Physiological V(D)J Recombination is Mediated by RAG Scanning of Loop-extruded Chromatin

Abstract RAG endonuclease initiates V(D)J recombination by cleaving paired V, D, and J gene segments flanked by complementary recombination signal sequences (RSSs). We address physiological relevance of RAG scanning and the underlying mechanisms through studies of the ~300 kb 3′-proximal IgH locus CBE-anchored loop domain. This domain contains nine Ds and four JHs within a 57-kb region between the upstream IGCR1 loop anchor and the V(D)J recombination center (RC). IgH D to JH-rearrangements occur in deletional orientation, mediated by convergent downstream D-RSSs and JH-RSSs, despite Ds having upstream RSSs in the same orientation as JH-RSSs that could promote inversion. Extra-chromosomal V(D)J recombination substrate studies attributed this deletional orientation-specific joining bias to D-RSS sequence differences. However, we find that deletional joining of 8 of 9 chromosomal IgH Ds, results from RAG linear scanning of chromatin upstream of the RAG-bound RC, a process that detects convergent D-RSSs but to which D-RSSs in the same orientation are invisible. The exceptional D employs an RSS-based mechanism to counteract proximal location to JHs in the RC. By deleting JHs, we reveal that RAG scans directionally downstream from 3′D-RSSs until reaching the IgH 3′CBE loop anchor. Targeting catalytic-dead Cas9 (dCas9) binding in this scanning path impedes RAG scanning in association with cohesin accumulation and loop formation between the block site and RC. Strong transcription through a repetitive downstream switch region blocks RAG scanning similarly to the dCas9 block. We demonstrate a critical role for RAG scanning in physiological V(D)J recombination and implicate cohesin-mediated loop extrusion as a driving force.