Intra- and interchromosomal contact mapping reveals the Igh locus has extensive conformational heterogeneity and interacts with B-lineage genes

Abstract

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (V_H), diversity (D_H), and joining (J_H) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the V_H genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.

Keywords: 3D immunoglobulin structure; CP: Immunology; Capture Hi-C; genome organization; immunoglobin locus; interchromosomal; polymer modeling.

Graphical abstract

Figure 1

Capture Hi-C method (A) Capture Hi-C (CHi-C) workflow. Top left: cross-linked chromatin was used to generate Hi-C libraries. Top right: BAC DNA was digested, sonicated, and in vitro transcribed to yield a library of biotinylated RNA baits. The Hi-C and RNA bait libraries were hybridized together and baited fragments captured with streptavidin beads. Captured sequences were amplified and paired-end sequenced. The resulting CHi-C libraries yielded intralocus Igh reads (bottom left) and interchromosomal reads (bottom right). (B) Genome-wide view of read coverage in HiCUP-processed CHi-C Rag1^Mom−/− pro-B 1 dataset. Reads were quantified in 100-bp bins and normalized per million reads. Read coverage is enriched at baited regions (blue circles). (C) Read enrichment over the Igh locus in CHi-C datasets. Five million randomly sampled reads from HiCUP-processed Hi-C and CHi-C datasets were quantified in 200-kb bins and visualized in the WashU Epigenome Browser. Arcs show interactions. Arc color indicates the number of interactions. One representative replicate is shown for each dataset type. (D) Previously reported interactions between the histone 1 (Hist1) clusters on chromosome 13 were detected in all CHi-C samples. A virtual 4C from the Hist1 baited region was performed on one and both ends in Scribler-processed datasets. Hist1 genes are in blue. Vomeronasal (Vmrn) genes are in pink. (E) The Igh locus interacts more frequently with regions outside the locus in cis in thymus than in pro-B cells. A virtual 4C was conducted from the baited Igh region in Scribler-processed datasets. Biological replicates were averaged. See also Figure S1 and Table S1.

Figure 2

All-to-all interaction matrices of the Igh locus Interaction matrices at 20-kb resolution for the Igh baited region for (A) pro-B and (B) thymus datasets. Each matrix entry is a coverage normalized interaction frequency value for the pairwise interaction between two 20-kb bins. Matrices from two biological replicates have been averaged to produce matrices (A) and (B). White lines are bins with too-low read coverage excluded from analysis by hypergeometric optimization of motif enrichment (HOMER). Arrow 1, 3′ CBEs; arrow 2, Eμ; arrow 3, IGCR1; arrow 4, most proximal V_H genes; arrow 5, 5′ of 7183 V_H gene family; arrow 6, 5′ of S107 V_H gene family; arrow 7, 3′ J606 V_H genes; arrow 8, start of distal subdomain; arrow 9, end of distal subdomain. The positions of subdomains in pro-B cells determined using HiCseg are indicated by gray rectangles. See also Figures S2 and S3 and Table S2.

Figure 3

The individual spatial conformations of the Igh locus are a heterogeneous ensemble in pro-B cells (A) A random selection of nine out of 5,001 simulated conformations of the Igh locus in pro-B cells. (B and C) Experimental and simulated heatmaps are shown for (B) pro-B and (C) thymus. Single structures generated by the polymer model were clustered using the root-mean-square difference in bead-to-bead distances as a dissimilarity score and the heatmap corresponding to the largest cluster is shown (B) and (C). (D and E) The average number of interacting partners (bead-to-bead distance <1.5a) across all conformations was calculated for each bead in (D) pro-B cells and (E) thymus. CTCF-binding level is indicated by the color of the data point. Vertical dotted lines indicate the position of the 3′ CBEs (left, orange), Eμ (middle, yellow), and IGCR1 (right, orange). (F) For each conformation in pro-B cells, the fraction of V_H beads interacting with at least one D_H bead was determined. In most conformations, over 20% of V_H beads contact a D_H bead. (G and H) The number of V_H-D_H interactions across the 5,001 pro-B conformations per D_H bead (G) and V_H bead (H). (I) The fraction of V_H gene beads contacted by each bead in each conformation, averaged across all 5,001 conformations for pro-B (orange) and thymus (green). Vertical dotted lines as for (D) above. See also Figure S4.

Figure 4

Distance from the CM reveals Igh structure is focused on the V distal region (A–D) Average distance from CM (a) across all conformations was calculated for each bead in pro-B cells (A) and thymus (B). Similarly, average distance from CMv (a) across all conformations was calculated for each bead in pro-B cells (C) and thymus (D). The color of V_H bead data points indicates the average recombination score of V_H genes in the bead. The positions of the 3′ CBEs (left, orange), Eμ (yellow), and IGCR1 (right, orange) are indicated; other non-V_H beads are gray. (E and F) Average distance from CMv (a) for each bead in the subset of structures in which it is interacting with each of the beads named top right (color corresponds to the color of the points). Gray line shows the median distance from CMv across the subsets of structures in which a given bead is interacting with each of the other beads; gray ribbon indicates the range encompassing 75% of these subsets of structures. (G–I) Correlation between distance of V_H bead from CMv and recombination score^4,66). (G) For all V_H beads, the correlation between distance of V_H bead from CMv and the average recombination score of all V_H genes in that bead was determined. (H) For V_H beads containing a single active V_H gene, correlation between distance of V_H bead from CMv and the recombination score of the V_H gene. (I) As in (H), but instead of using all conformations, the average distance in a subset of conformations where the V_H is implicated in a V_H-D_H interaction was used. cor, Spearman’s correlation coefficient, >0.4 equivalent to moderately strong correlation; p value < 0.05 statistically significant.

Figure 5

The Ig loci participate in shared, developmental-stage-specific interchromosomal interactions (A) Statistically significant (Z score > 3.5) interchromosomal interactions are plotted for each Ig viewpoint (Igh, top; Igκ, middle; Igλ, bottom) in pro-B cells (left), pre-B cells (middle), and thymocytes (right). Gray rectangles denote chromosomes and arcs denote trans interactions, with highest Z scores in red. Full list of Z score values is in Table S3B. (B and C) Log2(average Z score +1) was calculated for each 0.5-Mb bin genome-wide for the Igh viewpoint and was compared with the corresponding values for the Igκ viewpoint in (B) pro-B and (C) pre-B datasets. cor, Spearman correlation coefficient. Dotted red lines indicate the threshold of a statistically significant Z score. (D) 0.5-Mb bins were selected for hierarchical clustering if they had a significant interaction with any of the three Ig loci at any developmental stage. Clustering, based on average Z score values, was performed using the pheatmap package in R. (E) The 0.5-Mb bins were selected for hierarchical clustering if they had a significant interaction with any of the Ig loci at a developmental stage permissive for recombination or recombined. Clustering performed as in (D). (F–H) For the Igh (F), Igκ (G), and Igλ (H) viewpoints, boxplots show the A or B compartment strength for 0.5-Mb bins that are significant interaction hits at each developmental stage. Values > 0 indicate A compartments and values < 0 indicate B compartments. In (F) and (H), statistically significant differences were determined using a Kruskal-Wallis test followed by post hoc testing for pairwise comparisons using the dunn.test function with Bonferroni correction. For (G) a Mann- Whitney U test was used. In pro-B cells (I), and pre-B cells (J) the amount of transcription, measured by log2 (average RNA-seq read count normalized per million; Table S4) was compared between bins participating and not participating in significant interaction with the Igh locus. See also Figures S5 and S6.

Figure 6

The Igh locus is in spatial proximity to key B lineage genes more frequently in B cells than in thymocytes (A–H) Two-color 3D FISH probing of the Igh locus and either (A) Pax5, (B) Foxo1, (C) Ebf1, (D) Runx1, (E) Bach2, (F) Igκ, (G) Myc, or (H) Rag1/2 loci was performed in pro-B cells, pre-B cells, and thymocytes. Line graphs show cumulative distributions of distances <1 μm between the Igh locus and genes of interest in three cell types. The closest signal pairs in each cell were used. n = number of nuclei analyzed. Bar graphs show distribution of distances grouped into four brackets (<0.5, 0.5–0.8, 0.8–1, and >1 μm). Dashed line denotes the probability of a random interaction at a distance of <1 μm of 6.9%. The p values were calculated using the Fisher’s exact test with Bonferroni correction. Representative images of single nuclei are shown. The nuclear area was stained with DAPI, the Igh probes were labeled with Alexa Fluor 488 (green), and probes for genes of interest were labeled with Alexa Fluor 555 (red). The Igh (DJ) probe (BAC RP23-109B20) was used for pro-B and thymus, and the Igh (V) probe (BAC RP23-70F21) for pre-B (compared in Figure S7; discussed in STAR Methods). (I) Three-color 3D FISH for the Igh, Ebf1, and Foxo1 loci was performed in pro-B cells, pre-B cells, and thymocytes. The closest signal pairs in each cell were filtered to retain only cells in which at least two of the pairwise distances connecting the closest three signals were within 1 μm. Bar graph shows the distribution of distances (<0.5, 0.5–0.8, 0.8–1, and >1 μm). Dashed line denotes the probability of a random tripartite interaction at a distance of <1 μm of 0.48%. (J) Three-color 3D FISH for the Ebf1, Foxo1, and Bach2 loci was performed in pro-B cells as for (I) above. Dashed lines denote the probability of a random bipartite (upper line) or tripartite (lower line) interaction as above. See also Figure S7 and Table S5.

Figure 7

Peripheral regions of the Igh locus interact in trans with other loci (A) To identify trans contacts of the Igh locus, read pairs with at least one end mapping to a baited region and the other end mapping to a different chromosome were extracted. Virtual 4C interaction profiles were generated from selected 0.5-Mb bins among the top hits of interchromosomal interactions with the Igh in pro-B cells. Other ends were quantified in 40-kb bins with a 10-kb step over the Igh locus. The positions of FISH probes used in Figure 6 and Igh gene segments are indicated at the top. Vertical dotted yellow line, Eμ. (B) Organization of the Igh locus into sub-TADS (pink and blue circles) while a dynamic continuum of interaction occurs between the 3′ sub-TAD and the V_H region enabling diverse V_H-to-D_H recombination events (red dotted arrows). The peripheral regions of the Igh locus participate in interchromosomal interactions, for example with Ebf1 and Foxo1.