CTCF-binding elements mediate control of V(D)J recombination

Abstract

Immunoglobulin heavy chain (IgH) variable region exons are assembled from V(H), D and J(H) gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the V(H) and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of D(H)-proximal V(H) gene segments and promoting rearrangement of distal V(H) segments. IGCR1 maintains ordered and lineage-specific V(H)(D)J(H) recombination by suppressing V(H) joining to D segments not joined to J(H) segments, and V(H) to DJ(H) joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal V(H)-to-DJ(H) recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein.

Figure 1. Mutation of IGCR1 CBEs impairs B cell development

(a) Murine 129SV IgH locus (accession number: AJ851868) schematic showing 4.1 kb IGCR1 region in WT compared to IGCR1 deleted, loxP inserted, or CBE mutated configuration. (b) Flow cytometry analysis of IgM^- bone marrow (BM) and IgM⁺ splenic B cell populations in WT, loxP^I/I, and IGCR1/CBE^-/- mice. In BM the B220^intCD43⁺ pro-B and B220⁺CD43^- pre-B cell populations are indicated. (c) Expression of IgM^a and IgM^b allotypic markers in BM and spleen from WT IgM^a/IgM^a (pure 129SV), WT IgM^b/IgM^b (pure C57BL/6), WT F1 (IgM^a/IgM^b), and heterozygous mutant IGCR1/CBE^- IgM^a/ WT IgM^b mice.

Figure 2. IGCR1 mutations alter V_H usage, germline transcription and rearrangement order

(a) PCR analyses of indicated V_H family rearrangements in pro-B cells from indicated mice compared to a DLG5 loading control. Results are typical of four experiments. Bands corresponding to rearrangements to various J_Hs are indicated on right. (b) RT-PCR analysis of indicated germline V_H transcripts in three independent WT and IGCR1^-/- A-MuLV virus transformed RAG2^-/- pro-B cell lines. N=nonspliced sense/antisense and S=spliced sense. (c) ChIP-qPCR analyses of H3K4me2 and H3K9ac histone modifications at indicated V_Hs in 129SV Rag2^−/− (black) and Rag2^−/− IGCR1^−/− (red) A-MuLV-transformed pro-B lines. The 5′ region (5′), body (G) and 3′ region (3′) of V_H81X and body (G) of V_HQ52.2.4 were analyzed. Average values and standard deviations of three experiments with one line shown are representative of results from both. (d) Semi-quantitative PCR analyses of direct V_H-D rearrangements in sorted pro-B cells from indicated mice. The PCR assays utilized for panels a, b, and d are diagrammed in Supp. Figs. 6a, 7a, and 8a.

Figure 3. IGCR1/CBE Mutations lead to V_HDJ_H and V_HD rearrangements in thymocytes

(a) PCR analyses of V_H family rearrangements in sorted DP thymocytes and total splenic B cells from indicated mice with DLG5 as a loading control. Bands corresponding to rearrangements to various J_Hs are indicated on right. Igκ rearrangement (VκJκ) served as a control for B cell contamination. (b) Semi-quantitative PCR analyses of direct V_H-D rearrangements in sorted DP-T cells from indicated mice. Assays are diagrammed in Supp. Figs. 6a and 8a.

Figure 4. IGCR1 is required to allow feedback regulation of proximal V_H to DJ_H recombination

(a) Mean percentage of splenic B cells with V_HDJ_H rearrangements on both IgH alleles as determined by analyses of hybridomas from three independent sets of WT, IGCR1^+/-, and IGCR1^-/- mice (Supp. Table 5). Error bars represent standard deviation. p-values were calculated by student t-test. (b) IgH V_HDJ_H rearrangements in splenic B cells from two independent WT and VB1-8 knock-in mice carrying either a WT (IGCR1^+/+ VB1-8 KI) or an IGCR1 deleted (IGCR1^+/- VB1-8 KI) second allele. Bands corresponding to rearrangements to various J_Hs are indicated on right. DLG5 is the loading control.

Figure 5. IGCR1 mediates long distance IgH chromosomal loops

(a) Schematic of chromosome interactions between IGCR1-containing and 3′IgHCBE-containing KpnI restriction fragments in 3C assays. Interactions between IGCR1 and 3′IgHCBE locales in 129SV RAG2^-/- and RAG2^-/-IGCR1^-/- A-MuLV transformed pro-B cells were quantified by real time PCR (Taqman) using probe (P2) (left) and a probe (P1) (right). (b) Schematic of chromosome interactions between iEμ-containing KpnI restriction fragment and indicated KpnI restriction fragments in other IgH locales. Interactions between iEμ and IGCR1, iEμ and 3′IgHRR locales, iEμ and 3′IgHCBE locales in RAG2^-/- and RAG2^-/-IGCR1^-/- A-MuLV transformed pro-B cells were quantified by real time PCR using a probe (P3) from the iEμ locale. F1-F8 indicate primers used for PCR. K indicates KpnI sites. Red arcs indicate interactions detected in RAG2^-/- cells. The average association frequency of three independent 3C experiments with 2 independent A-MuLV transformed lines from each genotype is shown with standard deviation indicated.