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Understanding 3D genome organization by multidisciplinary methods

Abstract

Understanding how chromatin is folded in the nucleus is fundamental to understanding its function. Although 3D genome organization has been historically difficult to study owing to a lack of relevant methodologies, major technological breakthroughs in genome-wide mapping of chromatin contacts and advances in imaging technologies in the twenty-first century considerably improved our understanding of chromosome conformation and nuclear architecture. In this Review, we discuss methods of 3D genome organization analysis, including sequencing-based techniques, such as Hi-C and its derivatives, Micro-C, DamID and others; microscopy-based techniques, such as super-resolution imaging coupled with fluorescence in situ hybridization (FISH), multiplex FISH, in situ genome sequencing and live microscopy methods; and computational and modelling approaches. We describe the most commonly used techniques and their contribution to our current knowledge of nuclear architecture and, finally, we provide a perspective on up-and-coming methods that open possibilities for future major discoveries.

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Fig. 1: Multiple levels of genome organization and the methods to study them.
Fig. 2: Main C-based methods for interrogation of 3D genome organization.
Fig. 3: Main ligation-independent methods for interrogation of 3D genome organization.
Fig. 4: Microscopy and FISH-based methods for 3D genome investigation.
Fig. 5: Computational modelling of the 3D genome.
Fig. 6: Live microscopy using CRISPR–dCas9 to study the 4D genome.

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Acknowledgements

The authors thank M. Di Stefano for critical reading of the Computational analysis and modelling section, B. Schuttengrueber and F. Bantignies for input on figures, and Q. Szabo for help with and input on Fig. 4. I.J. was supported by an European Molecular Biology Organization (EMBO) Long-Term Fellowship (ALTF 559-2018) and the Laboratory of Excellence EpiGenMed. Research in the G.C. laboratory is supported by grants from the European Research Council (Advanced Grant 3DEpi, under grant agreement No 788972), the European Union’s Horizon 2020 research and innovation programme (MuG, under grant agreement No 676556 and ChromDesign, under the Martie Sklodowska-Curie grant agreement No 813327), the Agence Nationale de la Recherche (ANR-15-CE12-0006 EpiDevoMath), the Fondation pour la Recherche Médicale (DEI20151234396), the MSDAVENIR foundation (Project GENE-IGH), the INSERM and the French National Cancer Institute (INCa).

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Correspondence to Giacomo Cavalli.

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Nature Reviews Molecular Cell Biology thanks Zhijun Duan, Bing Ren, Juan Vaquerizas, who co-reviewed with Elizabeth Ing-Simmons, and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Glossary

First principles

Basic building blocks of knowledge that cannot be deduced from any other preposition used for mathematical modelling of polymer behaviours.

Loop-extrusion model

A model suggesting that motor protein complexes such as cohesin or condensin form around chromatin and use the energy of ATP to slide through it while extruding the intervening region.

Tyramide signal amplification

(TSA). A method enabling sensitive detection of low-abundance molecules in fluorescent immunocytochemistry applications.

Multiway contacts

Chromatin contacts involving more than two chromatin fragments.

Nuclear speckles

Nuclear foci enriched in pre-mRNA splicing factors.

Polycomb

An evolutionarily conserved group of proteins involved in the regulation of a large group of developmental (and other) genes.

Cajal bodies

Nuclear bodies 0.3–1 μm in size that contain RNAs and proteins and are involved in RNA metabolism-related processes.

PML bodies

Nuclear bodies 0.1–1 μm in size that contain many components, including the promyelocytic leukaemia protein (PML), and are frequently localized near Cajal bodies.

Lamina-associated domains

Chromosome domains associated with the nuclear lamina in the 3D nuclear space.

Airy diffraction pattern

A diffused circle surrounded by rings of decreasing intensity generated when a laser passes through a circular opening.

Point spread function

The response of an imaging system to a point object. If the object is below the microscope resolution, it will appear larger than it really is.

Sub-diffractive point spread function

A point spread function of smaller size than that generated by diffraction-limited systems.

Dendrimer crosslinking

A procedure in which formaldehyde crosslinking can be followed or replaced by crosslinking with dendrimers, which are highly ordered, branched polymeric molecules of different sizes.

Diffraction limit

The points where two airy patterns are too close to be distinguishable.

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Jerkovic´, I., Cavalli, G. Understanding 3D genome organization by multidisciplinary methods. Nat Rev Mol Cell Biol (2021). https://doi.org/10.1038/s41580-021-00362-w

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