RESUMEN
Studying the similarities and differences in genomic interactions between species provides fertile grounds for determining the evolutionary dynamics underpinning genome function and speciation. Here, we describe the principles of 3D genome folding in vertebrates and show how lineage-specific patterns of genome reshuffling can result in different chromatin configurations. We (1) identified different patterns of chromosome folding in across vertebrate species (centromere clustering versus chromosomal territories); (2) reconstructed ancestral marsupial and afrotherian genomes analyzing whole-genome sequences of species representative of the major therian phylogroups; (3) detected lineage-specific chromosome rearrangements; and (4) identified the dynamics of the structural properties of genome reshuffling through therian evolution. We present evidence of chromatin configurational changes that result from ancestral inversions and fusions/fissions. We catalog the close interplay between chromatin higher-order organization and therian genome evolution and introduce an interpretative hypothesis that explains how chromatin folding influences evolutionary patterns of genome reshuffling.
Asunto(s)
Evolución Molecular , Marsupiales , Animales , Cromosomas/genética , Mamíferos/genética , Genoma , Vertebrados/genética , Cromatina/genéticaRESUMEN
Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cromatina/química , Mutación con Pérdida de Función , Proteínas Nucleares/genética , Transcripción Genética , Neoplasias de la Vejiga Urinaria/genética , Secuencia de Bases , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Anotación de Secuencia Molecular , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The spatial folding of chromosomes inside the nucleus has regulatory effects on gene expression, yet the impact of genome reshuffling on this organization remains unclear. Here, we take advantage of chromosome conformation capture in combination with single-nucleotide polymorphism (SNP) genotyping and analysis of crossover events to study how the higher-order chromatin organization and recombination landscapes are affected by chromosomal fusions in the mammalian germ line. We demonstrate that chromosomal fusions alter the nuclear architecture during meiosis, including an increased rate of heterologous interactions in primary spermatocytes, and alterations in both chromosome synapsis and axis length. These disturbances in topology were associated with changes in genomic landscapes of recombination, resulting in detectable genomic footprints. Overall, we show that chromosomal fusions impact the dynamic genome topology of germ cells in two ways: (i) altering chromosomal nuclear occupancy and synapsis, and (ii) reshaping landscapes of recombination.
Asunto(s)
Cromatina/metabolismo , Cromosomas/metabolismo , Recombinación Genética , Espermatocitos/metabolismo , Animales , Evolución Biológica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Emparejamiento Cromosómico/genética , Segregación Cromosómica , Cromosomas/genética , Europa (Continente) , Fertilidad/genética , Técnicas de Genotipaje/métodos , Masculino , Ratones , Polimorfismo de Nucleótido Simple , Cultivo Primario de Células , Análisis de Semen , Espermatocitos/citologíaRESUMEN
Mammalian gametogenesis involves dramatic and tightly regulated chromatin remodeling, whose regulatory pathways remain largely unexplored. Here, we generate a comprehensive high-resolution structural and functional atlas of mouse spermatogenesis by combining in situ chromosome conformation capture sequencing (Hi-C), RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) of CCCTC-binding factor (CTCF) and meiotic cohesins, coupled with confocal and super-resolution microscopy. Spermatogonia presents well-defined compartment patterns and topological domains. However, chromosome occupancy and compartmentalization are highly re-arranged during prophase I, with cohesins bound to active promoters in DNA loops out of the chromosomal axes. Compartment patterns re-emerge in round spermatids, where cohesin occupancy correlates with transcriptional activity of key developmental genes. The compact sperm genome contains compartments with actively transcribed genes but no fine-scale topological domains, concomitant with the presence of protamines. Overall, we demonstrate how genome-wide cohesin occupancy and transcriptional activity is associated with three-dimensional (3D) remodeling during spermatogenesis, ultimately reprogramming the genome for the next generation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Genómica/métodos , Espermatogénesis/genética , Humanos , Masculino , Conformación Molecular , CohesinasRESUMEN
Cohesin exists in two variants carrying either STAG/SA1 or SA2. Here we have addressed their specific contributions to the unique spatial organization of the mouse embryonic stem cell genome, which ensures super-enhancer-dependent transcription of pluripotency factors and repression of lineage-specification genes within Polycomb domains. We find that cohesin-SA2 facilitates Polycomb domain compaction through Polycomb repressing complex 1 (PRC1) recruitment and promotes the establishment of long-range interaction networks between distant Polycomb-bound promoters that are important for gene repression. Cohesin-SA1, in contrast, disrupts these networks, while preserving topologically associating domain (TAD) borders. The diverse effects of both complexes on genome topology may reflect two modes of action of cohesin. One, likely involving loop extrusion, establishes overall genome arrangement in TADs together with CTCF and prevents excessive segregation of same-class compartment regions. The other is required for organization of local transcriptional hubs such as Polycomb domains and super-enhancers, which define cell identity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Animales , Sitios de Unión , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Elementos de Facilitación Genéticos , Masculino , Ratones , Células Madre Embrionarias de Ratones/citología , Proteínas del Grupo Polycomb/genética , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas , CohesinasRESUMEN
Nuclear architecture is decisive for the assembly of transcriptional responses. However, how chromosome organization is dynamically modulated to permit rapid and transient transcriptional changes in response to environmental challenges remains unclear. Here we show that hyperosmotic stress disrupts different levels of chromosome organization, ranging from A/B compartment changes to reduction in the number and insulation of topologically associating domains (TADs). Concomitantly, transcription is greatly affected, TAD borders weaken, and RNA Polymerase II runs off from hundreds of transcription end sites. Stress alters the binding profiles of architectural proteins, which explains the disappearance of local chromatin organization. These processes are dynamic, and cells rapidly reconstitute their default chromatin conformation after stress removal, uncovering an intrinsic organization. Transcription is not required for local chromatin reorganization, while compartment recovery is partially transcription-dependent. Thus, nuclear organization in mammalian cells can be rapidly modulated by environmental changes in a reversible manner.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Presión Osmótica , ARN Polimerasa II/metabolismo , Transcripción Genética , Línea Celular , HumanosRESUMEN
In breast cancer cells, some topologically associating domains (TADs) behave as hormonal gene regulation units, within which gene transcription is coordinately regulated in response to steroid hormones. Here we further describe that responsive TADs contain 20- to 100-kb-long clusters of intermingled estrogen receptor (ESR1) and progesterone receptor (PGR) binding sites, hereafter called hormone-control regions (HCRs). In T47D cells, we identified more than 200 HCRs, which are frequently bound by unliganded ESR1 and PGR. These HCRs establish steady long-distance inter-TAD interactions between them and organize characteristic looping structures with promoters in their TADs even in the absence of hormones in ESR1+-PGR+ cells. This organization is dependent on the expression of the receptors and is further dynamically modulated in response to steroid hormones. HCRs function as platforms that integrate different signals, resulting in some cases in opposite transcriptional responses to estrogens or progestins. Altogether, these results suggest that steroid hormone receptors act not only as hormone-regulated sequence-specific transcription factors but also as local and global genome organizers.
Asunto(s)
Receptor alfa de Estrógeno/biosíntesis , Estrógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Progesterona/farmacología , Receptores de Progesterona/biosíntesis , Elementos de Respuesta , Transducción de Señal/efectos de los fármacos , Receptor alfa de Estrógeno/genética , Humanos , Células MCF-7 , Receptores de Progesterona/genéticaRESUMEN
Lamins (A/C and B) are major constituents of the nuclear lamina (NL). Structurally conserved lamina-associated domains (LADs) are formed by genomic regions that contact the NL. Lamins are also found in the nucleoplasm, with a yet unknown function. Here we map the genome-wide localization of lamin B1 in an euchromatin-enriched fraction of the mouse genome and follow its dynamics during the epithelial-to-mesenchymal transition (EMT). Lamin B1 associates with actively expressed and open euchromatin regions, forming dynamic euchromatin lamin B1-associated domains (eLADs) of about 0.3 Mb. Hi-C data link eLADs to the 3D organization of the mouse genome during EMT and correlate lamin B1 enrichment at topologically associating domain (TAD) borders with increased border strength. Having reduced levels of lamin B1 alters the EMT transcriptional signature and compromises the acquisition of mesenchymal traits. Thus, during EMT, the process of genome reorganization in mouse involves dynamic changes in eLADs.
Asunto(s)
Lamina Tipo B/metabolismo , Animales , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Eucromatina/química , Eucromatina/genética , Eucromatina/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Lamina Tipo B/química , Lamina Tipo B/genética , RatonesRESUMEN
The three-dimensional conformation of genomes is an essential component of their biological activity. The advent of the Hi-C technology enabled an unprecedented progress in our understanding of genome structures. However, Hi-C is subject to systematic biases that can compromise downstream analyses. Several strategies have been proposed to remove those biases, but the issue of abnormal karyotypes received little attention. Many experiments are performed in cancer cell lines, which typically harbor large-scale copy number variations that create visible defects on the raw Hi-C maps. The consequences of these widespread artifacts on the normalized maps are mostly unexplored. We observed that current normalization methods are not robust to the presence of large-scale copy number variations, potentially obscuring biological differences and enhancing batch effects. To address this issue, we developed an alternative approach designed to take into account chromosomal abnormalities. The method, called OneD, increases reproducibility among replicates of Hi-C samples with abnormal karyotype, outperforming previous methods significantly. On normal karyotypes, OneD fared equally well as state-of-the-art methods, making it a safe choice for Hi-C normalization. OneD is fast and scales well in terms of computing resources for resolutions up to 5 kb.
Asunto(s)
Cariotipo Anormal , Animales , Composición de Base , Sesgo , Línea Celular , Aberraciones Cromosómicas , Biología Computacional/métodos , Biología Computacional/estadística & datos numéricos , Simulación por Computador , Variaciones en el Número de Copia de ADN , Técnicas Genéticas , Humanos , Cadenas de Markov , Ratones , Modelos Estadísticos , Reproducibilidad de los ResultadosRESUMEN
Chromosomal architecture is known to influence gene expression, yet its role in controlling cell fate remains poorly understood. Reprogramming of somatic cells into pluripotent stem cells (PSCs) by the transcription factors (TFs) OCT4, SOX2, KLF4 and MYC offers an opportunity to address this question but is severely limited by the low proportion of responding cells. We have recently developed a highly efficient reprogramming protocol that synchronously converts somatic into pluripotent stem cells. Here, we used this system to integrate time-resolved changes in genome topology with gene expression, TF binding and chromatin-state dynamics. The results showed that TFs drive topological genome reorganization at multiple architectural levels, often before changes in gene expression. Removal of locus-specific topological barriers can explain why pluripotency genes are activated sequentially, instead of simultaneously, during reprogramming. Together, our results implicate genome topology as an instructive force for implementing transcriptional programs and cell fate in mammals.
Asunto(s)
Reprogramación Celular/genética , Ensamble y Desensamble de Cromatina/genética , Estructuras Cromosómicas/genética , Genoma , Factores de Transcripción/fisiología , Animales , Sitios de Unión/genética , Células Cultivadas , Estructuras Cromosómicas/metabolismo , Compensación de Dosificación (Genética)/genética , Femenino , Regulación de la Expresión Génica , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Ratones , Ratones Transgénicos , Unión Proteica , Inactivación del Cromosoma X/genéticaRESUMEN
T47D_rep2 and b1913e6c1_51720e9cf were 2 Hi-C samples. They were born and processed at the same time, yet their fates were very different. The life of b1913e6c1_51720e9cf was simple and fruitful, while that of T47D_rep2 was full of accidents and sorrow. At the heart of these differences lies the fact that b1913e6c1_51720e9cf was born under a lab culture of Documentation, Automation, Traceability, and Autonomy and compliance with the FAIR Principles. Their lives are a lesson for those who wish to embark on the journey of managing high-throughput sequencing data.
Asunto(s)
Curaduría de Datos/normas , Genómica/normas , Guías de Práctica Clínica como Asunto , Análisis de Secuencia de ADN/normas , Curaduría de Datos/métodos , Genoma , Genómica/métodos , Humanos , Registros/normas , Análisis de Secuencia de ADN/métodosRESUMEN
Chromosomes are large polymer molecules composed of nucleotides. In some species, such as humans, this polymer can sum up to meters long and still be properly folded within the nuclear space of few microns in size. The exact mechanisms of how the meters long DNA is folded into the nucleus, as well as how the regulatory machinery can access it, is to a large extend still a mystery. However, and thanks to newly developed molecular, genomic and computational approaches based on the Chromosome Conformation Capture (3C) technology, we are now obtaining insight on how genomes are spatially organized. Here we review a new family of computational approaches that aim at using 3C-based data to obtain spatial restraints for modeling genomes and genomic domains.
Asunto(s)
Cromosomas/ultraestructura , Genoma , Modelos Genéticos , Animales , Cromosomas/genética , Simulación por Computador , Regulación de la Expresión Génica , Humanos , Conformación de Ácido NucleicoRESUMEN
BACKGROUND INFORMATION: Retromer is required for endosome-to-Golgi retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR), allowing delivery of hydrolases into lysosomes. It is constituted by a conserved heterotrimer formed by vacuolar protein sorting (Vps) gene products Vps26, Vps35 and Vps29, which is in charge of cargo selection, and a dimer of phosphoinositide-binding sorting nexins (SNXs), which has a structural role. Retromer has been implicated in sorting of additional cargo. Thus, retromer also promotes polymeric immunoglobulin A (pIgA) transcytosis by the pIgA receptor (pIgR) in polarised cells, and considerable evidence implicates retromer in controlling epithelial cell polarity. However, the precise localisation of retromer along the endocytic pathway of polarised cells has not been studied in detail. RESULTS: Our biochemical analysis using rat liver endosome fractions suggests a distinct distribution pattern. Although subunits of the cargo-selective complex were enriched in early endosomes (EEs), levels of SNX2 were greater in sorting endosomes. We then immunolocalised the retromer subunits in polarised Madin-Darby canine kidney (MDCK) cells by confocal microscopy. An estimated 25% of total Vps26 and SNX2 localised to EEs, with negligible portions in recycling endosomes as well as in late endosomes and lysosomes. Although Vps26 was in structures of more heterogeneous size and shape than SNX2, these markedly overlapped. In consequence, the two retromer subcomplexes mostly colocalised. When we analysed retromer overlap with its cargo, we found that structures retromer and pIgA(+) are independent of those structures retromer and CI-MPR(+) . Remarkably, retromer localised preferentially at the transcytotic pathway. Pharmacological inhibition of phosphoinositide 3-kinase affected the co-distribution of retromer with pIgA and the CI-MPR, delaying pIgA progress to the apical surface. CONCLUSIONS: In polarised MDCK cells, we found retromer associated with certain specialised EE-derived pathways. Our data imply that retromer is largely engaged in pIgA transcytosis in pIgR-expressing MDCK cells, as opposed to endosome-to-Golgi retrieval.
Asunto(s)
Transporte Biológico/fisiología , Polaridad Celular/fisiología , Endosomas/fisiología , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Perros , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/fisiología , Humanos , Lisosomas/metabolismo , Lisosomas/fisiología , Células de Riñón Canino Madin Darby , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas Sprague-Dawley , Nexinas de Clasificación/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
ß-Amyloid (Aß) peptides are generated from the successive proteolytic processing of the amyloid precursor protein (APP) by the ß-APP cleaving enzyme (BACE or ß-secretase) and the γ-secretase complex. Initial cleavage of APP by BACE leads into the amyloidogenic pathway, causing or exacerbating Alzheimer's disease. Therefore, their intracellular traffic can determine how easily and frequently BACE has access to and cleaves APP. Here, we have used polarized Madin-Darby canine kidney (MDCK) cells stably expressing APP and BACE to examine the regulation of their polarized trafficking by retromer, a protein complex previously implicated in their endosome-to-Golgi transport. Our data show that retromer interacts with BACE and regulates its postendocytic sorting in polarized MDCK cells. Depleting retromer, inhibiting retromer function, or preventing BACE interaction with retromer, alters trafficking of BACE, which thereby increases its localization in the early endocytic compartment. As a result, this slows endocytosis of apically localized BACE, promoting its recycling and apical-to-basolateral transcytosis, which increases APP/BACE interaction and subsequent cleavage of APP toward generation and secretion of Aß peptides.