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

. 2011 Sep 11;477(7365):424-30.

doi: 10.1038/nature10495.

Hye Suk Yoon, Andrew Franklin, Suvi Jain, Anja Ebert, Hwei-Ling Cheng, Erica Hansen, Orion Despo, Claudia Bossen, Christian Vettermann, Jamie G Bates, Nicholas Richards, Darienne Myers, Harin Patel, Michael Gallagher, Mark S Schlissel, Cornelis Murre, Meinrad Busslinger, Cosmas C Giallourakis, Frederick W Alt

Affiliations

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

Chunguang Guo et al. Nature. 2011.

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.

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Figures

Figure 1

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, loxPI/I, and IGCR1/CBE-/- mice. In BM the B220intCD43+ pro-B and B220+CD43- pre-B cell populations are indicated. (c) Expression of IgMa and IgMb allotypic markers in BM and spleen from WT IgMa/IgMa (pure 129SV), WT IgMb/IgMb (pure C57BL/6), WT F1 (IgMa/IgMb), and heterozygous mutant IGCR1/CBE- IgMa/ WT IgMb mice.

Figure 2

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

(a) PCR analyses of indicated VH 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 JHs are indicated on right. (b) RT-PCR analysis of indicated germline VH 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 VHs 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 VH81X and body (G) of VHQ52.2.4 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 VH-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

Figure 3. IGCR1/CBE Mutations lead to VHDJH and VHD rearrangements in thymocytes

(a) PCR analyses of VH 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 JHs are indicated on right. Igκ rearrangement (VκJκ) served as a control for B cell contamination. (b) Semi-quantitative PCR analyses of direct VH-D rearrangements in sorted DP-T cells from indicated mice. Assays are diagrammed in Supp. Figs. 6a and 8a.

Figure 4

Figure 4. IGCR1 is required to allow feedback regulation of proximal VH to DJH recombination

(a) Mean percentage of splenic B cells with VHDJH 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 VHDJH 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 JHs are indicated on right. DLG5 is the loading control.

Figure 5

Figure 5. IGCR1 mediates long distance IgH chromosomal loops

(a) Schematic of chromosome interactions between IGCR1-containing and 3′_IgHC_BE-containing KpnI restriction fragments in 3C assays. Interactions between IGCR1 and 3′_IgH_CBE 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′_IgH_RR locales, iEμ and 3′_IgH_CBE 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 _Kpn_I 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.

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