Contribution of the IGCR1 regulatory element and the 3 & PRIME;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 VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH- based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC -bound RAG presents Ds for joining to JHs to form a DJH-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 VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3 & PRIME;Igh- CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs seg-regate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor -B cells neutralizes CBEs, allowing DJH- RC- bound RAG to scan the VH domain and perform VH-to-DJH rearrange-ments. To elucidate potential roles of IGCR1- based CBEs and 3 & PRIME;Igh- CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D- to- JH to VH-to-DJH recombination, we tested effects of inverting and/or deleting IGCR1 or 3 & PRIME;Igh- CBEs in mice and/or progenitor -B cell lines. These studies revealed that nor-mal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3 & PRIME;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.