Cutting edge: developmental stage-specific recruitment of cohesin to CTCF sites throughout immunoglobulin loci during B lymphocyte development

Abstract

Contraction of the large Igh and Igkappa loci brings all V genes, spanning >2.5 Mb in each locus, in proximity to DJ(H) or J(kappa) genes. CCCTC-binding factor (CTCF) is a transcription factor that regulates gene expression by long-range chromosomal looping. We therefore hypothesized that CTCF may be crucial for the contraction of the Ig loci, but no CTCF sites have been described in any V loci. Using ChIP-chip, we demonstrated many CTCF sites in the V(H) and V(kappa) regions. However, CTCF enrichment in the Igh locus, but not the Igkappa locus, was largely unchanged throughout differentiation, suggesting that CTCF binding alone cannot be responsible for stage-specific looping. Because cohesin can colocalize with CTCF, we performed chromatin immunoprecipitation for the cohesin subunit Rad21 and found lineage and stage-specific Rad21 recruitment to CTCF in all Ig loci. The differential binding of cohesin to CTCF sites may promote multiple loop formation and thus effective V(D)J recombination.

FIGURE 1. CTCF binding sites in the Igh locus

A, ChIP-chip analysis of CTCF binding in pro-B and pre-B cells across chromosome 12 with the Igh locus underlined. Lines indicate sites of CTCF binding from two independent experiments using MAT software (see Extended Methods). B, Map of the murine Igh locus and CTCF ChIP-chip analysis in pro-B cells and pre-B cells. Primer locations (▴) and negative control primers (-) are indicated. C, qPCR of CTCF and RAD21 ChIPs with primer sets for CTCF sites in the Igh locus. Results represent the mean ± SEM of 2-4 ChIPs.

FIGURE 2. CTCF binding sites in the Igκ locus

A ChIP-chip analysis of CTCF binding in pro-B and pre-B cells across chromosome 6. The location of the Igκ locus is underlined. B, Map of the murine Igκ locus and ChIP-chip analysis of the kappa displaying the entire kappa locus (top) and a close-up of the Sis, Jκ, Cκ, Eκ region (bottom). C, qPCR of CTCF and RAD21 ChIPs with primer sets at CTCF sites in the Igκ locus. Results represent the mean ± SEM of 2-3 ChIPs.

FIGURE 3. CTCF binding sites in the Igλ locus

A, Map of the murine IGλ locus and ChIP-Chip analysis of CTCF binding sites in pro-B and pre-B cells. B, qPCR of CTCF and RAD21 ChIPs with primer sets at CTCF sites in the IGλ. Results represent the mean ± SEM of 2-3 ChIPs.