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. 2018 Oct 26;362(6413):eaau1783.
doi: 10.1126/science.aau1783.

Super-resolution Chromatin Tracing Reveals Domains and Cooperative Interactions in Single Cells

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Free PMC article

Super-resolution Chromatin Tracing Reveals Domains and Cooperative Interactions in Single Cells

Bogdan Bintu et al. Science. .
Free PMC article

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.

Conflict of interest statement

Competing interests: The authors have no competing interests.

Figures

Fig. 1.
Fig. 1.. Multiplex FISH imaging for high-resolution chromatin tracing allows de novo identification of TADs and sub-TADs.
(A) A scheme of the imaging approach. The genomic region of interest is partitioned into consecutive 30-kb segments and first hybridized with primary oligonucleotide probes that label all segments. These probes contained a readout sequence unique to each 30-kb segment. Each segment is labeled by ~300 probes but only one is shown. Readout probes complementary to the readout sequences are then added sequentially, allowing the imaging of individual 30-kb segments. (B) Composite 3D STORM images of 41 consecutive 30-kb chromatin segments in a 1.2-Mb region of Chromosome 21 (Chr21:28Mb-29.2Mb), in 41 pseudocolors, in one copy of Chr21 of an IMR90 cell. (C) 3D STORM images of two pairs of chromatin segments showing different degree of overlap, but similar distances between their center positions (marked by white dots). (D) Ensemble Hi-C contact frequency matrix for the 1.2-Mb genomic region binned at 30-kb resolution (data from (12)). (E, F) Mean spatial-overlap matrix (E) and median spatial-distance matrix (F) for the same region derived from multiplexed STORM imaging. Each element of the matrix corresponds to the mean value of the overlap fraction (E) and median value of the center-of-mass distance (F) between a pair of the chromatin segments across ~250 imaged chromosomes. (G) Correlation between the Hi-C contact frequency and the mean spatial overlap shown in (D) and (E), respectively. (H) Correlation between the Hi-C contact frequencies and median spatial distances shown in (D) and (F), respectively. (I) Median spatial-distance matrix for the same genomic region derived from multiplexed diffraction-limited imaging of ~1200 chromosomes. (J) Correlation between the Hi-C contact frequencies and median spatial distances shown in (D) and (I), respectively. The Pearson correlation coefficients (ρ) are 0.92, −0.92 and −0.96 in (G), (H) and (J) respectively. The red lines in (H) and (J) are power-law fits with scaling exponents (s) equal to −4.93 ± 0.07 and −4.99 ± 0.05 in (H) and (J), respectively.
Fig. 2.
Fig. 2.. Chromatin forms TAD-like domain structures with spatially segregated globular conformations in single cells.
(A) The spatial-overlap matrices of the 1.2-Mb genomic region (Chr21:28Mb-29.2Mb) imaged in one of the two copies of Chr21 from two individual IMR90 cells. The genomic regions marked in red, cyan, yellow, green, and purple correspond to the five sub-TADs observed at the population-average level. (B) Multiplexed 3D STORM images corresponding to the two chromosomes shown in (A). The chromatin segments comprising two pairs of ensemble sub-TADs marked as red and cyan or green and purple in (A) are pseudo-colored in the same color code. Only one pair of sub-TADs is highlighted in colors per image for the ease of visualization, and the other segments in the region of interest are displayed in gray. Each chromatin image is rotated independently to allow the best visualization of the color-highlighted chromatin regions. (C) Top: Ensemble Hi-C contact frequency map with sub-TAD boundaries indicated with black lines, shown together with the sites bound by CTCF (cyan squares) and cohesin (represented by RAD21, magenta circles), as determined by ChIP-seq in IMR90 cells (38). Middle: The probability (fraction of the ~250 imaged chromosomes) for each genomic location to appear as a single cell domain boundary. Bottom: The median separation score for each genomic location across the ~250 imaged chromosomes. Error bars indicate 95% confidence intervals derived by resampling (n ~ 250 chromosomes). The separation score is determined as shown in fig. S3. (D) The occurrence probability of CTCF/cohesin sites as a function of genomic distance from single-cell domain boundaries. Individual single-cell domain boundaries were aligned and the relative positions of CTCF ChIP peaks (that colocalize with RAD21 peaks) up to 150 kb on either side of the domain boundaries were histogrammed at 30kb resolution. The histograms were normalized by dividing by the total number of boundaries.
Fig. 3.
Fig. 3.. Single-cell TAD-like structures are formed across cell types.
(A-C) Median spatial-distance matrices for the 2-Mb genomic region of interest (Chr21:28Mb-30Mb) in three cell types: IMR90 lung fibroblast (A), K562 erythroleukemia (B) and A549 carcinomic epithelial cells (C). The number of chromosomes imaged (~3,000–14,000) is indicated above each matrix. (D-F) Single-cell spatial-distance matrices of the imaged region (upper) and the corresponding pseudo-colored images showing 3D positions of the chromatin segments in each chromosome (lower). Two example cells (and one chromosome copy from each cell) are shown for each of the three cell types (D: IMR90; E: K562; F: A549). (G-I) Top: The probability for each genomic position to be a boundary of a single-cell domain for each of the three cell types (G: IMR90; H: K562; I: A549). Bottom: The mean separation score for each genomic coordinate for each cell type. Error bars indicate 95% confidence intervals (n ~3,200, 14,000, and 4,000 chromosomes for IMP90, K652 and A549 cells, respectively). The binding sites of CTCF and cohesin (marked by RAD21) determined by ChIP-seq for each cell type (38) are indicated with squares and circles respectively.
Fig. 4.
Fig. 4.. Single-cell TAD-like structures are present in cells lacking a functional cohesin complex.
(A) Median spatial-distance matrices for the 2.5-Mb genomic region of interest (Chr21:34.6Mb-37.1Mb) in the transgenic HCT116 cell line without (left) or with (right) auxin treatment to induce cohesin degradation. (B) Example single-cell spatial-distance matrices without (left) and with (right) auxin treatment. (C) The distribution of boundary strengths in the imaged region for cells without (left) and with (right) auxin treatment. For each identified domain boundary on a single-cell spatial-distance matrix, the boundary strength describes how steeply the spatial distance changed cross the boundary position. The medians of the two distributions with and without auxin treatment differed by less than 1%. (D) The probability for each genomic position to be a single-cell domain boundary in cells without (left) or with (right) auxin treatment.
Fig. 5.
Fig. 5.. Cooperative three-way interactions between chromatin segments.
(A) 3D STORM images of a 1.2-Mb region of interest (Chr21:28Mb-29.2Mb) in one of the two copies of Chr21 in two different IMR90 cells. The entire genomic region is represented in gray and three specific 30-kb segments harboring CTCF sites – segments 18 (A1), 27 (B1) and 32 (C1) are highlighted in red, cyan and yellow, respectively. (B) Cooperative interactions between a specific triplet of segments A1, B1 and C1. Left: The mean spatial-overlap matrix in the subpopulation of chromosomes where segments A1 and B1 overlap. Right: The mean spatial-overlap matrix in the other subpopulation of chromosomes where segments A1 and B1 do not overlap. Circles indicate the matrix elements corresponding to segment pairs A1–B1 (red-cyan), B1–C1 (cyan-yellow) and A1–C1 (red-yellow). (C) Cooperative interactions among all possible CTCF-site triplets in the 1.2-Mb imaged region. Shown in the plot are probabilities with which segments B and C contact in individual IMR90 cells under the condition that segments A and B contact (red) or do not contact (blue) for all ordered combinations of CTCF triplets (~500 total) in the imaged region. “Ordered” means B lies between A and C along the genomic coordinate. Also plotted is the unconditioned probability of B and C contacting regardless of whether A and B contact (black). The index of the triplets is sorted such that the unconditional probability is displayed in ascending order. (D) As in (C) but for all ordered triplets of chromatin segments in an extended 2-Mb region of interest (Chr21:28Mb-30Mb) regardless of whether the segment contains CTCF sites. There are ~90,000 such triplets in total, among which only ~2,000 are CTCF-site triplets (i.e. all three segments containing CTCF-binding sites). (E) As in (D) but for the HCT116 cells without (left) or with (right) auxin treatment. There are ~90,000 such triplets in total, among which only ~700 are CTCF-site triplets. (F) The fraction of triplets of segments that show cooperative interactions (i.e. the triplets for which the probability with which segments B and C contact in individual cells under the condition that segments A and B contact is higher than the unconditioned probability of B and C contacting regardless of whether A and B contact) for each imaged region in various cell types and cohesin depletion conditions.

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