Contribution of IGCR1 and 3' CBE Super Anchor to Developmental Regulation of Igh V(D)J Recombination [HTGTS_VDJ_Seq]
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 orchestrates 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 DJHRC, from which RAG scans upstream for VHs to assemble VHDJH exons in an ordered recombination process.  Cohesin-mediated chromatin loop extrusion is impeded by CTCF-bound elements (CBEs), with extrusion between two CBEs, particularly if convergently-oriented, forming chromatin-contact loops genome-wide.  Igh has provocative CBE organization, with two divergently-oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, dozens of VH domain CBEs convergent to CBE1, and 10 3'Igh-CBEs convergent to CBE2. IGCR1 CBEs segregate D/JH and VH domains by impeding RAG-scanning.  After DJH formation, down-regulation of WAPL, a cohesin unloader, neutralizes IGCR1 and VH-domain CBEs, allowing RAG to scan the VH domain.  To elucidate potential mechanisms underlying the developmental transition from D-to-JH to VH-to-DJH recombination and potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning, we tested effects of deleting or inverting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments its RAG-scanning impediment activity and that the 3'Igh-CBEs reinforce RC activity as a dynamic loop extrusion impediment, thereby, increasing its ability to support V(D)J recombination.  Finally, our findings implicate a mechanism by which developmental WAPL down-regulation contributes to ordered V(D)J recombination. Overall design: We performed HTGTS-VDJ-Seq to determine the roles of IGCR1 and 3'Igh CBEs in v-abl cells, pro-B cells.