Research Article

Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells

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Science  26 Oct 2018:
Vol. 362, Issue 6413, eaau1783
DOI: 10.1126/science.aau1783

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Imaging chromatin spatial organization

The genome is organized within the nucleus as three-dimensional domains that modulate DNA-templated processes. Bintu et al. used high-throughput Oligopaint labeling and imaging to observe chromatin dynamics inside the nuclei of several different mammalian cell lines. After combining the datasets, single-cell matrices revealed chromatin arranged in topologically associating domains (TADs). Removing cohesin resulted in a loss of aggregate TADs among populations of cells, but specific TADs were still detected at the single-cell level. Furthermore, higher-order organization was detected, suggestive of cooperative interactions within the genome.

Science, this issue p. eaau1783

Structured Abstract

INTRODUCTION

Chromatin adopts an intricate three-dimensional (3D) organization in the nucleus that is critical for many genome processes, from gene regulation to genome replication. Our understanding of the chromatin organization remains relatively poor at the kilobase-to-megabase scale, which spans the sizes of individual genes and regulatory domains and is thus of critical importance to genome regulation. Recent Hi-C experiments revealed topologically associating domains (TADs) as a ubiquitous chromatin organization feature in many organisms. However, the basic properties of TADs, including whether TADs represent a fundamental unit of genome organization in individual cells or an emergent property from cell population averaging, and the formation mechanism of TADs, remain unclear. In addition, our understanding of genome organization is largely built on pairwise interactions, whereas relatively little is known about higher-order chromatin interactions. Methods that provide a high-resolution visualization of chromatin structure in individual cells will elucidate these and many other questions related to genome organization.

RATIONALE

We report a super-resolution chromatin tracing method that allows determination of both the structural features and their genomic coordinates with high resolution in single cells. We reason that if numerous chromatin loci could be identified and precisely localized in individual cells, connecting their positions would allow us to trace the chromatin conformation. However, typical multicolor imaging allows only a few loci to be simultaneously imaged. To overcome this challenge, we partitioned the targeted genomic region into numerous segments, each 30 kb in length, and imaged individual segments using sequential rounds of fluorescence in situ hybridization. This allowed us to generate a 3D super-resolution image of the chromatin in numerous pseudocolors, each reporting the position and structure of a 30-kb segment with nanometer-scale precision.

RESULTS

Our imaging data revealed an abundance of TAD-like domain structures with spatially segregated globular conformations in single cells. The domain boundaries varied from cell to cell, exhibiting nonzero probability of residing at any genome positions, but with a preference at CCCTC-binding factor (CTCF)- and cohesin-binding sites. Notably, cohesin depletion, which abolished TADs at the population-average level, did not alter the prevalence of TAD-like structures in single cells; only the preferential positioning of domain boundaries was lost, explaining the loss of population-level TADs. Our results suggest that cohesin is not required for the formation or maintenance of single-cell domain structures, but that their preferential boundary positions are influenced by cohesin-CTCF interaction.

In addition, we observed prevalent multiway interactions among triplets of chromatin loci. These higher-order interactions were cooperative, i.e., most three-way contacts were observed at higher frequencies than would be expected from the frequency of pairwise interactions. Notably, these multiway interactions were also retained after cohesin depletion.

CONCLUSION

Our imaging method offers a high-resolution physical view of chromatin conformation of targeted genomic regions in single cells, providing a powerful and complementary approach to sequencing-based genome-wide methods for interrogating genome organization. The TAD-like structures and multiway chromatin interactions observed in single cells add important constraints on genome folding and have implications for understanding the role of genome structure in diverse biological processes from enhancer-promoter communication to genome compartmentalization. We envision that future work will further improve the resolution and genomic coverage of this approach and will combine the imaging of chromatin with regulatory factors and/or expressed RNAs to reveal the underlying mechanism and functional implication of chromosome organization.

Super-resolution chromatin tracing reveals TAD-like domain structures in single cells.

Consecutive 30-kb segments of a chromatin region of interest were sequentially imaged with diffraction-limited or super-resolution fluorescence microscopy. The pseudocolored images of the positions of individual chromatin segments in single cells and the corresponding matrices of intersegment distances reveal TAD-like structures with a globular conformation in single cells. The population-average matrix reveals TADs at the ensemble level.

Abstract

The spatial organization of chromatin is pivotal for regulating genome functions. We report an imaging method for tracing chromatin organization with kilobase- and nanometer-scale resolution, unveiling chromatin conformation across topologically associating domains (TADs) in thousands of individual cells. Our imaging data revealed TAD-like structures with globular conformation and sharp domain boundaries in single cells. The boundaries varied from cell to cell, occurring with nonzero probabilities at all genomic positions but preferentially at CCCTC-binding factor (CTCF)- and cohesin-binding sites. Notably, cohesin depletion, which abolished TADs at the population-average level, did not diminish TAD-like structures in single cells but eliminated preferential domain boundary positions. Moreover, we observed widespread, cooperative, multiway chromatin interactions, which remained after cohesin depletion. These results provide critical insight into the mechanisms underlying chromatin domain and hub formation.

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