Identification of multi-loci hubs from 4C-seq demonstrates the functional importance of simultaneous interactions

Abstract

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3'Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.

Figure 1.

Workflow for extracting multi-loci chromatin interactions using 4C-seq data. (A) Cross-linking step. (B) Primary restriction enzyme digestion. (C) First ligation step. (D) Removal of crosslinks. (E) Secondary restriction enzyme digestion. (F) Second ligation step. (G) PCR amplification of fragments (tri-loci fragments are shown in the dotted box). (H) High throughput sequencing (tri-loci reads are shown in the dotted box). (I) Alignment of tri-loci reads. Each read is split into three segments at primary restriction enzyme cut sites that include the bait segment. These segments are aligned independently to the genome. (J) Tri-loci interactions along the genome (bottom) and their representation in a heatmap (colored squares indicate simultaneous interaction of the blue and orange genomic locations with the bait). (K) Heatmaps were generated by binning the genomic region of interest in a resolution dictated by the genomic length of the loci involved in the study.

Figure 2.

Frequency and genomic distance associated with multi-loci interactions. (A) Scheme showing the location of the four 4C-seq baits on chromosome 6. (B) Frequencies of tri-loci reads in 4C-seq experiments using baits Eβ, MiEκ, 3′Eκ, and REIR. (C) Frequencies of cis tri-loci fragments. (D) Distribution of the frequencies of multi-loci fragments at different distances from the bait region. (E) Correlation of pair-wise interactions between replicates and non-replicates. (F) Correlation of interactions using active versus inactive baits.

Figure 3.

4C-seq multi-loci interactions reflect known lineage and stage specific changes in Tcrb locus conformation. (A) The first principal component of pair-wise interactions in DN, DP, pre-B and Immature B cells using Eβ as bait. (B–E) Heatmap for tri-loci interactions (window size of 50kb) involving the Eβ bait in DN, DP, pre-B and Immature B cells across the Tcrb locus.

Figure 4.

4C-seq multi-loci interactions identify changes in contacts across Tcrb that result from deletion of Eβ. (A and B) Heatmap for tri-loci interactions (window size of 50kb) involving the Eβ bait in DN WT versus Eβ^−/− cells across the whole Tcrb locus. The ATAC-seq tracks display read density. (C and D) Heatmap for tri-loci interactions (window size of 2kb) involving the Eβ bait in DN WT versus Eβ^−/− cells across DJC of the Tcrb locus. The ATAC-seq tracks display read density.

Figure 5.

4C-seq multi-loci interactions reflect known lineage specific changes in Igk locus conformation. (A) Scatterplot of the first and second principal components of pair-wise interactions of DN, DP, pre-B and Immature B cells using MiEκ and 3′Eκ as baits. (B–E) Heatmaps for tri-loci interactions (window size of 200kb) involving the MiEκ bait in pre-B, Immature B, DN and DP cells across the Igk locus.

Figure 6.

Enhancer hubs and their impact on super-enhancer activity. (A–C) Heatmap for tri-loci interactions (window size of 2.5 kb) involving the MiEκ bait in WT versus MiEκ^−/− and 3′Eκ^−/− B cells across the 3′ end of the Igk locus. The intensity of interactions between the three regions in the neighborhoods of the three enhancers (which is proportional to the intensity of the blue color) is marked by a red dot in each of the heatmaps. (D–F) Heatmap for tri-loci interactions involving the 3′Eκ bait in WT versus MiEκ^−/− and 3′Eκ^−/− B cells across the 3′ end of the Igk locus. (G) Model showing the interactions in of the three enhancers in WT, MiEκ^−/− and 3′Eκ^−/− pre-B cells. (H) RNA-seq data showing the transcriptional output across the 3′ end of the Igk locus in WT, MiEκ^−/− and 3′Eκ^−/− pre-B cells.