Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project

Abstract

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

Keywords: 4D nucleome; cell cycle; chromosome folding; development; disease model; genomics technologies; imaging technologies; modeling; nuclear organization.

Figure 1.

Chromatin dynamics at different timescales. (A) Across differentiation, on a time scale involving several cell cycle stages and many days with iPS cells differentiating towards neurons, cortical organoids, cardiomyocytes, and hepatocytes. (B) Across the cell cycle on a scale of hours (~24 hours as a typical cell cycle). (C) Within the G1 phase (tens of minutes time scale), where an individual locus is traced in space over time to observe loop extrusion for example.

Figure 2.. FISH Omics methods can be utilized to map chromatin structures across multiple genomic scales.

FISH Omics methods (also known as Multiplexed FISH) can be subdivided into two general categories, Ball-and-Stick (A and C) and Volumetric (B) Chromatin Tracing. In the former, a targeted genomic segment is represented by the centroid of an imaged spot and reflects the totality of fluorescence from multiple fluorophores. Instead, in Volumetric Chromatin Tracing a targeted genomic segment is visualized as a cloud of localizations each representing one emission event of a single-molecule fluorophore and outlining the volume that is occupied by the entire chromatin trace. As a whole, the two approaches can be used to dissect genomic chromatin structure across multiple genomic scales that range from kilobases to genome-wide (top row). (A) When Ball-and-Stick Chromatin Tracing is performed to target genomic segments that are contiguous to one another along the chromosome (i.e., contiguous coverage), it is typically used to map structural features that unfold at the kilobase scale (e.g., Promoter-Enhancer interactions). (B) When Volumetric Chromatin Tracing is employed to map chromatin structures in the megabase range in either a contiguous or discontinuous manner it is typically used to understand chromatin compaction or intermingling of neighboring gene domains. Finally, (C) when Ball-and-Stick Chromatin Tracing is used with discontiguous coverage it can be employed to map the structure of full chromosomes as well as the organization of chromosome compartments across the whole genome. Presented here from top to bottom are typical mapping coverage (top row), schematic representations of the strategy of probe introduction (second row from the top), and the way in which data is typically displayed (second row from bottom). The bottom row displays example applications for each of the three representative cases as follows (left to right): contiguous coverage with 3 Kb target segments, 52 rounds of hybridization, 100 nm scale bar, adapted from; contiguous coverage with 0.36–1.8 Mb target segments, 9 rounds of hybridization, 500 nm scale bar, adapted from; discontiguous coverage with 100 Kb target segments (50 TADs), 40 rounds of hybridization, adapted from; discontiguous coverage with 25 Kb target segments, 80 rounds of hybridization (each targeting 2460 segments), 5 μm scale bar, data from Cell 102, adapted from.

Figure 3.. Tracking chromatin looping, enhancer-promoter interactions, and nascent transcription in real-time using live-cell imaging.

(A) Tracking and quantifying the dynamics of CTCF/cohesin-mediated loops with live-cell imaging. By fluorescently labeling the two CTCF sites that hold together a TAD or CTCF/cohesin loop, it is possible to estimate the duration and looped fraction from live-cell imaging using 3D distance as read-out as recently demonstrated in^,. (B) Tracking Enhancer-Promoter interactions and nascent transcription with live-cell imaging. Similarly, by fluorescently labeling an enhancer and a promoter and by using the MS2/PP7 systems to visualize nascent transcription, it is also possible to track enhancer-promoter interactions with live-cell imaging to understand their relationship with transcription as illustrated in 132,133.