RESUMEN
The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.
Asunto(s)
Núcleo Celular , Genoma , Genoma/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismoRESUMEN
Reactivation of the inactive X chromosome is a hallmark epigenetic event during reprogramming of mouse female somatic cells to induced pluripotent stem cells (iPSCs). This involves global structural remodeling from a condensed, heterochromatic into an open, euchromatic state, thereby changing a transcriptionally inactive into an active chromosome. Despite recent advances, very little is currently known about the molecular players mediating this process and how this relates to iPSC-reprogramming in general. To gain more insight, here we perform a RNAi-based knockdown screen during iPSC-reprogramming of mouse fibroblasts. We discover factors important for X chromosome reactivation (XCR) and iPSC-reprogramming. Among those, we identify the cohesin complex member SMC1a as a key molecule with a specific function in XCR, as its knockdown greatly affects XCR without interfering with iPSC-reprogramming. Using super-resolution microscopy, we find SMC1a to be preferentially enriched on the active compared with the inactive X chromosome and that SMC1a is critical for the decompacted state of the active X. Specifically, depletion of SMC1a leads to contraction of the active X both in differentiated and in pluripotent cells, where it normally is in its most open state. In summary, we reveal cohesin as a key factor for remodeling of the X chromosome from an inactive to an active structure and that this is a critical step for XCR during iPSC-reprogramming.
Asunto(s)
Células Madre Pluripotentes Inducidas , Femenino , Animales , Ratones , Reprogramación Celular , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Estructuras Cromosómicas , CohesinasRESUMEN
Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.
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Microscopía , Animales , Ratones , Microscopía/métodosRESUMEN
Fluorescence in situ hybridization (FISH) allows researchers to visualize the spatial position and quantity of nucleic acids in fixed samples. Recently, considerable progress has been made in developing oligonucleotide (oligo)-based FISH methods that have enabled researchers to study the three-dimensional organization of the genome at super-resolution and visualize the spatial patterns of gene expression for thousands of genes in individual cells. However, there are few existing computational tools to support the bioinformatics workflows necessary to carry out these experiments using oligo FISH probes. Here, we introduce paint server and homology optimization pipeline (PaintSHOP), an interactive platform for the design of oligo FISH experiments. PaintSHOP enables researchers to identify probes for their experimental targets efficiently, to incorporate additional necessary sequences such as primer pairs and to easily generate files documenting library design. PaintSHOP democratizes and standardizes the process of designing complex probe sets for the oligo FISH community.
Asunto(s)
Pintura Cromosómica/métodos , Biología Computacional/métodos , Genoma Humano , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos/química , Secuencias Repetitivas de Ácidos Nucleicos , Transcriptoma , HumanosRESUMEN
There is a need for methods that can image chromosomes with genome-wide coverage, as well as greater genomic and optical resolution. We introduce OligoFISSEQ, a suite of three methods that leverage fluorescence in situ sequencing (FISSEQ) of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how four rounds of sequencing are sufficient to produce 3D maps of 36 genomic targets across six chromosomes in hundreds to thousands of cells, implying a potential to image thousands of targets in only five to eight rounds of sequencing. We also use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunocytochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes.
Asunto(s)
Pintura Cromosómica/métodos , Cromosomas/química , Cromosomas/genética , Genoma Humano , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sondas de Oligonucleótidos , Mapeo Físico de CromosomaRESUMEN
Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct "territories." Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.
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Centrosoma/metabolismo , Eucromatina/genética , Heterocromatina/metabolismo , Animales , Estructuras Cromosómicas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eucromatina/metabolismo , Genoma/genética , Heterocromatina/genética , Heterocromatina/ultraestructura , Histonas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
Fluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here we introduce signal amplification by exchange reaction (SABER), which endows oligonucleotide-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplified RNA and DNA FISH signals (5- to 450-fold) in fixed cells and tissues. We also applied 17 orthogonal amplifiers against chromosomal targets simultaneously and detected mRNAs with high efficiency. We then used 10-plex SABER-FISH to identify in vivo introduced enhancers with cell-type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.
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ADN/análisis , Colorantes Fluorescentes/metabolismo , Hibridación Fluorescente in Situ/métodos , Oligonucleótidos/química , Imagen Óptica/métodos , ARN/análisis , Retina/metabolismo , Animales , Células Cultivadas , ADN/genética , ADN de Cadena Simple/química , Humanos , Ratones , ARN/genética , Retina/diagnóstico por imagenRESUMEN
Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.
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Centrómero/genética , Drosophila/genética , Retroelementos/genética , Animales , Proteína A Centromérica/genética , Cromatina/metabolismo , Elementos Transponibles de ADN/genética , ADN Satélite/genética , Drosophila/embriología , Proteínas de Drosophila/genética , Embrión no Mamífero/metabolismo , Genoma de los Insectos , Secuencias Repetidas Terminales/genéticaRESUMEN
Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/genética , Epigénesis Genética , Animales , Línea Celular , Posicionamiento de Cromosoma , Drosophila melanogaster/citología , Represión Epigenética , Fractales , Genoma/genética , Proteínas del Grupo Polycomb/metabolismo , Transcripción GenéticaRESUMEN
Fluorescence in situ hybridization (FISH) is a powerful single-cell technique that harnesses nucleic acid base pairing to detect the abundance and positioning of cellular RNA and DNA molecules in fixed samples. Recent technology development has paved the way to the construction of FISH probes entirely from synthetic oligonucleotides (oligos), allowing the optimization of thermodynamic properties together with the opportunity to design probes against any sequenced genome. However, comparatively little progress has been made in the development of computational tools to facilitate the oligos design, and even less has been done to extend their accessibility. OligoMiner is an open-source and modular pipeline written in Python that introduces a novel method of assessing probe specificity that employs supervised machine learning to predict probe binding specificity from genome-scale sequence alignment information. However, its use is restricted to only those people who are confident with command line interfaces because it lacks a Graphical User Interface (GUI), potentially cutting out many researchers from this technology. Here, we present OligoMinerApp (http://oligominerapp.org), a web-based application that aims to extend the OligoMiner framework through the implementation of a smart and easy-to-use GUI and the introduction of new functionalities specially designed to make effective probe mining available to everyone.
Asunto(s)
Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos , Programas Informáticos , Genoma , InternetRESUMEN
There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in â¼40-50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 µl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.
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ADN de Cadena Simple/síntesis química , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Reparación del ADN/efectos de los fármacos , ADN de Cadena Simple/química , Marcación de Gen/métodos , Células HEK293 , Humanos , Metanol/química , Metanol/farmacología , Mutagénesis Sitio-Dirigida/métodos , Polímeros/química , Factores de TiempoRESUMEN
Oligonucleotide (oligo)-based FISH has emerged as an important tool for the study of chromosome organization and gene expression and has been empowered by the commercial availability of highly complex pools of oligos. However, a dedicated bioinformatic design utility has yet to be created specifically for the purpose of identifying optimal oligo FISH probe sequences on the genome-wide scale. Here, we introduce OligoMiner, a rapid and robust computational pipeline for the genome-scale design of oligo FISH probes that affords the scientist exact control over the parameters of each probe. Our streamlined method uses standard bioinformatic file formats, allowing users to seamlessly integrate new and existing utilities into the pipeline as desired, and introduces a method for evaluating the specificity of each probe molecule that connects simulated hybridization energetics to rapidly generated sequence alignments using supervised machine learning. We demonstrate the scalability of our approach by performing genome-scale probe discovery in numerous model organism genomes and showcase the performance of the resulting probes with diffraction-limited and single-molecule superresolution imaging of chromosomal and RNA targets. We anticipate that this pipeline will make the FISH probe design process much more accessible and will more broadly facilitate the design of pools of hybridization probes for a variety of applications.
Asunto(s)
Genómica/métodos , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , Animales , Arabidopsis , ADN/genética , ADN/metabolismo , Minería de Datos , Humanos , Ratones , Modelos Genéticos , Sondas de Oligonucleótidos/metabolismoRESUMEN
Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method-integrative modeling of genomic regions (IMGR)-to increase the genomic resolution of our traces to 10 kb.
Asunto(s)
Paseo de Cromosoma/métodos , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 19/ultraestructura , Modelos Genéticos , Células Cultivadas , Pintura Cromosómica/métodos , Estructuras Cromosómicas/química , Estructuras Cromosómicas/genética , Estructuras Cromosómicas/ultraestructura , Cromosomas Humanos Par 19/química , Femenino , Colorantes Fluorescentes , Humanos , Imagenología Tridimensional , Hibridación Fluorescente in Situ/métodos , Masculino , Sondas de Oligonucleótidos , LinajeRESUMEN
Homolog pairing, which plays a critical role in meiosis, poses a potential risk if it occurs in inappropriate tissues or between nonallelic sites, as it can lead to changes in gene expression, chromosome entanglements, and loss-of-heterozygosity due to mitotic recombination. This is particularly true in Drosophila, which supports organismal-wide pairing throughout development. Discovered over a century ago, such extensive pairing has led to the perception that germline pairing in the adult gonad is an extension of the pairing established during embryogenesis and, therefore, differs from the mechanism utilized in most species to initiate pairing specifically in the germline. Here, we show that, contrary to long-standing assumptions, Drosophila meiotic pairing in the gonad is not an extension of pairing established during embryogenesis. Instead, we find that homologous chromosomes are unpaired in primordial germ cells from the moment the germline can be distinguished from the soma in the embryo and remain unpaired even in the germline stem cells of the adult gonad. We further establish that pairing originates immediately after the stem cell stage. This pairing occurs well before the initiation of meiosis and, strikingly, continues through the several mitotic divisions preceding meiosis. These discoveries indicate that the spatial organization of the Drosophila genome differs between the germline and the soma from the earliest moments of development and thus argue that homolog pairing in the germline is an active process as versus a passive continuation of pairing established during embryogenesis.
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Emparejamiento Cromosómico/genética , Células Germinativas/citología , Meiosis/genética , Células Madre/citología , Animales , Segregación Cromosómica/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Oocitos/citología , Recombinación GenéticaRESUMEN
A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide- and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.
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Pintura Cromosómica/métodos , Genoma/genética , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos/metabolismo , Animales , Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Cromosomas/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Biblioteca de Genes , Humanos , Interfase/genética , Metafase/genética , Ratones , Ovario/citología , Ovario/metabolismo , Coloración y EtiquetadoRESUMEN
Fluorescent in situ hybridization (FISH) enables the visualization of the position and abundance of nucleic acid molecules in fixed cell and tissue samples. Many FISH-based methods employ sets of synthetic, computationally designed DNA oligonucleotide (oligo) FISH probes, including massively multiplexed imaging spatial transcriptomics and spatial genomics technologies. Oligo probes can either be designed de novo or accessed from an existing database of pre-discovered probe sequences. This chapter describes the use of PaintSHOP, a user-friendly, web-based platform for the design of sets of oligo-based FISH probes. PaintSHOP hosts large collections of pre-discovered probes from many model organisms and also provides collections of functional sequences such as primers and readout domains and interactive tools to add these functional sequences to selected probes. Detailed examples are provided for three common experimental scenarios.
Asunto(s)
Genómica , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos/genética , Cartilla de ADNRESUMEN
Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate > 100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific probes against such regions presents significant technical challenges. Consequently, many open questions remain about the organization and function of highly repetitive sequences. Here, we introduce Tigerfish, a software tool for the genome-scale design of oligo probes against repetitive DNA intervals. We showcase Tigerfish by designing a panel of 24 interval-specific repeat probes specific to each of the 24 human chromosomes and imaging this panel on metaphase spreads and in interphase nuclei. Tigerfish extends the powerful toolkit of oligo-based FISH to highly repetitive DNA.
Asunto(s)
ADN , Secuencias Repetitivas de Ácidos Nucleicos , Humanos , Hibridación Fluorescente in Situ/métodos , ADN/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Sondas de Oligonucleótidos/genética , Sondas de ADN/genética , Oligonucleótidos/genéticaRESUMEN
The accuracy of crucial nuclear processes such as transcription, replication, and repair, depends on the local composition of chromatin and the regulatory proteins that reside there. Understanding these DNA-protein interactions at the level of specific genomic loci has remained challenging due to technical limitations. Here, we introduce a method termed "DNA O-MAP", which uses programmable peroxidase-conjugated oligonucleotide probes to biotinylate nearby proteins. We show that DNA O-MAP can be coupled with sample multiplexed quantitative proteomics and next-generation sequencing to quantify DNA-protein and DNA-DNA interactions at specific genomic loci.
RESUMEN
Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate >100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific probes against such regions presents significant technical challenges. Consequently, many open questions remain about the organization and function of highly repetitive sequences. Here, we introduce Tigerfish, a software tool for the genome-scale design of oligo probes against repetitive DNA intervals. We showcase Tigerfish by designing a panel of 24 interval-specific repeat probes specific to each of the 24 human chromosomes and imaging this panel on metaphase spreads and in interphase nuclei. Tigerfish extends the powerful toolkit of oligo-based FISH to highly repetitive DNA.