美国哈佛医学院Hongli Hu等研究人员合作揭示Igk与Igh V(D)J连接机制不同的分子基础。这一研究成果于2024年5月29日在线发表在国际学术期刊《自然》上。

研究人员表示，在发育中的B细胞中，V(D)J重组将Igh和Igk基因座上的数百个基因片段组装成编码IgH和Igκ可变区的外显子。V、D和J基因片段的两侧是保守的重组信号序列（RSS），其靶标是RAG内切酶。RAG在基于J_H-RSS的重组中心（RC）内捕获JH-RSS后，协调Igh V(D)J重组。J_H-RSS定向可使RAG扫描上游含D和V_H的染色质，这些染色质通过黏连蛋白介导的环挤压以线性方式呈现。在Igh扫描过程中，RAG只稳健地利用D-RSS或与J_H-RSS呈趋同（缺失）定向的V_H-RSS。然而，对于V_κ到J_κ的连接，RAG利用的是来自缺失方向和反转方向簇的V_κ-RSS，这与线性扫描不一致。

研究人员描述了V_κ到J_κ连接机制的特征。Igk会发生强烈的初级和次级重排，这就给扫描测定带来了困难。因此，研究人员对细胞进行了设计，使其只进行初级V_κ到J_κ重排，并发现从初级J_κ-RC开始的RAG扫描仅终止于CTCF位点的Sis元件上游8kb处。Sis和J_κ-RC几乎不与V_κ基因座相互作用，而Sis上游4kb处基于CTCF位点的Cer元件则与基因座上的各种环路挤压障碍相互作用。与V_H基因座反转相似，DJ_H基因座反转削弱了V_H到DJ_H的连接；而V_κ基因座或J_κ基因座反转则允许V_κ到J_κ的连接。

这些实验共同表明，环挤压使V_κ片段靠近Cer，从而被基于RC的RAG短程扩散介导的捕获。为了确定Igk与Igh中扩散性V(D)J重组的关键机制要素，研究人员检测了由定向染色体易位产生的杂交Igh-Igk基因座中的V_κ对J_H和D对J_κ重排，并确定了非常强的V_κ和J_κ RSS。事实上，与Igh-RSS相比，杂交或正常Igk和Igh基因座的RSS置换证实了Igk-RSS促进强健的扩散连接的能力。研究人员认为，Igk演化出了强大的RSS来介导V_κ到J_κ的扩散连接，而Igh则演化出了较弱的RSS，这是通过RAG扫描阻碍来调节V_H连接所需的。

附：英文原文

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

Author: Zhang, Yiwen, Li, Xiang, Ba, Zhaoqing, Lou, Jiangman, Gaertner, K. Elyse, Zhu, Tammie, Lin, Xin, Ye, Adam Yongxin, Alt, Frederick W., Hu, Hongli

Issue&Volume: 2024-05-29

Abstract: 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 JH-RSS within the JH-RSS-based recombination centre1,2,3 (RC). JH-RSS orientation programmes RAG to scan upstream D- and VH-containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion4,5,6,7. During Igh scanning, RAG robustly utilizes only D-RSSs or VH-RSSs in convergent (deletional) orientation with JH-RSSs4,5,6,7. 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 rearrangements9,10, 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 8kb 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 4kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to VH locus inversion7, DJH inversion abrogated VH-to-DJH 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-JH 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 VH joining by RAG-scanning impediments.

DOI: 10.1038/s41586-024-07477-y

Source: https://www.nature.com/articles/s41586-024-07477-y