Transcriptional control in large genomes often requires looping interactions between distal DNA elements, such as enhancers and target promoters. Current chromosome conformation capture techniques do not offer sufficiently high resolution to interrogate these regulatory interactions on a genomic scale. Here we use Capture Hi-C (CHi-C), an adapted genome conformation assay, to examine the long-range interactions of almost 22,000 promoters in 2 human blood cell types. We identify over 1.6 million shared and cell type–restricted interactions spanning hundreds of kilobases between promoters and distal loci. Transcriptionally active genes contact enhancer-like elements, whereas transcriptionally inactive genes interact with previously uncharacterized elements marked by repressive features that may act as long-range silencers. Finally, we show that interacting loci are enriched for disease-associated SNPs, suggesting how distal mutations may disrupt the regulation of relevant genes. This study provides new insights and accessible tools to dissect the regulatory interactions that underlie normal and aberrant gene regulation.
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We thank E. Darbo for assistance in data analysis and K. Tabbada for sequencing. This work was supported by a Leukaemia and Lymphoma Research Fellowship (C.S.O.), the Framework Programme 7 Epigenesys Network of Excellence (B.M., N.M.L.), Cancer Research UK (B.M., F.T.-C., N.M.L.), UCL (B.M., N.M.L.), OIST (F.T.-C., N.M.L.) and the Wellcome Trust (N.M.L.).
The Babraham Institute has filed a patent application relating to the content of this manuscript (PCT/GB2014/052664).
Integrated supplementary information
(a) Symmetric genome-wide contact matrix (log2 read count) from 45 million reads (chr. 1–chr. Y). Reads were grouped into 1-Mb bins. Chr. 1 is shown as a chromosome-wide example. (b) Distribution of di-tag read separation distances in cis, in Hi-C and CHi-C libraries from GM12878 and CD34+ cells. (c) Heat map of di-tag reads exhibiting conserved topological domain structure in Hi-C and CHi-C libraries. Reads were grouped into 40-kb bins. (d) Number of unique reads in Hi-C and CHi-C samples mapping to bait and non-bait fragments. The red dot denotes the mean.
(a–c) The sequencing reads pooled from three GM12878 CHi-C replicates were preprocessed by HiCUP and randomly downsampled to 10–90% of the total read counts. (a) The percentage of interactions identified using all reads also found in subsets of different sizes. (b) Box plots displaying the separation distances of the promoter interactions called in each subset of reads. (c) The percentage of interactions that are not present in the interaction list from all reads in each subset. (d) Overlap of the promoter interactions detected within three independently prepared GM12878 CHi-C libraries. (e) Overlap of the promoter interactions detected within two independently prepared CD34+ CHi-C libraries. (f) Overlap of the interaction lists using more stringent FDR thresholds.
(a) Overlap of the interactions to promoters detected by both CHi-C (this study) and 5C (Sanyal et al., 2012) in GM12878 cells. (b) Interaction profile between baited promoters and fragments containing a CTCF site (blue), enhancer (yellow) or non-baited promoter (red).
(a) Notch plot displaying the separation distances between active and inactive promoters in CD34+ cells and their interacting fragments. P values were derived by pairwise t test (Holm adjusted). (b) The separation distances of the interactions between two baited promoter fragments. (c) The interaction profiles up- and downstream of active and inactive promoters in CD34+ cells. The insets display a zoomed-in view of the interactions that are within 40 kb of the promoter, with light shading representing the 95% confidence interval. (d) The propensity of promoter interactions for the gene body relative to equally sized upstream sequences for active (red) and inactive (blue) genes. (e) Notch plot of the number of interactions of baited promoters in CD34+ cells with non-baited interacting fragments. P values were derived by pairwise t test (Holm adjusted). (f) Number of interactions of non-baited interacting fragments in CD34+ cells to baited promoters.
(a) Proportions of CD34+ interactions with an adjacent promoter and of those intervened by other promoters. (b) Examples of distal fragments (black) that interact with an active (green) or inactive (red) promoter in CD34+ cells, bridging across other genes. RNA-seq track indicates the gene activity. (c) Examples of promoter-other interactions near CTCF sites in GM12878 cells. Interactions originating data from an inactive KCNJ3 and active DOCK10 promoter bridge over CTCF sites (in red). (d) The proportion of CD34+ interactions between a baited fragment and a non-baited fragment that bridge CTCF sites. (e) The directionality of CD34+ interactions with respect to TAD boundary positions between baited promoter and non-baited fragments (brown) and between two baited promoters (green). (f) The interactions in GM12878 cells originating from an inactive ACB5 or active ZNF292 promoter are mainly directed away from the nearest TAD boundary. Green bars represent TADs, and vertical arrows highlight the boundaries.
(a) Percentage of fragments across the genome (black) and in promoter interacting fragments (yellow) that encompass a ChromHMM-annotated enhancer. (b) Relative enrichments of interacting fragments for histone modifications in CD34+ cells. Top, relative enrichments of interacting and non-interacting fragments. Below, segregation of interacting fragments into expression quartiles based upon the activity of the associated promoter. (c) Relative enrichments of interacting fragments for chromatin segments in CD34+ cells (left) and the emission parameters defining each state (right). (d) Relative enrichments of non-baited interacting fragments that do not contain canonical enhancers, in GM12878 (left) and CD34+ (right) samples. The enrichment of transcription-associated histone marks in fragments interacting with highly expressed promoters seen for all fragments is also observed in this subset. (e) Relative enrichments for histone modifications for CD34+ interactions between two baited promoters. (f) Relative enrichment of histone modifications for fragments that interact with H3K9me3- or H3K27me3-bound promoters.
Conserved interactions spanning less than 100 kb (top), 100–500 kb (middle) and more than 500 kb (bottom) for promoters that are active in both cell types (red), inactive in both cell types (blue) or have discordant expression in these cells (gray).
The promoter interaction map shows the promoter interaction profiles of (a) BCL6, (b) HOTTIP, (c) TMEM159, (d) SOX4 and (e) RFC3. The size and color of each interaction peak reflect the relative number of reads per fragment. ChromHMM functional annotation tracks are displayed below the interaction profiles. Graphs show the gel quantitation of interactions by 3C between the promoter fragment and non-interacting fragment (prom-neg; black columns) and between the promoter fragment and interacting fragment (prom-elem; gray columns). L, DNA ladder; Co, PCR control. Asterisks denote PCR primer positions. For BCL6, interactions were also assessed by three-dimensional DNA FISH, using BAC probes encompassing the BCL6 promoter (green, G) and non-interacting (purple, P) and +1.2-Mb interacting (red, R) regions, shown as bars below the map. Representative examples of signals in GM12878 and CD34+ cells are shown. The outline of DAPI counterstaining is shown as a dotted white line. Graphs show measurement of the distance between signals (green-red, GR; green-purple, GP; purple-red, PR); the number of loci analyzed is shown (n).
(a) The enhancer activity of interacting fragments that interact with active genes, relative to a minimal promoter plasmid. (b) The silencing activity of a fragment that interacts with the inactive BCL6 promoter, relative to a constitutively active promoter plasmid. Relative luciferase activity is displayed as the mean, with standard error of three independent experiments, each carried out with technical triplicates. Statistical significance was determined by t test (one-tailed).
Centered on the not-baited fragments that contain a GWAS SNP from the 8,808 SNPs analyzed. The graph shows the proportion of SNP-containing fragments and the 50 upstream and downstream fragments that interact with a baited promoter in GM12878 (left) and CD34+ (right) cells.
Supplementary Figure 11 Statistical significance of interacting and non-interacting GWAS SNP fragments.
The histogram shows the median P value of trait association for non-interacting GWAS SNPs divided by the median P value of trait association for interacting GWAS SNPs. A positive value indicates that interacting GWAS SNPs have a lower median P value than those that do not interact. A log10 scale is shown. Asterisks denote the statistical significance of the trend within each category (Mann-Whitney).
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Mifsud, B., Tavares-Cadete, F., Young, A. et al. Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C. Nat Genet 47, 598–606 (2015). https://doi.org/10.1038/ng.3286
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