. We report that upon CDK11 depletion, loss of sister chromatid cohesion occurs during mitosis but not during G2 phase. CDK11(p58) depletion prevents Bub1 and Shugoshin 1 recruitment but has no effect on the dimethylation of histone H3 lysine 4 at centromeres. We also report that a construct expressing a kinase dead version of CDK11(p58) fails to prevent CDK11 depletion-induced sister chromatid separation, showing that CDK11(p58) kinase activity is required for protection of sister chromatid cohesion at centromeres during mitosis. Thus, CDK11(p58) kinase activity appears to be involved in early events in the establishment of the centromere protection machinery.
Subject(s)
Centromere/metabolism , Chromatids/metabolism , Cyclin D3/metabolism , Mitosis , Sister Chromatid Exchange , Cell Cycle Proteins/metabolism , G2 Phase , HeLa Cells , Humans , Protein Serine-Threonine Kinases/metabolismABSTRACT
Proteins that recognize and bind specific sites in DNA are essential for regulation of numerous biological functions. Such proteins often require a negative supercoiled DNA topology to function correctly. In current research, short linear DNA is often used to study DNA-protein interactions. Although linear DNA can easily be modified, for capture on a surface, its relaxed topology does not accurately resemble the natural situation in which DNA is generally negatively supercoiled. Moreover, specific binding sequences are flanked by large stretches of non-target sequence in vivo. Here, we present a straightforward method for capturing negatively supercoiled plasmid DNA on a streptavidin surface. It relies on the formation of a temporary parallel triplex, using a triple helix forming oligonucleotide containing locked nucleic acid nucleotides. All materials required for this method are commercially available. Lac repressor binding to its operator was used as model system. Although the dissociation constants for both the linear and plasmid-based operator are in the range of 4 nM, the association and dissociation rates of Lac repressor binding to the plasmid-based operator are ~18 times slower than on a linear fragment. This difference underscores the importance of using a physiologically relevant DNA topology for studying DNA-protein interactions.
Subject(s)
DNA, Superhelical/chemistry , DNA/chemistry , Plasmids/genetics , DNA, Superhelical/metabolism , Lac Repressors/metabolism , Oligonucleotides/chemistry , Operator Regions, GeneticABSTRACT
MOTIVATION: The cell nucleus is a highly organized cellular organelle that contains the genetic material. The study of nuclear architecture has become an important field of cellular biology. Extracting quantitative data from 3D fluorescence imaging helps understand the functions of different nuclear compartments. However, such approaches are limited by the requirement for processing and analyzing large sets of images. RESULTS: Here, we describe Tools for Analysis of Nuclear Genome Organization (TANGO), an image analysis tool dedicated to the study of nuclear architecture. TANGO is a coherent framework allowing biologists to perform the complete analysis process of 3D fluorescence images by combining two environments: ImageJ (http://imagej.nih.gov/ij/) for image processing and quantitative analysis and R (http://cran.r-project.org) for statistical processing of measurement results. It includes an intuitive user interface providing the means to precisely build a segmentation procedure and set-up analyses, without possessing programming skills. TANGO is a versatile tool able to process large sets of images, allowing quantitative study of nuclear organization. AVAILABILITY: TANGO is composed of two programs: (i) an ImageJ plug-in and (ii) a package (rtango) for R. They are both free and open source, available (http://biophysique.mnhn.fr/tango) for Linux, Microsoft Windows and Macintosh OSX. Distribution is under the GPL v.2 licence. CONTACT: thomas.boudier@snv.jussieu.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Cell Nucleus/ultrastructure , Imaging, Three-Dimensional/methods , Software , Cell Nucleus/genetics , Genome , Microscopy, FluorescenceABSTRACT
A SELEX approach has been developed in order to select oligonucleotides that bind double-stranded DNA in the presence of a triplex-stabilizing agent, and was applied to a target sequence containing an oligopurine-oligopyrimidine stretch. After only seven rounds of selection, the process led to the identification of oligonucleotides that were able to form triple helices within the antiparallel motif. Inspection of the selected sequences revealed that, contrary to GC base pair which were always recognized by guanines, recognition of AT base pair could be achieved by either adenine or thymine, depending on the sequence context. While thymines are strongly preferred for several positions, some others can accommodate the presence of adenines. These results contribute to set the rules for designing oligonucleotides that form stable triple helices in the presence of triplex-stabilizing agents at physiological pH. They set the basis for further experiments regarding extension of potential target sequences for triple-helix formation or recognition of ligand-DNA complexes.
Subject(s)
Aptamers, Nucleotide/chemistry , DNA/chemistry , SELEX Aptamer Technique/methods , Base Sequence , Cloning, Molecular , Intercalating AgentsABSTRACT
BACKGROUND: Centromeric regions of human chromosomes contain large numbers of tandemly repeated α-satellite sequences. These sequences are covered with constitutive heterochromatin which is enriched in trimethylation of histone H3 on lysine 9 (H3K9me3). Although well studied using artificial chromosomes and global perturbations, the contribution of this epigenetic mark to chromatin structure and genome stability remains poorly known in a more natural context. RESULTS: Using transcriptional activator-like effectors (TALEs) fused to a histone lysine demethylase (KDM4B), we were able to reduce the level of H3K9me3 on the α-satellites repeats of human chromosome 7. We show that the removal of H3K9me3 affects chromatin structure by increasing the accessibility of DNA repeats to the TALE protein. Tethering TALE-demethylase to centromeric repeats impairs the recruitment of HP1α and proteins of Chromosomal Passenger Complex (CPC) on this specific centromere without affecting CENP-A loading. Finally, the epigenetic re-writing by the TALE-KDM4B affects specifically the stability of chromosome 7 upon mitosis, highlighting the importance of H3K9me3 in centromere integrity and chromosome stability, mediated by the recruitment of HP1α and the CPC. CONCLUSION: Our cellular model allows to demonstrate the direct role of pericentromeric H3K9me3 epigenetic mark on centromere integrity and function in a natural context and opens interesting possibilities for further studies regarding the role of the H3K9me3 mark.
Subject(s)
Centromere , Chromatin , Chromatin/genetics , Chromosomal Instability , DNA , Epigenesis, Genetic , Humans , Jumonji Domain-Containing Histone DemethylasesABSTRACT
The restriction endonuclease EcoRV can rapidly locate a short recognition site within long non-cognate DNA using 'facilitated diffusion'. This process has long been attributed to a sliding mechanism, in which the enzyme first binds to the DNA via nonspecific interaction and then moves along the DNA by 1D diffusion. Recent studies, however, provided evidence that 3D translocations (hopping/jumping) also help EcoRV to locate its target site. Here we report the first direct observation of sliding and jumping of individual EcoRV molecules along nonspecific DNA. Using fluorescence microscopy, we could distinguish between a slow 1D diffusion of the enzyme and a fast translocation mechanism that was demonstrated to stem from 3D jumps. Salt effects on both sliding and jumping were investigated, and we developed numerical simulations to account for both the jump frequency and the jump length distribution. We deduced from our study the 1D diffusion coefficient of EcoRV, and we estimated the number of jumps occurring during an interaction event with nonspecific DNA. Our results substantiate that sliding alternates with hopping/jumping during the facilitated diffusion of EcoRV and, furthermore, set up a framework for the investigation of target site location by other DNA-binding proteins.
Subject(s)
DNA-Binding Proteins/chemistry , Deoxyribonucleases, Type II Site-Specific/chemistry , DNA/chemistry , Diffusion , Microscopy, Fluorescence , Protein Binding , Sodium Chloride/pharmacologyABSTRACT
Fluorescence microscopy provides a powerful method to directly observe single enzymes moving along a DNA held in an extended conformation. In this work, we present results from single EcoRV enzymes labeled with quantum dots which interact with DNA manipulated by double optical tweezers. The application of quantum dots facilitated accurate enzyme tracking without photobleaching whereas the tweezers allowed us to precisely control the DNA extension. The labeling did not affect the biochemical activity of EcoRV checked by directly observing DNA digestion on the single molecule level. We used this system to demonstrate that during sliding, the enzyme stays in close contact with the DNA. Additionally, slight overstretching of the DNA resulted in a significant decrease of the 1D diffusion constant, which suggests that the deformation changes the energy landscape of the sliding interaction. Together with the simplicity of the setup, these results demonstrate that the combination of optical tweezers with fluorescence tracking is a powerful tool for the study of enzyme translocation along DNA.
Subject(s)
DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , Microscopy, Fluorescence/methods , Optical Tweezers , Quantum Dots , DNA/chemistry , Diffusion , Nucleic Acid ConformationABSTRACT
We report here the first realization of an artificial branched DNA template where a single wall carbon nanotube is positioned with the necessary geometry of an individually gated field effect transistor.
Subject(s)
DNA/chemistry , Nanotechnology/instrumentation , Nanotubes, Carbon/chemistry , Biomimetics , DNA/metabolism , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Streptavidin/chemistry , Streptavidin/metabolism , Transistors, ElectronicABSTRACT
DNA combing is a useful strategy for manipulating single DNA molecules and has a wide range of applications in genetics, single molecule studies, and nanobiotechnology. Visualization of combed DNA molecules is usually performed by using DNA binding organic dyes. Such dyes are not suitable in all circumstances, especially because of their photoreactivity. We have developed a method for the detection of combed DNA molecules by fluorescence microscopy that avoids the use of DNA-staining agents and does not perturb the structure of the DNA molecule. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both ends of a DNA molecule via sequence-specific hybridization and subsequent ligation. After the modified DNA molecules have been combed on a polystyrene-coated surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots.
Subject(s)
DNA/analysis , Quantum Dots , Benzoxazoles , Biotin , DNA/isolation & purification , Digoxigenin , Fluorescent Dyes , Microscopy, Fluorescence , Quinolinium CompoundsABSTRACT
Pericentromeric heterochromatin plays important roles in controlling gene expression and cellular differentiation. Fluorescent pyrrole-imidazole polyamides targeting murine pericentromeric DNA (major satellites) can be used for the visualization of pericentromeric heterochromatin foci in live mouse cells. New derivatives targeting human repeated DNA sequences (α-satellites) were synthesized and their interaction with target DNA was characterized. The possibility to use major satellite and α -satellite binding polyamides as tools for staining pericentromeric heterochromatin was further investigated in fixed and living mouse and human cells. The staining that was previously observed using the mouse model was further characterized and optimized, but remained limited regarding the fluorophores that can be used. The promising results regarding the staining in the mouse model could not be extended to the human model. Experiments performed in human cells showed chromosomal DNA staining without selectivity. Factors limiting the use of fluorescent polyamides, in particular probe aggregation in the cytoplasm, were investigated. Results are discussed with regards to structure and affinity of probes, density of target sites and chromatin accessibility in both models.
Subject(s)
Cell Tracking/methods , Chromatin/isolation & purification , Fluorescent Dyes/chemistry , Staining and Labeling/methods , Animals , Binding Sites , Cell Line , Chromatin/chemistry , Humans , Imidazoles/chemistry , Mice , Nylons/chemistry , Pyrroles/chemistryABSTRACT
Alpha satellite is the major repeated DNA element of primate centromeres. Specific evolutionary mechanisms have led to a great diversity of sequence families with peculiar genomic organization and distribution, which have till now been studied mostly in great apes. Using high throughput sequencing of alpha satellite monomers obtained by enzymatic digestion followed by computational and cytogenetic analysis, we compare here the diversity and genomic distribution of alpha satellite DNA in two related Old World monkey species, Cercopithecus pogonias and Cercopithecus solatus, which are known to have diverged about 7 Ma. Two main families of monomers, called C1 and C2, are found in both species. A detailed analysis of our data sets revealed the existence of numerous subfamilies within the centromeric C1 family. Although the most abundant subfamily is conserved between both species, our fluorescence in situ hybridization (FISH) experiments clearly show that some subfamilies are specific for each species and that their distribution is restricted to a subset of chromosomes, thereby pointing to the existence of recurrent amplification/homogenization events. The pericentromeric C2 family is very abundant on the short arm of all acrocentric chromosomes in both species, pointing to specific mechanisms that lead to this distribution. Results obtained using two different restriction enzymes are fully consistent with a predominant monomeric organization of alpha satellite DNA that coexists with higher order organization patterns in the C. pogonias genome. Our study suggests a high dynamics of alpha satellite DNA in Cercopithecini, with recurrent apparition of new sequence variants and interchromosomal sequence transfer.
Subject(s)
Centromere/genetics , Cercopithecus/genetics , DNA, Satellite/genetics , Animals , Base Sequence , Cercopithecidae/genetics , Consensus Sequence , Evolution, Molecular , In Situ Hybridization, Fluorescence , Karyotype , Male , Minisatellite Repeats , Sequence Analysis, DNAABSTRACT
Observation of DNA-protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA-protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.
Subject(s)
DNA/analysis , Microscopy, Fluorescence/methods , Quantum Dots , Biotinylation , Color , Digoxigenin/chemistryABSTRACT
Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem-loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 +/- 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.
Subject(s)
DNA/chemistry , Fluorescent Dyes/chemistry , Bacteriophage lambda/genetics , Base Sequence , Benzoxazoles/chemistry , DNA/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Microscopy, Fluorescence , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/genetics , Quinolinium Compounds/chemistryABSTRACT
Labeling of a covalently closed circular double-stranded DNA was achieved using a so-called 'padlock oligonucleotide'. The oligonucleotide was targeted to a sequence which is present in the replication origin of phage f1 and thus in numerous commonly used plasmids. After winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, a biotinylated oligonucleotide was circularized using T4 DNA ligase and in this way became catenated to the plasmid. A gel shift assay was developed to measure the extent of plasmid modification by the padlock oligonucleotide. A similar assay showed that a modified supercoiled plasmid was capable of binding one streptavidin molecule thanks to the biotinylated oligonucleotide and that this binding was quantitative. The catenated complex was visualized by electron and atomic force microscopies using streptavidin conjugates or single strand-binding proteins as protein tags for the padlock oligonucleotide. This method provides a versatile tool for plasmid functionalization which offers new perspectives in the physical study of supercoiled DNA and in the development of improved vectors for gene therapy.
Subject(s)
DNA, Superhelical/chemistry , DNA, Superhelical/metabolism , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Base Sequence , Biotinylation , Coliphages/genetics , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA/ultrastructure , DNA Ligases/metabolism , DNA, Superhelical/genetics , DNA, Superhelical/ultrastructure , Deoxyuracil Nucleotides/metabolism , Electrophoretic Mobility Shift Assay , Genetic Therapy/methods , Indicators and Reagents/metabolism , Microscopy, Atomic Force , Microscopy, Electron , Oligonucleotides/genetics , Plasmids/chemistry , Plasmids/genetics , Plasmids/metabolism , Plasmids/ultrastructure , Replication Origin/genetics , Streptavidin/metabolism , Substrate SpecificityABSTRACT
We report on a sequence-specific double-stranded DNA labelling strategy in which a stem-loop triplex forming oligonucleotide (TFO) is able to encircle its DNA target. Ligation of this TFO to either a short hairpin oligonucleotide or a long double-stranded DNA fragment leads to the formation of a topological complex. This process requires the hybridization of both extremities of the TFO to each other on a few base pairs. The effects of different factors on the formation of these complexes have been investigated. Efficient complex formation was observed using both GT or TC TFOs. The stem-loop structure enhances the specificity of the complex. The topologically linked TFO remains associated with its target even under conditions that do not favour triple-helix formation. This approach is sufficiently sensitive for detection of a 20-bp target sequence at the subfemtomolar level. This study provides new insights into the mechanics and properties of stem-loop TFOs and their complexes with double-stranded DNA targets. It emphasizes the interest of such molecules in the development of new tools for the specific labelling of short DNA sequences.
Subject(s)
DNA/analysis , Oligonucleotide Probes/chemistry , Staining and Labeling/methods , AT Rich Sequence/genetics , Base Sequence , DNA/chemistry , DNA/genetics , DNA, Circular/chemistry , DNA, Circular/genetics , Electrophoresis, Agar Gel , GC Rich Sequence/genetics , Isotope Labeling , Molecular Sequence Data , Nucleic Acid Conformation , Nucleic Acid Hybridization , Oligonucleotide Probes/genetics , Plasmids/chemistry , Plasmids/geneticsABSTRACT
Triplex-forming oligonucleotides (TFOs) provide useful tools for the artificial regulation of gene expression at the transcriptional level. They can become topologically linked to their DNA target upon circularization, thereby forming very stable triple helical structures. These "padlock oligonucleotides" are able to interfere with transcription elongation when their target site is located in the transcribed region of a gene. In vitro transcription experiments showed that a bacterial RNA polymerase was stopped at the site of triple-helix formation, whereas expression of a reporter gene was inhibited in live cells. In both cases, the padlock oligonucleotide was more efficient at inhibiting transcription elongation than a linear TFO, and the inhibition was observed only in the presence of a triplex stabilizing agent. These results provide new insights into the ligand-modulated locking of padlock oligonucleotides around their DNA target.
Subject(s)
DNA/chemistry , Oligonucleotides/chemistry , Oligonucleotides/pharmacology , Transcription, Genetic/drug effects , Bacteria/enzymology , Bacteria/genetics , Base Sequence , DNA/drug effects , DNA-Directed RNA Polymerases/antagonists & inhibitors , Gene Expression Regulation/drug effects , HeLa Cells/drug effects , Humans , Ligands , Molecular Sequence Data , Molecular StructureABSTRACT
The cell nucleus is a highly organized structure and plays an important role in gene regulation. Understanding the mechanisms that sustain this organization is therefore essential for understanding genome function. Centromeric regions (CRs) of chromosomes have been known for years to adopt specific nuclear positioning patterns, but the significance of this observation is not yet completely understood. Here, using a combination of fluorescence in situ hybridization and immunochemistry on fixed human cells and high-throughput imaging, we directly and quantitatively investigated the nuclear positioning of specific human CRs. We observe differential attraction of individual CRs toward both the nuclear border and the nucleoli, the former being enhanced in nonproliferating cells and the latter being enhanced in proliferating cells. Similar positioning patterns are observed in two different lymphoblastoid cell lines. Moreover, the positioning of CRs differs from that of noncentromeric regions, and CRs display specific orientations within chromosome territories. These results suggest the existence of not-yet-characterized mechanisms that drive the nuclear positioning of CRs and therefore pave the way toward a better understanding of how CRs affect nuclear organization.