Contribution of the IGCR1 regulatory element and the 3' Igh CTCF-binding elements to regulation of Igh V(D)J recombination.

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from V H , D, and J H gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a J H -based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to J H s to form a DJ H -RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the V H and D/J H domains, over 100 CBEs across the V H domain convergent to CBE1, and 10 clustered 3' Igh -CBEs convergent to CBE2 and V H CBEs. IGCR1 CBEs segregate D/J H and V H domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJ H -RC-bound RAG to scan the V H domain and perform V H -to-DJ H rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3' Igh -CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-J H to V H -to-DJ H recombination, we tested effects of inverting and/or deleting IGCR1 or 3' Igh -CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3' Igh -CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.