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 Eμ 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 Eμ 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 Eμ enhancer controls V_H-DJ_H recombination.