Enhancers of the PAIR4 regulatory module promote distal V_H gene recombination at the Igh locus

While extended loop extrusion across the entire Igh locus controls V-H-DJ(H) recombination, local regulatory sequences, such as the PAIR elements, may also activate V-H gene recombination in pro-B-cells. Here, we show that PAIR-associated V(H)8 genes contain a conserved putative regulatory element (V8E) in their downstream sequences. To investigate the function of PAIR4 and its V8.7E, we deleted 890 kb containing all 14 PAIRs in the Igh 5 ' region, which reduced distal V-H gene recombination over a 100-kb distance on either side of the deletion. Reconstitution by insertion of PAIR4-V8.7E strongly activated distal V-H gene recombination. PAIR4 alone resulted in lower induction of recombination, indicating that PAIR4 and V8.7E function as one regulatory unit. The pro-B-cell-specific activity of PAIR4 depends on CTCF, as mutation of its CTCF-binding site led to sustained PAIR4 activity in pre-B and immature B-cells and to PAIR4 activation in T-cells. Notably, insertion of V8.8E was sufficient to activate V-H gene recombination. Hence, enhancers of the PAIR4-V8.7E module and V8.8E element activate distal V-H gene recombination and thus contribute to the diversification of the BCR repertoire in the context of loop extrusion.