RESUMEN
Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin, and Smc5/6. This organization regulates different DNA-related processes along the cell cycle, such as transcription, recombination, segregation, and DNA repair. During the G2 stage, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here, we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion along G2/M Saccharomyces cerevisiae chromosomes.
Asunto(s)
Proteínas Cromosómicas no Histona , Proteínas de Saccharomyces cerevisiae , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Cohesinas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Viruses that transcribe their DNA within the nucleus have to adapt to the existing cellular mechanisms that govern transcriptional regulation. Recent technological breakthroughs have highlighted the highly hierarchical organization of the cellular genome and its role in the regulation of gene expression. This review provides an updated overview on the current knowledge on how the hepatitis B virus interacts with the cellular 3D genome and its consequences on viral and cellular gene expression. We also briefly discuss the strategies developed by other DNA viruses to co-opt and sometimes subvert cellular genome spatial organization.
Asunto(s)
ADN Circular/genética , ADN Viral/genética , Virus de la Hepatitis B/genética , Animales , Carcinoma Hepatocelular/virología , Genoma Viral , Hepatitis B/virología , Hepatitis B Crónica/virología , Humanos , Replicación ViralRESUMEN
Over the last decade, genomic proximity ligation approaches have reshaped our vision of chromosomes 3D organizations, from bacteria nucleoids to larger eukaryotic genomes. The different protocols (3Cseq, Hi-C, TCC, MicroC [XL], Hi-CO, etc.) rely on common steps (chemical fixation digestion, ligation ) to detect pairs of genomic positions in close proximity. The most common way to represent these data is a matrix, or contact map, which allows visualizing the different chromatin structures (compartments, loops, etc.) that can be associated to other signals such as transcription, protein occupancy, etc. as well as, in some instances, to biological functions.In this chapter we present and discuss the filtering of the events recovered in proximity ligation experiments as well as the application of the balancing normalization procedure on the resulting contact map. We also describe a computational tool for visualizing normalized contact data dubbed Scalogram.The different processes described here are illustrated and supported by the laboratory custom-made scripts pooled into "hicstuff," an open-access python package accessible on github ( https://github.com/koszullab/hicstuff ).
Asunto(s)
Cromosomas , Cromatina/genética , Genoma , GenómicaRESUMEN
Chromosomes of all species studied so far display a variety of higher-order organisational features, such as self-interacting domains or loops. These structures, which are often associated to biological functions, form distinct, visible patterns on genome-wide contact maps generated by chromosome conformation capture approaches such as Hi-C. Here we present Chromosight, an algorithm inspired from computer vision that can detect patterns in contact maps. Chromosight has greater sensitivity than existing methods on synthetic simulated data, while being faster and applicable to any type of genomes, including bacteria, viruses, yeasts and mammals. Our method does not require any prior training dataset and works well with default parameters on data generated with various protocols.
Asunto(s)
Cromosomas/genética , Computadores , Reconocimiento de Normas Patrones Automatizadas , Algoritmos , Cromosomas Fúngicos/genética , Cromosomas Humanos/genética , Genoma Fúngico , Humanos , Saccharomyces cerevisiae/genética , Flujo de TrabajoRESUMEN
Cohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferasas/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Mitosis , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT ('facilitates chromatin transcription') in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Elongación Transcripcional/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Mapas de Interacción de Proteínas , Saccharomyces cerevisiae/citología , CohesinasRESUMEN
Whether non-integrated viral DNAs distribute randomly or target specific positions within the higher-order architecture of mammalian genomes remains largely unknown. Here we use Hi-C and viral DNA capture (CHi-C) in primary human hepatocytes infected by either hepatitis B virus (HBV) or adenovirus type 5 (Ad5) virus to show that they adopt different strategies in their respective positioning at active chromatin. HBV contacts preferentially CpG islands (CGIs) enriched in Cfp1 a factor required for its transcription. These CGIs are often associated with highly expressed genes (HEG) and genes deregulated during infection. Ad5 DNA interacts preferentially with transcription start sites (TSSs) and enhancers of HEG, as well as genes upregulated during infection. These results show that DNA viruses use different strategies to infiltrate genomic 3D networks and target specific regions. This targeting may facilitate the recruitment of transcription factors necessary for their own replication and contribute to the deregulation of cellular gene expression.
Asunto(s)
Cromatina/metabolismo , Genoma Humano , Virus de la Hepatitis B/fisiología , Secuencia de Bases , Islas de CpG/genética , ADN Viral/genética , Regulación de la Expresión Génica , Células Hep G2 , Hepatocitos/virología , Humanos , Modelos Biológicos , Plásmidos/metabolismo , Transactivadores/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética , Regulación hacia Arriba/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The mammalian cell nucleus contains numerous discrete suborganelles named nuclear bodies. While recruitment of specific genomic regions into these large ribonucleoprotein (RNP) complexes critically contributes to higher-order functional chromatin organization, such regions remain ill-defined. We have developed the high-salt-recovered sequences-sequencing (HRS-seq) method, a straightforward genome-wide approach whereby we isolated and sequenced genomic regions associated with large high-salt insoluble RNP complexes. By using mouse embryonic stem cells (ESCs), we showed that these regions essentially correspond to the most highly expressed genes, and to cis-regulatory sequences like super-enhancers, that belong to the active A chromosomal compartment. They include both cell-type-specific genes, such as pluripotency genes in ESCs, and housekeeping genes associated with nuclear bodies, such as histone and snRNA genes that are central components of Histone Locus Bodies and Cajal bodies. We conclude that HRSs are associated with the active chromosomal compartment and with large RNP complexes including nuclear bodies. Association of such chromosomal regions with nuclear bodies is in agreement with the recently proposed phase separation model for transcription control and might thus play a central role in organizing the active chromosomal compartment in mammals.
Asunto(s)
Cromosomas/química , Ribonucleoproteínas/química , Animales , Células Cultivadas , Fraccionamiento Químico/métodos , Cromosomas/metabolismo , Células Madre Embrionarias/metabolismo , Ratones , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Ribonucleoproteínas/metabolismo , SalinidadRESUMEN
As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to â¼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.
Asunto(s)
Adenosina Trifosfatasas , Cromosomas Bacterianos , Proteínas de Unión al ADN , Escherichia coli K12 , Complejos Multiproteicos , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/ultraestructura , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , Cromosomas Bacterianos/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Escherichia coli K12/ultraestructura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Estructura Cuaternaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The biochemical activities of microbial communities, or microbiomes, are essential parts of environmental and animal ecosystems. The dynamics, balance, and effects of these communities are strongly influenced by phages present in the population. Being able to characterize bacterium-phage relationships is therefore essential to investigate these ecosystems to the full extent of their complexity. However, this task is currently limited by (i) the ability to characterize complete bacterial and viral genomes from a complex mix of species and (ii) the difficulty to assign phage sequences to their bacterial hosts. We show that both limitations can be circumvented using meta3C, an experimental and computational approach that exploits the physical contacts between DNA molecules to infer their proximity. In a single experiment, dozens of bacterial and phage genomes present in a complex mouse gut microbiota were assembled and scaffolded de novo. The phage genomes were then assigned to their putative bacterial hosts according to the physical contacts between the different DNA molecules, opening new perspectives for a comprehensive picture of the genomic structure of the gut flora. Therefore, this work holds far-reaching implications for human health studies aiming to bridge the virome to the microbiome.
Asunto(s)
Bacterias , Bacteriófagos , Microbioma Gastrointestinal , Genoma Bacteriano , Genoma Viral , Animales , Bacterias/genética , Bacterias/metabolismo , Bacterias/virología , Bacteriófagos/genética , Bacteriófagos/metabolismo , Masculino , RatonesRESUMEN
Eukaryotic chromosomes undergo movements that are involved in the regulation of functional processes such as DNA repair. To better understand the origin of these movements, we used fluorescence microscopy, image analysis and chromosome conformation capture to quantify the actin contribution to chromosome movements and interactions in budding yeast. We show that both the cytoskeletal and nuclear actin drive local chromosome movements, independently of Csm4, a putative LINC protein. Inhibition of actin polymerization reduces subtelomere dynamics, resulting in more confined territories and enrichment in subtelomeric contacts. Artificial tethering of actin to nuclear pores increased both nuclear pore complex (NPC) and subtelomere motion. Chromosome loci that were positioned away from telomeres exhibited reduced motion in the presence of an actin polymerization inhibitor but were unaffected by the lack of Csm4. We further show that actin was required for locus mobility that was induced by targeting the chromatin-remodeling protein Ino80. Correlated with this, DNA repair by homologous recombination was less efficient. Overall, interphase chromosome dynamics are modulated by the additive effects of cytoskeletal actin through forces mediated by the nuclear envelope and nuclear actin, probably through the function of actin in chromatin-remodeling complexes.
Asunto(s)
Actinas/fisiología , Cromosomas Fúngicos/fisiología , Saccharomyces cerevisiae/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de la Membrana/fisiología , Poro Nuclear/metabolismo , Multimerización de Proteína , Transporte de Proteínas , Reparación del ADN por Recombinación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Telómero/genética , Telómero/metabolismoRESUMEN
Genome-wide derivatives of the chromosome conformation capture (3C) technique are now well-established approaches to study the multiscale average organization of chromosomes from bacteria to mammals. However, the experimental parameters of the protocol have to be optimized for different species, and the downstream experimental products (i.e., pair-end sequences) are influenced by these parameters. Here, we describe a complete pipeline to generate 3C-seq libraries and compute chromosomal contact maps of yeast species.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas Fúngicos/genética , Genoma Fúngico/genética , Cromatina/genética , Saccharomyces cerevisiaeRESUMEN
The potential roles of the numerous repetitive elements found in the genomes of multi-cellular organisms remain speculative. Several studies have suggested a role in stabilizing specific 3D genomic contacts. To test this hypothesis, we exploited inter-chromosomal contacts frequencies obtained from Hi-C experiments and show that the folding of the human, mouse and Drosophila genomes is associated with a significant co-localization of several specific repetitive elements, notably many elements of the SINE family. These repeats tend to be the oldest ones and are enriched in transcription factor binding sites. We propose that the co-localization of these repetitive elements may explain the global conservation of genome folding observed between homologous regions of the human and mouse genome. Taken together, these results support a contribution of specific repetitive elements in maintaining and/or reshaping genome architecture over evolutionary times.
Asunto(s)
Genoma , Conformación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Sitios de Unión , Núcleo Celular , Mapeo Cromosómico , Cromosomas , Biología Computacional/métodos , Drosophila , Células Madre Embrionarias/metabolismo , Evolución Molecular , Humanos , Ratones , Anotación de Secuencia Molecular , Especificidad de Órganos/genética , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: The spatiotemporal behavior of chromatin is an important control mechanism of genomic function. Studies in Saccharomyces cerevisiae have broadly contributed to demonstrate the functional importance of nuclear organization. Although in the wild yeast survival depends on their ability to withstand adverse conditions, most of these studies were conducted on cells undergoing exponential growth. In these conditions, as in most eukaryotic cells, silent chromatin that is mainly found at the 32 telomeres accumulates at the nuclear envelope, forming three to five foci. RESULTS: Here, combining live microscopy, DNA FISH and chromosome conformation capture (HiC) techniques, we report that chromosomes adopt distinct organizations according to the metabolic status of the cell. In particular, following carbon source exhaustion the genome of long-lived quiescent cells undergoes a major spatial re-organization driven by the grouping of telomeres into a unique focus or hypercluster localized in the center of the nucleus. This change in genome conformation is specific to quiescent cells able to sustain long-term viability. We further show that reactive oxygen species produced by mitochondrial activity during respiration commit the cell to form a hypercluster upon starvation. Importantly, deleting the gene encoding telomere associated silencing factor SIR3 abolishes telomere grouping and decreases longevity, a defect that is rescued by expressing a silencing defective SIR3 allele competent for hypercluster formation. CONCLUSIONS: Our data show that mitochondrial activity primes cells to group their telomeres into a hypercluster upon starvation, reshaping the genome architecture into a conformation that may contribute to maintain longevity of quiescent cells.
Asunto(s)
Fase de Descanso del Ciclo Celular/genética , Telómero/ultraestructura , Carbono/metabolismo , Centrómero , Cromosomas Fúngicos , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: The opportunistic pathogen Candida glabrata is a member of the Saccharomycetaceae yeasts. Like its close relative Saccharomyces cerevisiae, it underwent a whole-genome duplication followed by an extensive loss of genes. Its genome contains a large number of very long tandem repeats, called megasatellites. In order to determine the whole replication program of the C. glabrata genome and its general chromosomal organization, we used deep-sequencing and chromosome conformation capture experiments. RESULTS: We identified 253 replication fork origins, genome wide. Centromeres, HML and HMR loci, and most histone genes are replicated early, whereas natural chromosomal breakpoints are located in late-replicating regions. In addition, 275 autonomously replicating sequences (ARS) were identified during ARS-capture experiments, and their relative fitness was determined during growth competition. Analysis of ARSs allowed us to identify a 17-bp consensus, similar to the S. cerevisiae ARS consensus sequence but slightly more constrained. Megasatellites are not in close proximity to replication origins or termini. Using chromosome conformation capture, we also show that early origins tend to cluster whereas non-subtelomeric megasatellites do not cluster in the yeast nucleus. CONCLUSIONS: Despite a shorter cell cycle, the C. glabrata replication program shares unexpected striking similarities to S. cerevisiae, in spite of their large evolutionary distance and the presence of highly repetitive large tandem repeats in C. glabrata. No correlation could be found between the replication program and megasatellites, suggesting that their formation and propagation might not be directly caused by replication fork initiation or termination.
Asunto(s)
Candida glabrata/genética , Cromosomas Fúngicos , Replicación del ADN , Genoma Fúngico , Ciclo Celular/genética , Genes FúngicosRESUMEN
Chromosomes of a broad range of species, from bacteria to mammals, are structured by large topological domains whose precise functional roles and regulatory mechanisms remain elusive. Here, we combine super-resolution microscopies and chromosome-capture technologies to unravel the higher-order organization of the Bacillus subtilis chromosome and its dynamic rearrangements during the cell cycle. We decipher the fine 3D architecture of the origin domain, revealing folding motifs regulated by condensin-like complexes. This organization, along with global folding throughout the genome, is present before replication, disrupted by active DNA replication, and re-established thereafter. Single-cell analysis revealed a strict correspondence between sub-cellular localization of origin domains and their condensation state. Our results suggest that the precise 3D folding pattern of the origin domain plays a role in the regulation of replication initiation, chromosome organization, and DNA segregation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Bacillus subtilis/metabolismo , Bacillus subtilis/ultraestructura , Cromosomas Bacterianos/ultraestructura , Replicación del ADN , ADN Superhelicoidal , Microscopía , Modelos Moleculares , Imagen Óptica , Origen de RéplicaRESUMEN
Closing gaps in draft genome assemblies can be costly and time-consuming, and published genomes are therefore often left 'unfinished.' Here we show that genome-wide chromosome conformation capture (3C) data can be used to overcome these limitations, and present a computational approach rooted in polymer physics that determines the most likely genome structure using chromosomal contact data. This algorithm--named GRAAL--generates high-quality assemblies of genomes in which repeated and duplicated regions are accurately represented and offers a direct probabilistic interpretation of the computed structures. We first validated GRAAL on the reference genome of Saccharomyces cerevisiae, as well as other yeast isolates, where GRAAL recovered both known and unknown complex chromosomal structural variations. We then applied GRAAL to the finishing of the assembly of Trichoderma reesei and obtained a number of contigs congruent with the know karyotype of this species. Finally, we showed that GRAAL can accurately reconstruct human chromosomes from either fragments generated in silico or contigs obtained from de novo assembly. In all these applications, GRAAL compared favourably to recently published programmes implementing related approaches.
Asunto(s)
Algoritmos , Cromosomas Fúngicos , Cromosomas Humanos , Mapeo Contig/estadística & datos numéricos , Genoma , Modelos Estadísticos , Mapeo Contig/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Cariotipo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Trichoderma/genéticaRESUMEN
Genomic analyses of microbial populations in their natural environment remain limited by the difficulty to assemble full genomes of individual species. Consequently, the chromosome organization of microorganisms has been investigated in a few model species, but the extent to which the features described can be generalized to other taxa remains unknown. Using controlled mixes of bacterial and yeast species, we developed meta3C, a metagenomic chromosome conformation capture approach that allows characterizing individual genomes and their average organization within a mix of organisms. Not only can meta3C be applied to species already sequenced, but a single meta3C library can be used for assembling, scaffolding and characterizing the tridimensional organization of unknown genomes. By applying meta3C to a semi-complex environmental sample, we confirmed its promising potential. Overall, this first meta3C study highlights the remarkable diversity of microorganisms chromosome organization, while providing an elegant and integrated approach to metagenomic analysis.
Asunto(s)
Bacterias/genética , Cromosomas Bacterianos/genética , Cromosomas Fúngicos/genética , Metagenómica/métodos , Levaduras/genética , Genoma Bacteriano , Procesamiento de Imagen Asistido por Computador , Plásmidos/metabolismo , Duplicaciones Segmentarias en el GenomaRESUMEN
A computational challenge raised by chromosome conformation capture (3C) experiments is to reconstruct spatial distances and three-dimensional genome structures from observed contacts between genomic loci. We propose a two-step algorithm, ShRec3D, and assess its accuracy using both in silico data and human genome-wide 3C (Hi-C) data. This algorithm avoids convergence issues, accommodates sparse and noisy contact maps, and is orders of magnitude faster than existing methods.
Asunto(s)
Cromosomas Fúngicos , Cromosomas Humanos , Simulación por Computador , Genoma Fúngico , Genoma Humano , Imagenología Tridimensional/métodos , Levaduras/genética , Algoritmos , Humanos , Conformación de Ácido Nucleico , Programas InformáticosRESUMEN
MOTIVATIONS: De novo sequencing of genomes is followed by annotation analyses aiming at identifying functional genomic features such as genes, non-coding RNAs or regulatory sequences, taking advantage of diverse datasets. These steps sometimes fail at detecting non-coding functional sequences: for example, origins of replication, centromeres and rDNA positions have proven difficult to annotate with high confidence. Here, we demonstrate an unconventional application of Chromosome Conformation Capture (3C) technique, which typically aims at deciphering the average 3D organization of genomes, by showing how functional information about the sequence can be extracted solely from the chromosome contact map. RESULTS: Specifically, we describe a combined experimental and bioinformatic procedure that determines the genomic positions of centromeres and ribosomal DNA clusters in yeasts, including species where classical computational approaches fail. For instance, we determined the centromere positions in Naumovozyma castellii, where these coordinates could not be obtained previously. Although computed centromere positions were characterized by conserved synteny with neighboring species, no consensus sequences could be found, suggesting that centromeric binding proteins or mechanisms have significantly diverged. We also used our approach to refine centromere positions in Kuraishia capsulata and to identify rDNA positions in Debaryomyces hansenii. Our study demonstrates how 3C data can be used to complete the functional annotation of eukaryotic genomes. AVAILABILITY AND IMPLEMENTATION: The source code is provided in the Supplementary Material. This includes a zipped file with the Python code and a contact matrix of Saccharomyces cerevisiae. CONTACT: romain.koszul@pasteur.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.