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

Abstract

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 <i>Igh</i> 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_Hs to form a DJ_H-RC. <i>Igh</i> has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, <i>Igh</i> 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'<i>Igh</i>-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'<i>Igh</i>-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'<i>Igh</i>-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'<i>Igh</i>-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.