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The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50-300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10-200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.

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BACKGROUND: Genome assembly into chromosomes facilitates several analyses including cytogenetics, genomics and phylogenetics. Despite rapid development in bioinformatics, however, assembly beyond scaffolds remains challenging, especially in species without closely related well-assembled and available reference genomes. So far, four draft genomes of Rangifer tarandus (caribou or reindeer, a circumpolar distributed cervid species) have been published, but none with chromosome-level assembly. This emblematic northern species is of high interest in ecological studies and conservation since most populations are declining. RESULTS: We have designed specific probes based on Oligopaint FISH technology to upgrade the latest published reindeer and caribou chromosome-level genomes. Using this oligonucleotide-based method, we found six mis-assembled scaffolds and physically mapped 68 of the largest scaffolds representing 78% of the most recent R. tarandus genome assembly. Combining physical mapping and comparative genomics, it was possible to document chromosomal evolution among Cervidae and closely related bovids. CONCLUSIONS: Our results provide validation for the current chromosome-level genome assembly as well as resources to use chromosome banding in studies of Rangifer tarandus.

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For decades, biochemical methods for the analysis of genome structure and function provided cell-population-averaged data that allowed general principles and tendencies to be disclosed. Microscopy-based studies, which immanently involve single-cell analysis, did not provide sufficient spatial resolution to investigate the particularly small details of 3D genome folding. Nevertheless, these studies demonstrated that mutual positions of chromosome territories within cell nuclei and individual genomic loci within chromosomal territories can vary significantly in individual cells. The development of new technologies in biochemistry and the advent of super-resolution microscopy in the last decade have made possible the full-scale study of 3D genome organization in individual cells. Maps of the 3D genome build based on C-data and super-resolution microscopy are highly consistent and, therefore, biologically relevant. The internal structures of individual chromosomes, loci, and topologically associating domains (TADs) are resolved as well as cell-cycle dynamics. 3D modeling allows one to investigate the physical mechanisms underlying genome folding. Finally, joint profiling of genome topology and epigenetic features will allow 3D genomics to handle complex cell-to-cell heterogeneity. In this review, we summarize the present state of studies into 3D genome organization in individual cells, analyze the technical problems of single-cell studies, and outline perspectives of 3D genomics.

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The genome tridimensional (3D) organization and its role towards the regulation of key cell processes such as transcription is currently a main question in biology. Interphase chromosomes are spatially segregated into "territories," epigenetically-defined large domains of chromatin that interact to form "compartments" with common transcriptional status, and insulator-flanked domains called "topologically associating domains" (TADs). Moreover, chromatin organizes around nuclear structures such as lamina, speckles, or the nucleolus to acquire a higher-order genome organization. Due to recent technological advances, the different hierarchies are being solved. Particularly, advances in microscopy technologies are shedding light on the genome structure at multiple levels. Intriguingly, more and more reports point to high variability and stochasticity at the single-cell level. However, the functional consequences of such variability in genome conformation are still unsolved. Here, I will discuss the implication of the cell-to-cell heterogeneity at the different scales in the context of newly developed imaging approaches, particularly multiplexed Fluorescence in situ hybridization methods that enabled "chromatin tracing." Extensions of these methods are now combining spatial information of dozens to thousands of genomic loci with the localization of nuclear features such as the nucleolus, nuclear speckles, or even histone modifications, creating the fast-moving field of "spatial genomics." As our view of genome organization shifts the focus from ensemble to single-cell, new insights to fundamental questions begin to emerge.

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Current methods for chromosome painting via fluorescence in situ hybridization (FISH) are costly, time-consuming, and limited in complexity. In contrast to conventional sources of probe, Oligopaints are computationally designed, synthesized on microarrays, and amplified by PCR. This approach allows for precise control over the sequences they target, which can range from a few kilobases to entire chromosomes with the same basic protocol. We have utilized the flexibility and scalability of Oligopaints to generate low-cost and renewable chromosome paints for Drosophila, mouse, and human chromosomes. These Oligopaint libraries can be customized to label any genomic feature(s) in a chromosome-wide manner. Additionally, this method is compatible with sequential FISH to label entire genomes with a single denaturation step. Here, we outline a protocol and considerations to scale the Oligopaint technology for fluorescent labeling of whole chromosomes.

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Eukaryotic DNA is highly organized within nuclei and this organization is important for genome function. Fluorescent in situ hybridization (FISH) approaches allow 3D architectures of genomes to be visualized. Scalable FISH technologies, which can be applied to whole animals, are needed to help unravel how genomic architecture regulates, or is regulated by, gene expression during development, growth, reproduction, and aging. Here, we describe a multiplexed DNA FISH Oligopaint library that targets the entire Caenorhabditis elegans genome at chromosome, three megabase, and 500 kb scales. We describe a hybridization strategy that provides flexibility to DNA FISH experiments by coupling a single primary probe synthesis reaction to dye conjugated detection oligos via bridge oligos, eliminating the time and cost typically associated with labeling probe sets for individual experiments. The approach allows visualization of genome organization at varying scales in all/most cells across all stages of development in an intact animal model system.

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A multiplexed approach to DNA FISH experiments has been used to visualize the three-dimensional organization of chromosomes and specific chromosomal regions in C. elegans.

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Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome.

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OligoSTORM and OligoDNA-PAINT meld the Oligopaint technology for fluorescent in situ hybridization (FISH) with, respectively, Stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) to enable in situ single-molecule super-resolution imaging of nucleic acids. Both strategies enable ≤20 nm resolution and are appropriate for imaging nanoscale features of the genomes of a wide range of species, including human, mouse, and fruit fly (Drosophila).

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Fluorescence in situ hybridization (FISH) is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes, and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains, and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbias and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH) allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH) using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells DNA and RNA FISH techniques have visualized intra-nuclear genomic structure and sub-cellular transcriptional dynamics of many genes and revealed their functions in various biological processes.

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Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

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Oligopaint probes are fluorescently labeled, single-stranded DNA oligonucleotides that can be used to visualize genomic regions ranging in size from tens of kilobases to many megabases. This unit details how Oligopaint probes can be synthesized using basic molecular biological techniques, and provides protocols for FISH, 3D-FISH, and sample preparation.

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