Flexible long-range loops in the VH gene region of the Igh locus facilitate the generation of a diverse antibody repertoire

Abstract

The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH-rearranged gene segment in the 3' proximal Igh domain. By using high-resolution mapping of long-range interactions, we demonstrate that local interaction domains established the three-dimensional structure of the extended Igh locus in lymphoid progenitors. In pro-B cells, these local domains engaged in long-range interactions across the Igh locus, which depend on the regulators Pax5, YY1, and CTCF. The large VH gene cluster underwent flexible long-range interactions with the more rigidly structured proximal domain, which probably ensures similar participation of all VH genes in VH-DJH recombination to generate a diverse antibody repertoire. These long-range interactions appear to be an intrinsic feature of the VH gene cluster, because they are still generated upon mutation of the Eμ enhancer, IGCR1 insulator, or 3' regulatory region in the proximal Igh domain.

Figure 1. High-Resolution Analysis of Long-Range Interactions within the Igh Locus

(A) Interaction of the HS3B-HD viewpoint (red) with chromosome 12 sequences in cultured Pax5^−/−Rag2^−/− and Rag2^−/− pro-B cells. The 20 kb running mean values of the 4C-seq reads were plotted as reads per million mapped sequence reads (RPMs). A black bar denotes the Igh locus. (B) Relative distribution of the 4C-seq reads (HS3B-HD viewpoint) in the Igh locus, rest of chromosome 12 (Chr.12 – Igh), or entire genome minus chromosome 12 (Genome – Chr.12). (C) Pax5-dependent long-range interactions of the HS3B region in ex vivo sorted and in vitro cultured cells of the indicated genotypes. (D) 4C-seq patterns of the HS8-BN and HS8-ED viewpoints in cultured Pax5^−/−Rag2^−/− and Rag2^−/− pro-B cells. A 20 kb running mean mappability track (gray) indicates to what degree (on a scale from 0 to 2) the end sequences of HindIII (C), BglII (D), and EcoRI (D) fragments can be unambiguously mapped to genomic Igh sequences. One representative 4C-seq experiment is shown for each viewpoint and cell type (C and D). (E) Annotation of the C57BL/6 Igh locus. The distinct V_H gene families (different colors) in the distal, middle, and proximal V_H gene regions (Johnston et al., 2006) as well as the D_H (gray) and C_H (blue) elements and Eµ and 3′RR enhancers (red) in the 3′ proximal Igh domain are shown together with the PAIR elements (orange) and the mm9 genomic coordinates of mouse chromosome 12. The indicated DNase I hypersensitive (DHS) regions and CTCF- and Pax5-binding sites were determined in Rag2^−/− pro-B cells by paired-end sequencing and peak calling (MACS; p value < 10⁻¹⁰) (Ebert et al., 2011; Revilla-I-Domingo et al., 2012). See also Figure S1.

Figure 2. Loop Formation in the 3′ Proximal Igh Region

(A–C) 4C-seq interaction patterns revealed by the indicated viewpoints (red) in short-term cultured Pax5^−/−Rag2^−/− and Rag2^−/− pro-B cells. Vertical lines indicate the positions of relevant V_H, D_H, and C_H gene segments as well as IGCR1, Eµ, and DNase I hypersensitive sites constituting the 3′RR (HS1–HS4) and 3′CBE (HS5–HS8) regions. Previously known and newly identified interactions are highlighted in green and orange, respectively. See legend of Figure 1 for further explanations. The data in (A) are based on average values of three independent experiments, and the results shown in (B) and (C) are derived from one experiment. (D) Characterization of the Cγ1-Cγ2b region. The sequences between the Cγ1 and Cγ2b gene segments were analyzed for binding of the indicated transcription factors and the presence of DHS sites and active histone modifications (H3K4me2 and H3K9ac) by deep sequencing of Pax5^−/−Rag2^−/− (red) and Rag2^−/− (black) pro-B cells (Revilla-I-Domingo et al., 2012). See also Figure S2.

Figure 3. Local Interactions within the Distal V_H Gene Cluster in the Absence of Locus Contraction

(A) 4C-seq interaction profiles at the Igh 5′ end in cultured Pax5^−/−Rag2^−/− and Rag2^−/− pro-B cells. Vertical lines indicate the positions of PAIR elements (yellow) and selected V_H genes (blue). (B) 4C-seq interaction patterns detected with the indicated PAIR viewpoints (red) in the distal V_H gene region. See legend of Figure 1 for further explanations. One 4C-seq experiment was performed with viewpoints Zfp386-HD and J558.88-ED, and the data of all other viewpoints are based on average values of three experiments. See also Figure S3.

Figure 4. Flexible Long-Range Interactions along the Distal V_H Gene Region in the Presence of Locus Contraction

(A–C) Long-range interactions of the indicated proximal viewpoints (red) with the distal V_H gene region in cultured Pax5^−/−Rag2^−/− and Rag2^−/− pro-B cells. See legend of Figure 1 for further explanations. The data of the HS3B-HD and Eµ-HD viewpoints (A) are average values of three experiments, and all other viewpoints (B, C) were analyzed once. (D) Comparison of the 4C-seq patterns obtained with random BAC libraries and Rag2^−/− pro-B cells (analyzed from HS3B-HD). The sequencing pattern of each BAC (with its size indicated below) was normalized by setting the highest peak to an RPM value of 10,000 (Figure S4B) prior to merging the patterns of the two BAC libraries (indicated in black and gray). Abbreviation: Del., internal BAC deletion. See also Figure S4.

Figure 5. Long-Range Interactions of the Distal V_H Gene Region with the Proximal Igh Domain

(A) 4C-seq interaction pattern of PAIR8 with sequences across the Igh locus in cultured Pax5^−/−Rag2^−/− and Rag2^−/− pro-B cells. (B) Long-range interactions of the indicated distal viewpoints (red) with sequences of the Igh 3′ region in the indicated cultured pro-B cell types. See legends of Figures 1 and 2 for further explanations. The data of all viewpoints in (A) and (B) are average values of three experiments except for the PAIR4-HD analysis of Pax5^−/−Rag2^−/− pro-B cells (average of two experiments). (C) Comparison of the 4C-seq patterns between random BAC libraries and Rag2^−/− pro-B cells (analyzed from J558.89-HD). See legend of Figure 4D for normalization of the BAC sequencing pattern. Yellow shading denotes regions with a 4C-seq pattern that differs between the random BAC libraries and Rag2^−/− pro-B cells. The specificity of the 4C-seq interactions in the IGCR1/D_H region (asterisk) of Rag2^−/− pro-B cells compared to the random BAC library was confirmed by 3C-qPCR (Figure S4E).

Figure 6. Role of CTCF and 3′ Regulatory Elements in Long-Range Igh Interactions

(A) 4C-seq analysis of cultured 129Sv pro-B cells of the indicated genotypes with the HS8-ED viewpoint (red). The homozygous J_HT deletion eliminates the D_HQ52, J_H, and Eµ elements (Gu et al., 1993). The 129Sv sequence reads were mapped to genomic C57BL/6 sequences except for the available 129S1 Igh sequences (see Supplemental Experimental Procedures). A 120 kb deletion (Δ) was present in the E14 (129Ola) ESCs used to generate the J_HT and 3′RR mutant alleles. 4C-seq experiments were performed once with Rag2^−/− 3′RR (–/–) mutant, Rag2^−/− Eµ (–/–) mutant, and J_HT-deficient pro-B cells and twice with 129Sv Rag2^−/−, Pax5^−/−Rag2^−/−, and Rag2^−/− IGCR1/CBE (–/–) mutant pro-B cells. (B) Two-color 3D DNA-FISH analysis with Igh BAC probes (indicated in A). Dot plots show the distances measured between the two DNA signals of individual Igh alleles together with the average distance determined for each genotype. (C) 3C-qPCR analysis of the IGCR1 interaction with distal PAIR4, PAIR8, and V_HJ558.89 sequences in cultured 129Sv pro-B cells of the indicated genotypes. A total of 12 3C templates of each genotype were used to determine the relative crosslinking frequency (see Supplemental Experimental Procedures), which was set to 1 for Rag2^−/− pro-B cells and is shown with the standard error of the mean. (D) Coprecipitation of CTCF with Pax5-Bio by streptavidin (SA) pull-down of nuclear extracts prepared from Abl-MLV-transformed pro-B cells of the Pax5^Bio/Bio (Pax5-Bio) or control Rosa26^BirA/BirA (BirA) genotype. The input (In; 1/100), supernatant (Sn; 1/100), and streptavidin-bound (B) precipitate were analyzed by immunoblotting (WB) with CTCF and Pax5 antibodies. (E) Coimmunoprecipitation of Pax5 from a nuclear extract of Rag2^−/− pro-B cells with CTCF antibodies followed by immunoblotting with a biotinylated rat anti-Pax5 mAb (detected with streptavidin-coupled horse radish peroxidase). Input (In; 1/100) and rabbit IgG were used as controls. Where indicated, DNA was digested with the endonuclease Benzonase during nuclear extract preparation. (F) Direct Pax5-CTCF protein interaction. The hexahistidine-tagged CTCF and GST-Pax5 proteins (schematically depicted to the right) were affinity purified from baculovirus-infected Sf9 cells (Figure S5B) and used for in vitro binding and GST pull-down assays followed by immunoblotting of the precipitates with CTCF and GST antibodies (see Supplemental Experimental Procedures). A protein size marker (in kilodaltons) and input (In; 1/20) are shown. Empty glutathione-sepharose beads (−) and GST-GFP were used as negative controls. Abbreviations are as follows: G, GST; P5, Pax5; PD, paired domain; OP, octapeptide; HD, partial homeodomain; TAD, transactivation domain; ID, inhibitory domain. See also Figure S5.

Figure 7. YY1 Controls PAIR Function and Long-Range Interactions across the Igh Locus

(A) 4C-seq analysis of ex vivo sorted Rag1^Cre/+Yy1^fl/fl pro-B cells (Figures S6B and S6C), control Rag2^−/− pro-B cells, and thymocytes with the viewpoint Eµ-HD (red). The Eµ-3′CBE interaction is highlighted in orange. A red asterisk indicates the loss of D_H sequences by D_H-J_H rearrangements in the recombination-proficient Rag1^Cre/+Yy1^fl/fl pro-B cells. (B) Identification of YY1 peaks in the Igh locus by Bio-ChIP-sequencing of cultured pro-B cells from Yy1^ihCd2/+Rosa26^BirA/BirARag2^−/− mice. Vertical bars below the Bio-ChIP-seq track identify significant YY1 peaks that were called by MACS with a p value of <10⁻¹⁰. For comparison, the Pax5-binding and DHS site patterns of Rag2^−/− pro-B cells are shown (Revilla-I-Domingo et al., 2012). (C) YY1 binding at PAIR elements and associated V_H3609 genes. (D and E) Analysis of YY1-dependent gene expression. The expression values (RPKM) of selected genes (D) and RNA-seq profiles of the antisense transcripts originating from PAIR4 and PAIR6 (E) are shown for Rag1^Cre/+Yy1^fl/fl (red) and control Rag1^Cre/+Yy1^fl/+ (black) pro-B cells together with the standard error of the mean (based on two RNA-seq experiments for each genotype). See also Figure S6.