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 lack of relevant methodologies, major technological breakthroughs in genome-wide mapping of chromatin contacts and advances in imaging technologies in the 21 st 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 fluorescent in situ hybridization (FISH), multiplex FISH, in situ genome sequencing and live microscopy methods; and computational and modeling 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.Simultaneously with the development of the C-based techniques, ligation-independent techniques were invented to assay not only chromosome conformation in general, but also the nuclear position of chromatin contacts (tyramide signal amplification (TSA), DNA adenine methyltransferase identification (DamID), split-pool recognition of interactions by tag extension (SPRITE)) and multi-way contacts (SPRITE and genome architecture mapping (GAM)), which are not assayed effectively using ligation-based techniques [34][35][36][37][38][39][40][41][42] . Finally, the recent advancement of superresolution microscopy and imaging techniques allowed us to investigate chromatin conformation of single cells at extremely high resolution and at a higher throughput than ever before 12,14,15,[43][44][45][46][47][48] . In addition to improvements in spatial resolution, live-imaging in combination with genome-engineering using CRISPR-Cas9 systems facilitated and improved the study of chromatin-contact dynamics [49][50][51] .Owing to these methodological and technological advancements, it is not surprising that the past decade has provided major revelations in 3D genome organization and function. Most notably is the finding that chromosomes in interphase predominantly fold into two compartments, A and B, which respectively consist of predominantly gene-active and gene-inactive regions 27 (Figure 1). Furthermore, parts of compartments, from the same or different chromosomes, can come together and create hubs, which are connected by multiple chromatin interactions, thereby sharing a common function (for example, gene repression) and coalescing around different nuclear bodies such as nuclear speckles 35,37,52 . On a scale below the compartments, chromatin interactions were found to be enriched within domains of 100 kb to 1 Mb in length termed topologically associating domains (TADs); these partially insulated domains are subdivided into smaller chromatin nanodomains (CNDs) 43,53-57 ...

Chromatin Organization and Function in Drosophila

“…In Drosophila, CRISPR/Cas9 screens have recently been used to identify factors involved in genome architecture, including chromatin-binding proteins and functional cis-regulatory elements, such as enhancers and boundary elements or insulators [198]. Furthermore, the development of new methods based on a nuclease-dead Cas9 (dCas9) coupled with live imaging is allowing researchers to study the 4D genome, changes in 3D chromatin structure over time [199]. In the CRISPR/dCas9 system, there is specific recruitment of a fluorescently labelled dCas9 to the genomic region of interest, which allows the tracking of the contact and folding dynamics of this region by live microscopy [199].…”

“…Furthermore, the development of new methods based on a nuclease-dead Cas9 (dCas9) coupled with live imaging is allowing researchers to study the 4D genome, changes in 3D chromatin structure over time [199]. In the CRISPR/dCas9 system, there is specific recruitment of a fluorescently labelled dCas9 to the genomic region of interest, which allows the tracking of the contact and folding dynamics of this region by live microscopy [199].…”

Chromatin lncRNA Platr10 controls stem cell pluripotency by coordinating an intrachromosomal regulatory network

“…However, the resolution of Hi-C is too low to delineate the promoter-enhancer intrachromosomal loop at the Oct4 locus. Thus, it will be important to delineate this lncRNA-chromatin interaction in greater detail using a high-resolution approach, like Micro-C, enhanced Hi-C, and Capture-C [ 32 ].…”

Current Understanding of Molecular Phase Separation in Chromosomes

“…Live-cell imaging is used widely to monitor condensates and study the characteristics of condensates inside a cell [19,105]. Conversely, genomic analyses, such as sequencing techniques and Hi-C, have been used to study chromosome organization, where phase separation can play a role, as discussed earlier [106]. In addition, computer simulations can provide another perspective on the principles of phase separation in model systems [107][108][109][110].…”