Molecular basis for differential Igk versus Igh V(D)J joining mechanisms

In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease¹. RAG orchestrates Igh V(D)J recombination upon capturing a J_H-RSS within the J_H-RSS-based recombination centre^1-3 (RC). J_H-RSS orientation programmes RAG to scan upstream D- and V_H-containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion^4-7. During Igh scanning, RAG robustly utilizes only D-RSSs or V_H-RSSs in convergent (deletional) orientation with J_H-RSSs^4-7. However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters⁸, inconsistent with linear scanning². Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements^9,10, which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element¹¹. Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element¹² 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to V_H locus inversion⁷, DJ_H inversion abrogated V_H-to-DJ_H joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vκ-to-J_H and D-to-Jκ rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk-RSSs to promote robust diffusional joining compared with Igh-RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating V_H joining by RAG-scanning impediments.