Contribution of the IGCR1 regulatory element and the 3’IghCBEs to Regulation ofIghV(D)J Recombination

Abstract

ABSTRACTImmunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JHgene segments located in separate clusters across theIghlocus. 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.Ighhas a provocative number and organization of CTCF-binding-elements (CBEs) that can impede loop extrusion. Thus,Ighhas two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VHand D/JHdomains, over 100 CBEs across the VHdomain convergent to CBE1, and 10 clustered 3’Igh-CBEs convergent to CBE2 and VHCBEs. IGCR1 CBEs segregate D/JHand VHdomains by impeding loop extrusion-mediated RAG-scanning. Down-regulation 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 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-JHto VH-to-DJHrecombination, 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 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 down-regulation mechanism in progenitor B cells as opposed to a strict developmental switch.SIGNIFICANCE STATEMENTTo counteract diverse pathogens, vertebrates evolved adaptive immunity to generate diverse antibody repertoires through a B lymphocyte-specific somatic gene rearrangement process termed V(D)J recombination. Tight regulation of the V(D)J recombination process is vital to generating antibody diversity and preventing off-target activities that can predispose the oncogenic translocations. Recent studies have demonstrated V(D)J rearrangement is driven by cohesin-mediated chromatin loop extrusion, a process that establishes genomic loop domains by extruding chromatin, predominantly, between convergently-oriented CTCF looping factor-binding elements (CBEs). By deleting and inverting CBEs within a critical antibody heavy chain gene locus developmental control region and a loop extrusion chromatin-anchor at the downstream end of this locus, we reveal how these elements developmentally contribute to generation of diverse antibody repertoires.