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

• 成果类型: Article
• 署名作者: Zhang, Yiwen; Li, Xiang; Ba, Zhaoqing; Lou, Jiangman; Gaertner, K. Elyse; Zhu, Tammie; Lin, Xin; Ye, Adam Yongxin; Alt, Frederick W.; Hu, Hongli
• 署名单位: Howard Hughes Medical Institute; Harvard University; Harvard University Medical Affiliates; Boston Children's Hospital; Program in Cellular & Molecular Medicine (PCMM); Harvard University; Harvard Medical School; National Institute of Biological Sciences, Beijing; University of Copenhagen; Georgetown University
• 刊物名称: Nature
• ISSN/ISSBN: 0028-3821
• DOI: 10.1038/s41586-024-07477-y
• 发表日期: 2024-06-06
• 关键词: recombination signal sequences; repertoire; transcription; locus; identification; rearrangement; deletion; element; genome; genes

摘要

In developing B cells, V(D)J recombination assembles exons encoding IgH and Ig kappa 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 endonuclease(1). 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 V kappa-to-J kappa joining, RAG utilizes V kappa-RSSs from deletional- and inversional-oriented clusters(8), inconsistent with linear scanning(2). Here we characterize the V kappa-to-J kappa joining mechanism. Igk undergoes robust primary and secondary rearrangements(9,10), which confounds scanning assays. We therefore engineered cells to undergo only primary V kappa-to-J kappa rearrangements and found that RAG scanning from the primary J kappa-RC terminates just 8kb upstream within the CTCF-site-based Sis element(11). Whereas Sis and the J kappa-RC barely interacted with the V kappa locus, the CTCF-site-based Cer element(12) 4kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to V-H locus inversion(7), DJ(H) inversion abrogated V-H-to-DJ(H) joining; yet V kappa locus or J kappa inversion allowed robust V kappa-to-J kappa joining. Together, these experiments implicated loop extrusion in bringing V kappa 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 V kappa-to-J(H) and D-to-J kappa rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong V kappa and J kappa 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 V kappa-to-J kappa joining, whereas Igh evolved weaker RSSs requisite for modulating V-H joining by RAG-scanning impediments. Experiments in mouse models, and in cell lines that only allow primary V kappa-to-J kappa rearrangements, enable characterization of the mechanisms of V(D)J recombination.