CTCF-binding elements 1 and 2 in the Igh intergenic control region cooperatively regulate V(D)J recombination

Ig heavy chain (IgH) variable region exons are assembled from V, D, and J gene segments during early B-lymphocyte differentiation. A several megabase region at the distal end of the mouse IgH locus (Igh) contains hundreds of V(H)s, separated by an intergenic region from Igh Ds, J(H)s, and constant region exons. Diverse primary Igh repertoires are generated by joining Vs, Ds, and Js in different combinations, with a given B cell productively assembling only one combination. The intergenic control region 1 (IGCR1) in the V-H-to-D intergenic region regulates Igh V(D) J recombination in the contexts of developmental order, lineage specificity, and feedback from productive rearrangements. IGCR1 also diversifies IgH repertoires by balancing proximal and distal V-H use. IGCR1 functions in all these regulatory contexts by suppressing predominant rearrangement of D-proximal VHs. Such IGCR1 functions were neutralized by simultaneous mutation of two CCCTC-binding factor (CTCF)-binding elements (CBE1 and CBE2) within it. However, it was unknown whether only one CBE mediates IGCR1 functions or whether both function in this context. To address these questions, we generated mice in which either IGCR1 CBE1 or CBE2 was replaced with scrambled sequences that do not bind CTCF. We found that inactivation of CBE1 or CBE2 individually led to only partial impairment of various IGCR1 functions relative to the far greater effects of inactivating both binding elements simultaneously, demonstrating that they function cooperatively to achieve full IGCR1 regulatory activity. Based on these and other findings, we propose an orientation-specific looping model for synergistic CBE1 and CBE2 functions.