Advances in Chromatin Imaging at Kilobase-Scale Resolution
Authors: Alistair Boettiger, Sedona Murphy
Journal: Trends in Genetics (2020)

Microscopy now allows the measurement of the polymeric structure of chromatin in intact, fixed nuclei, with a resolution of a few kilobases.Multiway contact interactions and physical separation of domains can be probed directly.Structural information can be combined with measurements of nascent transcription, nuclear position, cellular position, and cellular morphology.Single-cell imaging validates many of the features previously identified by bulk, proximity-based approaches, and challenges existing models of chromatin structure.In situ super-resolution imaging offers new potential for multiomic single-cell analyses. It is now widely appreciated that the spatial organization of the genome is nonrandom, and its complex 3D folding has important consequences for many genome processes. Recent developments in multiplexed, super-resolution microscopy have enabled an unprecedented view of the polymeric structure of chromatin – from the loose folds of whole chromosomes to the detailed loops of cis-regulatory elements that regulate gene expression. Facilitated by the use of robotics, microfluidics, and improved approaches to super-resolution, thousands to hundreds of thousands of individual cells can now be analyzed in an individual experiment. This has led to new insights into the nature of genomic structural features identified by sequencing, such as topologically associated domains (TADs), and the nature of enhancer–promoter interactions underlying transcriptional regulation. We review these recent improvements. It is now widely appreciated that the spatial organization of the genome is nonrandom, and its complex 3D folding has important consequences for many genome processes. Recent developments in multiplexed, super-resolution microscopy have enabled an unprecedented view of the polymeric structure of chromatin – from the loose folds of whole chromosomes to the detailed loops of cis-regulatory elements that regulate gene expression. Facilitated by the use of robotics, microfluidics, and improved approaches to super-resolution, thousands to hundreds of thousands of individual cells can now be analyzed in an individual experiment. This has led to new insights into the nature of genomic structural features identified by sequencing, such as topologically associated domains (TADs), and the nature of enhancer–promoter interactions underlying transcriptional regulation. We review these recent improvements. Work in recent decades has shown that the spatial organization of the genome is nonrandom, and its complex 3D folding has important consequences for all genome processes including replication, segregation, repair, and transcriptional regulation [1.Szabo Q. et al.Principles of genome folding into topologically associating domains.Sci. Adv. 2019; 5eaaw1668Crossref PubMed Scopus (14) Google Scholar, 2.Yu M. Ren B. The three-dimensional organization of mammalian genomes.Annu. Rev. Cell Dev. Biol. 2017; 33: 265-289Crossref PubMed Scopus (65) Google Scholar, 3.Rowley M.J. Corces V.G. Organizational principles of 3D genome architecture.Nat. Rev. Genet. 2018; 19: 789-800Crossref PubMed Scopus (93) Google Scholar, 4.Spielmann M. et al.Structural variation in the 3D genome.Nat. Rev. Genet. 2018; 7: 85-97Google Scholar, 5.Spitz F. Gene regulation at a distance: from remote enhancers to 3D regulatory ensembles.Semin. Cell Dev. 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In place of pairwise or three-way interaction measurements, new highly multiplexed methods allow thousands of potential interactions to be probed simultaneously in each cell for unbiased image-based discovery. Facilitated by robotics and microfluidics, in conjunction with tricks to accelerate super-resolution imaging, thousands to hundreds of thousands of individual cells can be analyzed in an individual experiment, thereby providing robust statistical power to these methods. These advances have given us new insights into the relationships between chromatin structure and epigenetics [13.Fabre P.J. et al.Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states.Proc. Natl. Acad. Sci. U. S. 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In the process, we will also explain a labeling paradox – why a small-volume protocol outperforms a larger-volume alternative – and an imaging paradox – why some super-resolution techniques such as photoblinking fluors can worsen resolution in certain high-resolution chromatin imaging experiments. With this appreciation of recent technical developments, we will describe new insights into nuclear architecture revealed by these imaging techniques, highlighting some of the ways these newly revealed structures differ from the textbook picture and earlier reviews based on bulk approaches. Finally, we touch on exciting emerging frontiers and new challenges for the field. 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The advent of low-cost, high-complexity oligonucleotide synthesis, enabled by the proliferation of microarrays, has allowed synthetic, rationally and computationally designed oligonucleotides to circumvent many of the limitations of cloning-dependent approaches [20.Beliveau B.J. et al.Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 21301-21306Crossref PubMed Scopus (0) Google Scholar,32.Huber D. et al.Fluorescence in situ hybridization (FISH): history, limitations and what to expect from micro-scale FISH?.Nanoscale Microscale Thermophys. Eng. 2018; 1: 15-24Google Scholar,50.Müller P. et al.COMBO-FISH enables high precision localization microscopy as a prerequisite for nanostructure analysis of genome loci.Int. J. Mol. 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Computational tools identify the best way to tile the oligonucleotides across the target to maximize their number and uniformity of hybridization properties, while avoiding potential off-target binding. Thus, although genomic targets vary substantially in features which affect hybridization, the computationally designed probes reduce this variation, enabling increasingly high-throughput and multitarget applications. The use of array-based oligonucleotide probes was introduced by several groups [20.Beliveau B.J. et al.Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 21301-21306Crossref PubMed Scopus (0) Google Scholar,51.Boyle S. et al.Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis.Chromosom. 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This was achieved by relying on in vitro transcription (IVT) as a key amplification step, followed by reverse transcription (RT) to convert probes back to more stable DNA [24.Chen K.H. et al.Spatially resolved, highly multiplexed RNA profiling in single cells.Science. 2015; 348: 412Crossref Scopus (390) Google Scholar,55.Murgha Y.E. et al.Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.PLoS One. 2014; 9e94752Crossref PubMed Scopus (23) Google Scholar,56.Murgha Y. et al.Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.Biotechniques. 2015; 58: 301-307Crossref PubMed Scopus (6) Google Scholar]. How does a smaller volume reaction help to increase yield, and how can it be more cost-and time-efficient to convert from DNA to RNA and back, rather than cleaning up a labeled PCR product? The key insight is that IVT and RT reactions can be carried out at a very high concentration of substrate and product, unlike PCR. Amplification by IVT reduces the loss of diversity inherent in competitive exponential growth of PCR. RT also produces single-stranded DNA, removing the need for enzymatic or gel-based separation. Small-volume (high-concentration) protocols dramatically reduce enzyme and purification costs, and enable 96-well plate parallel processing. 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The use of adapter/bridge oligonucleotides to link the unique barcode to a common fluorescently-labeled oligonucleotide can further increase the flexibility and decrease the cost of labeling [17.Nir G. et al.Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling.PLoS Genet. 2018; 14e1007872Crossref PubMed Scopus (25) Google Scholar, 18.Bintu B. et al.Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells.Science. 2018; 362eaau1783Crossref PubMed Scopus (107) Google Scholar, 19.Mateo L.J. et al.Visualizing DNA folding and RNA in embryos at single-cell resolution.Nature. 2019; 568: 49-54Crossref PubMed Scopus (29) Google Scholar,54.Fields B.D. et al.A multiplexed DNA FISH strategy for assessing genome architecture in Caenorhabditis elegans.Elife. 2019; 8e42823Crossref PubMed Scopus (4) Google Scholar]. 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