Abstract
Determining how chromosomes are positioned and folded within the nucleus is critical to understanding the role of chromatin topology in gene regulation. Several methods are available for studying chromosome architecture, each with different strengths and limitations. Established imaging approaches and proximity ligation-based chromosome conformation capture (3C) techniques (such as DNA-FISH and Hi-C, respectively) have revealed the existence of chromosome territories, functional nuclear landmarks (such as splicing speckles and the nuclear lamina) and topologically associating domains. Improvements to these methods and the recent development of ligation-free approaches, including GAM, SPRITE and ChIA-Drop, are now helping to uncover new aspects of 3D genome topology that confirm the nucleus to be a complex, highly organized organelle.
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Acknowledgements
The authors thank the Helmholtz Association (Germany) for support, C. Thieme (our laboratory) for help plotting GAM and SPRITE contact maps in Figs 1 and 5a,b, and S. Quinodoz (M. Guttman laboratory) for sharing SPRITE contact cluster data (Figs 1 and 5b). A.P. acknowledges support from the National Institutes of Health Common Fund 4D Nucleome Program grant U54DK107977. The authors apologize to the many scientists whose studies were not discussed in our review due to length constraints.
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A.P. has filed a patent application on GAM: Pombo, A., Edwards, P. A. W., Nicodemi, M., Beagrie, R. A. & Scialdone, A. Patent application on ‘Genome Architecture Mapping’. Patent PCT/EP2015/079413 (2015).
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Glossary
- Chromosome territories
-
The nuclear volumes occupied by each specific chromosome. Chromosomes tend to interact predominantly within themselves and occupy distinct regions within the interphase nucleus.
- Chromosomal compartments
-
Chromosomes fold into distinct subcompartments, which correlate with transcriptional activity (A compartment) or repression (B compartment). The A and B compartments are defined by Hi-C contact frequencies.
- Topologically associating domains
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(TADs). Chromosomal regions that fold into self-associating domains, with high internal interaction frequencies, demarcated by a clear drop of local interactions with neighbouring regions at their boundaries.
- Chromatin loops
-
Local regions of high interaction frequency between two genomic loci indicate that these regions form the basis of a DNA loop. Loops often form between regions with divergent CCCTC-binding factor (CTCF) sites, or between enhancers and their target promoters.
- CCCTC-binding factor
-
(CTCF). A transcription factor with 11 conserved zinc-finger (ZF) domains. This nuclear protein is able to use different combinations of the ZF domains to bind different DNA target sequences and proteins. CTCF is enriched at topologically associating domain (TAD) borders, where its binding can be important to specify TAD border definition.
- Chromatin
-
The combination of DNA, RNA and protein that constitutes the chromosomes in eukaryotic cells. Broadly, heterochromatin is associated with transcriptional repression and euchromatin is associated with transcriptional activity.
- Nuclear lamina
-
A protein mesh, consisting of lamins and other membrane-associated proteins, at the inner nuclear membrane that contributes to nuclear structure and function. Chromatin in the proximity of the lamina tends to be heterochromatic and transcriptionally repressed.
- Fluorescence in situ hybridization
-
A technique that can be used to visualize the location of nucleic acid sequences within the nucleus using sequence-specific fluorescent probes that hybridize to the regions of interest, combined with microscopy.
- Chromosome conformation capture
-
(3C). A technique used to detect the frequency of interactions between any specified two loci in the genome. Interactions between loci are captured by formaldehyde fixation, followed by restriction enzyme digestion and ligation. The frequencies of interactions between loci are determined by quantitative real-time PCR.
- Hi-C
-
(High-throughput chromosome conformation capture). A genome-wide version of chromosome conformation capture that allows all chromatin interactions in the genome to be mapped simultaneously. The frequencies of interactions between loci are determined by paired end sequencing.
- Proximity ligation
-
Fixation of cells, followed by fragmentation of chromatin and ligation of nearby, crosslinked DNA fragments.
- Genome architecture mapping
-
(GAM). A genome-wide approach to detect chromatin contacts based on their physical distances within the nucleus. DNA loci are detected in thin nuclear slices by DNA extraction and sequencing. Chromatin contacts are inferred from co-segregation frequencies of pairs of DNA loci across a large (400–1,000) collection of nuclear slices.
- Split-pool recognition of interactions by tag extension
-
(SPRITE). A ligation-free approach to detect chromatin interactions by tagging crosslinked chromatin complexes. The DNA (and RNA) molecules within an individual chromatin complex are identified after sequencing by their unique combination of barcodes that have been sequentially added using a split-pool strategy.
- Chromatin immunoprecipitation
-
(ChIP). A method used to determine whether a given protein binds to, or is localized to, specific chromatin loci in vivo, detected after (native or crosslinked) chromatin purification and immunoprecipitation, followed by DNA detection by PCR, microarray hybridization or sequencing.
- Genomic resolution
-
The size of the window (often in the range of kilobases) when, for most assays, reads after sequencing are mapped to the genome and then binned into equally sized genomic windows (bins).
- Sequencing depth
-
The average number of reads representing a given nucleotide in the reconstructed sequence. A 10× sequence depth means that each nucleotide of the transcript was sequenced, on average, 10 times.
- Nuclear bodies
-
Membrane-less compartments in the nucleus with high concentrations of DNA binding proteins, chromatin modifiers or RNAs that can be involved in shaping chromatin structure and modulating gene regulation. Nuclear bodies include the nucleolus, splicing speckles and Polycomb bodies.
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Kempfer, R., Pombo, A. Methods for mapping 3D chromosome architecture. Nat Rev Genet 21, 207–226 (2020). https://doi.org/10.1038/s41576-019-0195-2
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DOI: https://doi.org/10.1038/s41576-019-0195-2
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