RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a J_H-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs^1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning³. Scanning of RAG from the DJ_H-RC-RSS to upstream convergent V_H-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)^2,3,4,5. Primary progenitor B cells undergo a mechanistically undefined contraction of the V_H locus that is proposed to provide distal V_Hs access to the DJ_H-RC^6,7,8,9. Here we report that an inversion of the entire 2.4-Mb V_H locus in mouse primary progenitor B cells abrogates rearrangement of both V_H-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic V_H-RSSs that are normally in opposite orientation and RAG scanning beyond the V_H locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the V_H locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL)¹⁰, a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the V_H locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.

RAG 核酸内切酶通过在重组中心 (RC) 内结合 JH 重组信号序列 (RSS) 启动_{祖细胞 B 细胞中的}Igh V(D)J 重组，然后线性扫描上游染色质，由粘连蛋白介导的环挤压呈现，用于收敛D-RSS ^1,2。收敛导向 RSS 和隐秘 RSS 的使用是远程 RAG 扫描³所固有的。D-近端 CTCF 结合元素 (CBE) ^2,3,4,5阻碍了从 DJ _H -RC-RSS 到上游收敛 V _H -RSS 的 RAG 扫描。原代祖 B 细胞经历 VH 的机械不确定_收缩建议提供远端 V _H访问 DJ _H -RC ^6,7,8,9的轨迹。在这里，我们报告了小鼠原代祖细胞 B 细胞中整个 2.4-Mb V _H基因座的倒置消除了 V _H -RSS 和正常收敛的隐性 RSS 的重排，即使基因座收缩仍然发生。此外，这种倒置激活了通常处于相反方向的神秘 VH -RSS 的利用和 VH 基因座之外的 RAG 扫描，_通过几个会聚的 CBE 域到端粒。总之，这些发现意味着初级祖 B 细胞中 CBE 障碍的广泛解除管制促进了 VH 的 RAG_扫描由循环挤压介导的轨迹。我们进一步发现，与缺乏收缩和 RAG 扫描的v-Abl转化的祖 B 细胞系相比，wings apart-like protein homologue (WAPL) ¹⁰ （一种粘连蛋白卸载因子）的表达在原代祖 B 细胞中较低。VH_轨迹。相应地， v-Abl转化线中 WAPL 的耗尽激活了这两个过程，进一步暗示了基因座收缩机制中的环挤压。