Wapl repression by Pax5 promotes loop extrusion and V gene recombination at the Igh locus

The generation of a diverse antibody repertoire is essential for humoral immunity and requires the participation of all V genes in V(D)J recombination, which depends on the Pax5-regulated contraction of the 2.8-Mb long immunoglobulin heavy-chain (Igh) locus. How Pax5 controls Igh contraction in pro-B cells is, however, not known. Here, we demonstrate that locus contraction is caused by cohesin-mediated chromatin loop extrusion across the entire Igh locus. Notably, expression of the cohesin-release factor Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, which facilitates extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the Polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations of the three-dimensional chromatin architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells. 58 VDJ-Seq samples, 8 experiments: 66820-66825; 69945-69950; 69951-69954; 79219-79224; 81580-81586; 82659-82668; 86490-86494; 9775-97760; 28 3C-Seq samples, 6 experiments, 2 replicates per condition: 97747-97752 (control:97747-97748, experiments:97749-97750,97751-97752); 95730-95733 (control:95730-95731, experiment:95732-95733); 95734-95737 (control:95734-95735, experiment:95736-95737); 108691-108694 (control:108691-108692, experiments: 108693-108694); 108695-108698 (control:108695-108696, experiments: 108697-108698); 112065-112070 (control:112065-112066, experiments: 112067-112068,112069-112070) 28 ChIP-Seq samples, 8 CTCF (control:84325-84326(mm9), experiments:84327-84328(mm9.890-inv),89019-89020(mm9.CBE),89021-89022(mm9.CBE-inv)); 8 H3K27me3 (control:102349-102350, experiments:102351-102352,35325,29940,21478-21479)); 4 Pax5 Antibody (control:102353-102354, experiments:102355-1023536)); 1 Pax5 Bio-ChIP (9158); 3 Ezh2 (23056,35320,25442); 7 ATAC-Seq samples, various cell types and genotypes 44 RNA-Seq samples, 7 experiments: 52052-52055 (control: 52052-52053, experiments:52054-52055); 73767-73772 (control: 73767,73770, experiments: 73768-73769,73771-73772 ); 71445-71451 (control: 71445,71458,714459, experiments: 71446-71447|71450-71451); 60666-60671 (control: 60666,60667, experiments: 60669-60671); 72347-72354 (control:72347-72358|72351-72352, experiments: 72349-72350|72353-72354); 13020,13021,23354-23356,38485-38488 (control: 13020, experiments: 23354-23356|13021,38485-38488); 38492-38494,38496-38497 (control: 38493-38494, experiments: 38492,38496-38497) 7 HiC-Seq samples, various cell types and geno types, 3x2 replicates (86421,85436; 79197,78382; 79196,75860) and 1 replicate (86422)