Linear RAG scanning mediates editing of Igκ variable region repertoires | Nature

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Subjects

- Chromatin structure

- VDJ recombination

Abstract

V(D)J recombination-mediated Igκ light chain variable region exon assembly in precursor (pre)-B cells involves recombination activating gene (RAG) endonuclease-orchestrated cleavage between and joining of paired Vκ and Jκ gene segments and flanking RAG-targeting recombination signal sequences (RSSs)1,2,3. The 3.1-megabase Igk contains 4 Jκs (Jκ1, 2, 4, 5) and 100-plus Vκs in clusters oriented for deletional or inversional joining2. Vκ-to-Jκ joining is ordered, with primary Vκ-to-Jκ1 rearrangements occurring first, followed by secondary rearrangements of upstream Vκs that replace primary VκJκ1s by joining to Jκ2-5 (refs. 4,5). Loop extrusion moves deletional-oriented and inversional-oriented, locus-wide Vκs past the Cer/Sis CTCF-binding element-based diffusion platform for short-range diffusional presentation to Jκ1-bound RAG in the primary recombination centre (RC). To achieve diffusion-mediated Vκ-to-Jκ1 joining, Igk evolved powerful Vκ-associated and Jκ-associated RSSs3. Secondary Igk rearrangements replace non-functional or autoreactive primary VκJκ1 rearrangements, expanding the Igκ repertoire and mediating central tolerance by means of receptor editing4,6,7,8,9,10,11. Here we describe studies that elucidate the physiologically critical secondary Igk recombination mechanism. Primary deletional and inversional VκJκ1 joins, respectively, delete or displace Cer/Sis, creating a pre-B cell population that harbours secondary VκJκ1-based RCs across the Vκ locus and leaves most unrearranged Vκs immediately upstream of secondary RCs in deletional orientation. High-throughput assays demonstrated that RAG scanning from secondary VκJκ1-based RCs, collectively, extends linearly across the Vκ locus in primary pre-B cell populations. Correspondingly, studies of induced pluripotent stem (iPS) cell-generated mouse models or cell lines with physiological VκJκ-rearrangements further revealed that deletional and, originally, inversional Vκs are mostly captured by Jκ2-5-based secondary RCs in deletional orientation by means of linear RAG scanning. Strong Vκ-RSSs contribute to restricting secondary rearrangements, including potential editing rearrangements, to Vκs immediately upstream of a given secondary RC and support, at a lower level, linear scanning-based inversional Vκ-to-Jκ rearrangements. Our findings implicate Cer/Sis deletion and/or displacement as a developmental switch that converts the two-loop-based diffusional primary Igk rearrangement mechanism into a one-loop-based linear scanning secondary rearrangement mechanism.

Main

Igh and Igk V(D)J recombination is orchestrated by the RAG1–RAG2 heterotetramer bound to a recombination centre (RC) formed around intronic enhancers and J segments of each locus1,12. Vs, Ds and Js are flanked by bona fide recombination signal sequences (RSSs), consisting of conserved CACAGTG heptamer, a 12-base pair (bp) or 23-bp spacer, and an AT-rich nonamer that target recombination activating gene (RAG) endonuclease activity1,12. RAG cleaves robustly only paired gene segments flanked by bona fide RSSs with, respectively, 12-bp and 23-bp spacers (12RSSs and 23RSSs), which must be properly aligned in parallel in the active sites of the two RAG1 proteins in the RAG complex13,14. Cohesin-mediated chromatin loop extrusion contributes to pairing widely separated Igh and Igk RSSs for V(D)J recombination3,14,15,16,17,18,19,20. For Igh, impeded downstream loop extrusion at the RC leads to continued extrusion of upstream chromatin through the impeded cohesin ring, allowing JH-23RSS-bound RAG to linearly scan the upstream 2.5 megabases (Mb) of chromatin for D-12RSSs and, ultimately, VH-23RSSs for assembly of complete VH(D)JH exons14,15,16,17,18,19,20. This ‘single loop-based’ linear scanning mechanism only recognizes complementary RSSs in convergent orientation15,18,19,20 (Supplementary Video 1 and Extended Data Fig. 1a). The mouse Igk has 4 Jκs with upstream-oriented 23RSSs and more than 100 Vκs with 12RSSs, which lie in three clusters of mostly downstream (deletional) oriented distal Vκs, mostly upstream (inversional) oriented middle Vκs and both downstream and upstream-oriented proximal Vκs2,3 (Fig. 1a). Vκs across the 3.1 Mb locus are robustly used by the Vκ-to-Jκ1 joining process that generates initial (primary) VκJκ1 repertoires4. Vκs in inversional orientation could not be joined by the single loop-based linear RAG-scanning mechanism used by Igh. Indeed, for primary Vκ to Jκ1 joining, cohesin-mediated loop extrusion allows a Jκ1-RC-bound RAG to linearly scan just 8 kilobases (kb) upstream to the Sis CTCF-binding elements (CBEs) of Cer/Sis diffusion platform3. Upstream Vκs are moved through a second cohesin ring impeded for downstream extrusion at the Cer CBEs just upstream of Sis3. This ‘two-loop-based’ mechanism allows Vκs to be presented to a RAG-bound Jκ1 RC by short-range diffusion, allowing proper alignment