Insertion of an imprinted insulator into the IgH locus reveals developmentally regulated, transcription-dependent control of V(D)J recombination.

The assembly of antigen receptor loci requires a developmentally regulated and lineage-specific recombination between variable (V), diversity (D), and joining (J) segments through V(D) J recombination. The process is regulated by accessibility control elements, including promoters, insulators, and enhancers. The IgH locus undergoes two recombination steps, D-J(H) and then V-H-DJ(H), but it is unclear how the availability of the DJ(H) substrate could influence the subsequent V-H-DJ(H) recombination step. The E mu enhancer plays a critical role in V(D)J recombination and controls a set of sense and antisense transcripts. We epigenetically perturbed the early events at the IgH locus by inserting the imprinting control region (ICR) of the Igf2/H19 locus or a transcriptional insulator devoid of the imprinting function upstream of the E mu enhancer. The insertions recapitulated the main epigenetic features of their endogenous counterparts, including differential DNA methylation and binding of CTCF/cohesins. Whereas the D-J(H) recombination step was unaffected, both the insulator insertions led to a severe impairment of V-H-DJ(H) recombination. Strikingly, the inhibition of V-H-DJ(H) recombination correlated consistently with a strong reduction of DJ(H) transcription and incomplete demethylation. Thus, developmentally regulated transcription following D-J(H) recombination emerges as an important mechanism through which the E mu enhancer controls V-H-DJ(H) recombination.