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.