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Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains - PubMed

Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains

A Marieke Oudelaar et al. Nat Genet. 2018 Dec.

Abstract

The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact in single cells is not understood. To address this we developed Tri-C, a new chromosome conformation capture (3C) approach, to characterize concurrent chromatin interactions at individual alleles. Analysis by Tri-C identifies heterogeneous patterns of single-allele interactions between CTCF boundary elements, indicating that the formation of chromatin domains likely results from a dynamic process. Within these domains, we observe specific higher-order structures that involve simultaneous interactions between multiple enhancers and promoters. Such regulatory hubs provide a structural basis for understanding how multiple cis-regulatory elements act together to establish robust regulation of gene expression.

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Conflict of interest statement

Competing Financial Interests Statement

The authors declare no competing financial interests.

Figures

Figure 1:
Figure 1:. Characterization of the interaction landscape of the regulatory elements of the α-globin locus.

High-resolution Capture-C interaction profiles of the α-globin locus from the viewpoints (indicated by blue arrows) of the α-globin promoters, the R2 enhancer, and CTCF-binding sites HS-39 and HS+48 in erythroid (red) and ES (grey) cells. Profiles represent the mean number of normalized unique interactions per restriction fragment in 3 replicates. Statistically significant differential interactions (derived using DESeq2) between erythroid and ES cells are highlighted in bold colors. Gene annotation (α-globin genes highlighted in red), erythroid DNaseI hypersensitivity (DHS) and CTCF occupancy are shown at the top, with arrows indicating the orientation of the CTCF-binding motifs. Coordinates (mm9): chr11:32,050,000–32,250,000.

Figure 2:
Figure 2:. Structural conformation of the active and inactive α-globin locus.

Contact matrices (4 kb resolution) of the α-globin locus derived from Capture-C experiments with viewpoints closely spaced across the domain in erythroid (top) and ES (bottom) cells. Matrices represent the mean number of normalized unique interactions in 3 replicates. Gene annotation (α-globin genes highlighted in red), erythroid DNaseI hypersensitivity (DHS) and CTCF occupancy are shown in the middle. Coordinates (mm9): chr11:32,000,000–32,300,000.

Figure 3:
Figure 3:. Overview of the experimental procedure and data output of Tri-C.

(a) Overview of Tri-C. 3C libraries are generated by fixation, digestion and proximity ligation of chromatin. De-crosslinked 3C concatemers are sonicated to ~450 bp fragments and ligated to sequencing adapters, after which fragments containing viewpoints of interest are enriched by oligonucleotide capture. Small viewpoint restriction fragments are targeted to allow for efficient detection of multiple ligation junctions within single sonicated 3C fragments using Illumina sequencing platforms. (b) Number of unique reads containing pair-wise and multi-way interactions generated by Tri-C for viewpoints in the α-globin locus.

Figure 4:
Figure 4:. Analysis of multi-way interactions between enhancers and promoters in the α-globin locus.

Tri-C matrices represent the mean number of normalized, unique interaction counts in 10 erythroid or 7 ES replicates, displayed at 500 bp resolution, with proximity contacts around the R2 viewpoint excluded (grey diagonal). Matrices are annotated with gene positions (α-globin genes highlighted in red), erythroid DNaseI hypersensitivity (DHS) and/or CTCF occupancy. Coordinates (mm9): chr11:32,120,000–32,240,000. (a) Contact matrix showing multi-way interactions with R2 in erythroid cells. (b) Contact matrix showing multi-way interactions with R2 in ES cells. (c) Contact matrix showing differential multi-way interactions with R2 that are enriched in erythroid (red) or ES (blue) cells. Statistically significant enriched interactions in erythroid cells (derived using DESeq2) are highlighted. (d) Venn diagram illustrating the proportion of R2-R1-Hba triplets relative to all triplets containing pair-wise R2-R1 and R2-Hba interactions in erythroid and ES cells. Numbers represent normalized counts of unique reads containing the specified three-way or pair-wise interactions. (e) Comparison of the absolute numbers of R2-R1-Hba triplets (shown on the y-axis) and the proportion of these relative to all triplets containing pairwise R2-R1 and R2-Hba interactions (represented as percentages in the bar graphs) in erythroid and ES cells. Bar graphs represent average normalized unique read counts in 10 erythroid and 7 ES replicates, with individual data points overlaid as dot plots. Both absolute and relative R2-R1-Hba triplet counts are significantly enriched in erythroid cells (~4-fold, p = 0.0002 and ~3.5-fold, p = 0.0005, respectively; statistics derived using unpaired, two-tailed t-tests). (f) Contact matrix (4 kb resolution) showing enrichment of multi-way interactions with R2 in erythroid cells over pair-wise contact frequencies derived from multiplexed Capture-C data in 3 replicates. Statistically significant enriched multi-way interactions (derived using unpaired, two-tailed t-tests, corrected for multiple comparisons) are highlighted. (g) Comparison of a double-anchored interaction profile containing fragments interacting with both the R2 and R1 enhancers (red) to the conventional R2 interaction profile (grey) in erythroid cells. Enrichment of multi-way R2-R1 interactions over pair-wise R2 interactions are shown in the differential profile at the bottom (black), with the R2-R1-Hba interactions highlighted (magenta). Profiles represent windowed, normalized, unique interaction counts in 10 replicates. Statistically significant enriched multi-way interactions with cis-regulatory elements of α-globin (derived using DESeq2) are indicated. Note that the double-anchored interaction profile has a relatively high signal-to-noise ratio and therefore contains some spurious peaks, including a strong, yet insignificant, enrichment of an unusually large (> 1.5 kb) NlaIII fragment containing many repeats in between the two α-globin promoters.

Figure 5:
Figure 5:. Analysis of multi-way interactions between CTCF-binding sites in the α-globin locus.

Tri-C matrices represent the mean number of normalized, unique interaction counts in 10 erythroid or 7 ES replicates, displayed at 500 bp resolution, with proximity contacts around the HS-39 viewpoint excluded (grey diagonal). Matrices are annotated with gene positions (α-globin genes highlighted in red), erythroid DNaseI hypersensitivity (DHS) and CTCF occupancy, with arrows indicating the orientation of CTCF-binding motifs. Coordinates (mm9): chr11:32,040,000–32,240,000. (a) Contact matrix showing multi-way interactions with HS-39 in erythroid cells. (b) Contact matrix showing multi-way interactions with HS-39 in ES cells. (c) Contact matrix showing differential multi-way interactions with HS-39 that are enriched in erythroid (red) or ES (blue) cells. Although the diffuse interactions with the region spanning the other side of the α-globin domain are enriched in erythroid cells (highlighted), there are no significant enrichments of specific interactions between multiple CTCF-binding sites (p > 0.5; statistics derived using DESeq2).

Figure 6:
Figure 6:. Graphical summary.

Our data are supportive of a loop extrusion mechanism contributing to the formation of compartmentalized chromatin domains. Complex higher-order structures, in which multiple enhancers and promoters interact, are formed within these domains by tissue-specific mechanisms, likely involving interactions between multi-protein complexes bound at these cis-regulatory elements.

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