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
Zhuoyi Liang, Lijuan Zhao, Adam Yongxin Ye, Sherry Lin, Yiwen Zhang, Chunguang Guo, Hai‐Qiang Dai, Zhaoqing Ba, Frederick W. Alt
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 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.