The fundamental role of chromatin loop extrusion in physiological V(D)J recombination.

Abstract

RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates1. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain2 at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1)3, the DHs, and a RAG-bound recombination center (RC)4. The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iEμ”)5. Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs)6. Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs6. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare7,8. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs9, as opposed to putative linear scanning mechanisms10,11. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops4,12-14, potentially formed by cohesin-mediated loop extrusion15-18, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.