Three-way contact analysis characterizes the higher order organization of the Tcra locus

Abstract The generation of highly diverse antigen receptors in T and B lymphocytes relies on V(D)J recombination. The enhancer Eα has been implicated in regulating the accessibility of Vα and Jα genes through long-range interactions during rearrangements of the T-cell antigen receptor gene Tcra. However, direct evidence for Eα physically mediating the interaction of Vα and Jα genes is still lacking. In this study, we utilized the 3C-HTGTS assay, a chromatin interaction technique based on 3C, to analyze the higher order chromatin structure of the Tcra locus. Our analysis revealed the presence of sufficient information in the 3C-HTGTS data to detect multiway contacts. Three-way contact analysis of the Tcra locus demonstrated the co-occurrence of the proximal Jα genes, Vα genes and Eα in CD4+CD8+ double-positive thymocytes. Notably, the INT2–TEAp loop emerged as a prominent structure likely to be responsible for bringing the proximal Jα genes and the Vα genes into proximity. Moreover, the enhancer Eα utilizes this loop to establish physical proximity with the proximal Vα gene region. This study provides insights into the higher order chromatin structure of the Tcra locus, shedding light on the spatial organization of chromatin and its impact on V(D)J recombination.


Figure S6 Related to Figure 4b
Bait-SOI plots displaying co-occurrence contacts of sequences in the locus with the combination of the TEAp bait and the SOIs of the V genes.The green line represents the observed co-occurrence frequency of sequences, and the gray line the expected (mean ± s.d.) co-occurrence frequency of sequences.The z-scores are shown in the bottom rectangles, with dark blue indicating significant enrichment and dark red indicating significant lack of a given site.

Figure S7 Related to Figure 4f
Bait-SOI plot displaying co-occurrence contacts of sequences in the locus with the combination of the Trav17 bait and the SOI sliding windows (4kb bin and 2kb step) from TEAp to Traj18.The green line represents the observed co-occurrence frequency of sequences, and the gray line represents the expected (mean ± s.d.) co-occurrence frequency of sequences.
Figure     Bait-SOI plots displaying co-occurrence contacts of sequences in the locus with the combination of the TEAp bait and the SOIs of the V genes.The green line represents the observed co-occurrence frequency of sequences, and the gray line the expected (mean ± s.d.) co-occurrence frequency of sequences.The z-scores are shown in the bottom rectangles, with dark blue indicating significant enrichment and dark red indicating significant lack of a given site.

Figure
Figure S1 Related to figure 1. a) The 3C-HTGTS (teal) and 4C (red) tracks display read density.Normalized signals represent pairwise interactions captured with the baits of Trav17, INT2, or TEAp in anti-CD3-induced DP thymocytes of Rag1 -/-or Rag2 -/-mice.The pink-filled rectangles highlight the bait position.Representative tracks are from two or three independent experiments.Normalized CTCF, Rad21 and Nipbl ChIP-seq profiles in DP cells are shown below the 4C tracks.b) Correlation of 3C-HTGTS and 4C experimental replicates.INT2 and TEAp are used as baits, respectively.c) Overview of the experimental procedure and acquisition of 3C-HTGTS libraries, including crosslinking, digestion, ligation, crosslink reversal, and sonication.DNA fragments containing the bait sequence is linearly amplified using biotin-labeled primers.Following capture with streptavidin beads, DNA fragments are ligated with adaptors for library amplification.Libraries are sequenced on an Illumina sequencing platform.d) Boxplot showing frequencies of cis-triplet fragments in 3C-HTGTS data.e) Heatmaps showing three-way contacts in the second and third replicates in the Eα bait.Annotation is shown below the heatmaps.Resolution: 5kb; Coordinates (mm10): chr14:53740000-54300000.

Figure
Figure S2 Related to figure 2 a) Bar graph showing frequencies of reads containing multi-way contacts in anti-CD3-induced DP thymocytes of Rag1 -/-(WT) and EACBE -/-Rag1 -/-(KO) mice.b) Heatmaps showing three-way contacts in the baits of Eα, TEAp, Trav17, and INT2 in liver cells of the Rag1 -/-mice.The red arrows highlight the bait positions.Points represent the mean of normalized unique interactions for each restriction fragment in two replicates.Annotation and coordinates are shown below the heatmaps.Resolution: 5kb; Coordinates (mm10): chr14:53740000-54300000. c) Subtraction heatmaps showing the difference in three-way contact between DP thymocytes and liver cells from the four baits.Points represent the mean of normalized unique interactions per restriction fragment in three (DP cells) or two (liver cells) experimental replicates.d) Representative stripes detected using Stripenn are delineated on three-way contact matrices of 3C-HTGTS in the four baits, respectively.Stripes are marked with teal-lined rectangles, and the p-value for each stripe is shown.

Figure S3
Figure S3 Related to Figure 3a and 3bBait-SOI plots illustrating the co-occurrence contacts of sequences in the locus with the combination of the Eα bait and the sliding windows (4kb bin and 2kb step) of SOIs from TEAp to Traj18.The green line represents the observed co-occurrence frequencies of sequences, while the gray line represents the expected co-occurrence frequencies (mean ± s.d.) across the locus.The z-scores indicating the significance of enrichment or lack of a given site are displayed in the bottom rectangles.Dark blue color indicates significant enrichment, while dark red color indicates a significant lack of a given site.

Figure S4 The
Figure S4 The Eα promotes the proximity of the proximal Jα genes to the Vα region in DP thymocytes.a) 3C-HTGTS signal counts (teal) of the 3' portion of the Tcra-Tcrd locus, from the Eα -containing fragment (bait2).The normalized CTCF ChIP-seq profile (blue) in DP cells is shown below the 3C-HTGTS track.b) Heatmap showing three-way contacts of the 3' portion of the Tcra-Tcrd locus.Resolution: 5kb; Coordinates (mm10): chr14:53740000-54300000 c) Bait-SOI plot displaying the co-occurrence contacts of sequences in the locus with the combination of the Eα bait and the SOI of the Traj61-to-Traj56 region.d) and e) Bar graphs displaying the co-occurrence unique read counts in the combination of the Eα bait2 and the SOI of (d) the sequence of the proximal Jα region or (e) the sequences containing Trav3-4, Trav12-4, Trav17, Trav19 , or Trdv2-2, respectively.The third points represent the five Vα genes or the proximal Jα region.The green bars represent the enrichment of the given third site in bait-SOI co-occurrence, and the gray bars represent the enrichment of the given third site in bait contacts without SOI.The data represent mean ±s.d.

Figure S5
Figure S5The proximal Jα region is disfavored in contacts with the combination of the Eα bait and SOIs of V genes.a) Bait-SOI plots displaying the co-occurrence contacts of sequences in the locus with the combination of the Eα bait and the SOIs of Vα genes.The green line represents the observed co-occurrence frequency of sequences, and the gray line represents the expected (mean ± s.d.) co-occurrence frequency of sequences across the locus.b) Heatmap showing the z-score landscape of sequences in the locus in co-occurrence contacts with the combination of the Eα bait and the SOIs of V genes.Rectangles in the heatmap represent the value of z-score.

Figure
Figure S1 Related to figure 1. a) The 3C-HTGTS (teal) and 4C (red) tracks display read density.Normalized signals represent pairwise interactions captured with the baits of Trav17, INT2, or TEAp in anti-CD3-induced DP thymocytes of Rag1 -/-or Rag2 -/-mice.The pink-filled rectangles highlight the bait position.Representative tracks are from two or three independent experiments.Normalized CTCF, Rad21 and Nipbl ChIP-seq profiles in DP cells are shown below the 4C tracks.b) Correlation of 3C-HTGTS and 4C experimental replicates.INT2 and TEAp are used as baits, respectively.c) Overview of the experimental procedure and acquisition of 3C-HTGTS libraries, including crosslinking, digestion, ligation, crosslink reversal, and sonication.DNA fragments containing the bait sequence is linearly amplified using biotin-labeled primers.Following capture with streptavidin beads, DNA fragments are ligated with adaptors for library amplification.Libraries are sequenced on an Illumina sequencing platform.d) Boxplot showing frequencies of cis-triplet fragments in 3C-HTGTS data.e) Heatmaps showing three-way contacts in the second and third replicates in the Eα bait.Annotation is shown below the heatmaps.Resolution: 5kb; Coordinates (mm10): chr14:53740000-54300000.
Figure S4 The Eα promotes the proximity of the proximal Jα genes to the Vα region in DP thymocytes.a) 3C-HTGTS signal counts (teal) of the 3' portion of the Tcra-Tcrd locus, from the Eα -containing fragment (bait2).The normalized CTCF ChIP-seq profile (blue) in DP cells is shown below the 3C-HTGTS track.b) Heatmap showing three-way contacts of the 3' portion of the Tcra-Tcrd locus.Resolution: 5kb; Coordinates (mm10): chr14:53740000-54300000 c) Bait-SOI plot displaying the co-occurrence contacts of sequences in the locus with the combination of the Eα bait and the SOI of the Traj61-to-Traj56 region.d) and e) Bar graphs displaying the co-occurrence unique read counts in the combination of the Eα bait2 and the SOI of (d) the sequence of the proximal Jα region or (e) the sequences containing Trav3-4, Trav12-4, Trav17, Trav19 , or Trdv2-2, respectively.The third points represent the five Vα genes or the proximal Jα region.The green bars represent the enrichment of the given third site in bait-SOI co-occurrence, and the gray bars represent the enrichment of the given third site in bait contacts without SOI.The data represent mean ±s.d.

Figure S5
Figure S5The proximal Jα region is disfavored in contacts with the combination of the Eα bait and SOIs of V genes.a) Bait-SOI plots displaying the co-occurrence contacts of sequences in the locus with the combination of the Eα bait and the SOIs of Vα genes.The green line represents the observed co-occurrence frequency of sequences, and the gray line represents the expected (mean ± s.d.) co-occurrence frequency of sequences across the locus.b) Heatmap showing the z-score landscape of sequences in the locus in co-occurrence contacts with the combination of the Eα bait and the SOIs of V genes.Rectangles in the heatmap represent the value of z-score.
Figure S6 Related to Figure 4bBait-SOI plots displaying co-occurrence contacts of sequences in the locus with the combination of the TEAp bait and the SOIs of the V genes.The green line represents the observed co-occurrence frequency of sequences, and the gray line the expected (mean ± s.d.) co-occurrence frequency of sequences.The z-scores are shown in the bottom rectangles, with dark blue indicating significant enrichment and dark red indicating significant lack of a given site.

to Figure 3a and 3b
Bait-SOI plots illustrating the co-occurrence contacts of sequences in the locus with the combination of the Eα bait and the sliding windows (4kb bin and 2kb step) of SOIs from TEAp to Traj18.The green line represents the observed co-occurrence frequencies of sequences, while the gray line represents the expected co-occurrence frequencies (mean ± s.d.) across the locus.The z-scores indicating the significance of enrichment or lack of a given site are displayed in the bottom rectangles.Dark blue color indicates significant enrichment, while dark red color indicates a significant lack of a given site.
p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p = 0.033 Bait-SOI plot displaying co-occurrence contacts of sequences in the locus with the combination of the Trav17 bait and the SOI sliding windows (4kb bin and 2kb step) from TEAp to Traj18.The green line represents the observed co-occurrence frequency of sequences, and the gray line represents the expected (mean ± s.d.) co-occurrence frequency of sequences.