RESUMO
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.
Assuntos
Adenosina Trifosfatases , Cromossomos Bacterianos , Proteínas de Ligação a DNA , Escherichia coli K12 , Complexos Multiproteicos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Escherichia coli K12/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Estrutura Quaternária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
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.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas de Saccharomyces cerevisiae , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Coesinas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Mitose , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Bacillus subtilis/metabolismo , Bacillus subtilis/ultraestrutura , Cromossomos Bacterianos/ultraestrutura , Replicação do DNA , DNA Super-Helicoidal , Microscopia , Modelos Moleculares , Imagem Óptica , Origem de ReplicaçãoRESUMO
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.
Assuntos
Cromossomos/química , Ribonucleoproteínas/química , Animais , Células Cultivadas , Fracionamento Químico/métodos , Cromossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Ribonucleoproteínas/metabolismo , SalinidadeRESUMO
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.
Assuntos
Actinas/fisiologia , Cromossomos Fúngicos/fisiologia , Saccharomyces cerevisiae/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Membrana/fisiologia , Poro Nuclear/metabolismo , Multimerização Proteica , Transporte Proteico , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Telômero/genética , Telômero/metabolismoRESUMO
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.
Assuntos
Genoma , Conformação de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Animais , Sítios de Ligação , Núcleo Celular , Mapeamento Cromossômico , Cromossomos , Biologia Computacional/métodos , Drosophila , Células-Tronco Embrionárias/metabolismo , Evolução Molecular , Humanos , Camundongos , Anotação de Sequência Molecular , Especificidade de Órgãos/genética , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Cromossomos Fúngicos , Cromossomos Humanos , Simulação por Computador , Genoma Fúngico , Genoma Humano , Imageamento Tridimensional/métodos , Leveduras/genética , Algoritmos , Humanos , Conformação de Ácido Nucleico , SoftwareRESUMO
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.
Assuntos
Candida glabrata/genética , Cromossomos Fúngicos , Replicação do DNA , Genoma Fúngico , Ciclo Celular/genética , Genes FúngicosRESUMO
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.
Assuntos
Genoma Fúngico/genética , Genômica/métodos , Anotação de Sequência Molecular/métodos , Saccharomycetales/genética , Centrômero/genética , Sequência Consenso , DNA Ribossômico/genética , Loci Gênicos/genética , SinteniaRESUMO
Transcriptional regulation is at the heart of biological functions such as adaptation to a changing environment or to new carbon sources. One of the mechanisms which has been found to modulate transcription, either positively (activation) or negatively (repression), involves the formation of DNA loops. A DNA loop occurs when a protein or a complex of proteins simultaneously binds to two different sites on DNA with looping out of the intervening DNA. This simple mechanism is central to the regulation of several operons in the genome of the bacterium Escherichia coli, like the lac operon, one of the paradigms of genetic regulation. The aim of this review is to gather and discuss concepts and ideas from experimental biology and theoretical physics concerning DNA looping in genetic regulation. We first describe experimental techniques designed to show the formation of a DNA loop. We then present the benefits that can or could be derived from a mechanism involving DNA looping. Some of these are already experimentally proven, but others are theoretical predictions and merit experimental investigation. Then, we try to identify other genetic systems that could be regulated by a DNA looping mechanism in the genome of Escherichia coli. We found many operons that, according to our set of criteria, have a good chance to be regulated with a DNA loop. Finally, we discuss the proposition recently made by both biologists and physicists that this mechanism could also act at the genomic scale and play a crucial role in the spatial organization of genomes.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Transcrição Gênica , DNA Bacteriano/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon Lac , Conformação de Ácido NucleicoRESUMO
BACKGROUND: Chromatin organization has been increasingly studied in relation with its important influence on DNA-related metabolic processes such as replication or regulation of gene expression. Since its original design ten years ago, capture of chromosome conformation (3C) has become an essential tool to investigate the overall conformation of chromosomes. It relies on the capture of long-range trans and cis interactions of chromosomal segments whose relative proportions in the final bank reflect their frequencies of interactions, hence their spatial proximity in a population of cells. The recent coupling of 3C with deep sequencing approaches now allows the generation of high resolution genome-wide chromosomal contact maps. Different protocols have been used to generate such maps in various organisms. This includes mammals, drosophila and yeast. The massive amount of raw data generated by the genomic 3C has to be carefully processed to alleviate the various biases and byproducts generated by the experiments. Our study aims at proposing a simple normalization procedure to minimize the influence of these unwanted but inevitable events on the final results. RESULTS: Careful analysis of the raw data generated previously for budding yeast S. cerevisiae led to the identification of three main biases affecting the final datasets, including a previously unknown bias resulting from the circularization of DNA molecules. We then developed a simple normalization procedure to process the data and allow the generation of a normalized, highly contrasted, chromosomal contact map for S. cerevisiae. The same method was then extended to the first human genome contact map. Using the normalized data, we revisited the preferential interactions originally described between subsets of discrete chromosomal features. Notably, the detection of preferential interactions between tRNA in yeast and CTCF, PolII binding sites in human can vary with the normalization procedure used. CONCLUSIONS: We quantitatively reanalyzed the genomic 3C data obtained for S. cerevisiae, identified some of the biases inherent to the technique and proposed a simple normalization procedure to analyse them. Such an approach can be easily generalized for genomic 3C experiments in other organisms. More experiments and analysis will be necessary to reach optimal resolution and accuracies of the maps generated through these approaches. Working with cell population presenting highest levels of homogeneity will prove useful in this regards.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Fúngicos/genética , Cromossomos Humanos/genética , Genoma , Saccharomyces cerevisiae/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Circular , Humanos , RNA de Transferência/genética , RNA de Transferência/metabolismo , Curva ROC , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
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.
Assuntos
DNA Circular/genética , DNA Viral/genética , Vírus da Hepatite B/genética , Animais , Carcinoma Hepatocelular/virologia , Genoma Viral , Hepatite B/virologia , Hepatite B Crônica/virologia , Humanos , Replicação ViralRESUMO
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 ).
Assuntos
Cromossomos , Cromatina/genética , Genoma , GenômicaRESUMO
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.
Assuntos
Cromossomos/genética , Computadores , Reconhecimento Automatizado de Padrão , Algoritmos , Cromossomos Fúngicos/genética , Cromossomos Humanos/genética , Genoma Fúngico , Humanos , Saccharomyces cerevisiae/genética , Fluxo de TrabalhoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Mapas de Interação de Proteínas , Saccharomyces cerevisiae/citologia , CoesinasRESUMO
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.
Assuntos
Cromatina/metabolismo , Genoma Humano , Vírus da Hepatite B/fisiologia , Sequência de Bases , Ilhas de CpG/genética , DNA Viral/genética , Regulação da Expressão Gênica , Células Hep G2 , Hepatócitos/virologia , Humanos , Modelos Biológicos , Plasmídeos/metabolismo , Transativadores/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica , Regulação para Cima/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
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.
Assuntos
Bactérias , Bacteriófagos , Microbioma Gastrointestinal , Genoma Bacteriano , Genoma Viral , Animais , Bactérias/genética , Bactérias/metabolismo , Bactérias/virologia , Bacteriófagos/genética , Bacteriófagos/metabolismo , Masculino , CamundongosRESUMO
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.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Fúngicos/genética , Genoma Fúngico/genética , Cromatina/genética , Saccharomyces cerevisiaeRESUMO
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.