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核酸内切酶通过结合重组中心（RC）内的J _H重组信号序列（RSS），然后线性扫描上游染色质（由粘着蛋白介导的环挤出法）以收敛祖细胞B中的Igh V（D）J重组。D-RSS ^1,2。融合定向RSS和隐式RSS的使用是远程RAG扫描³固有的。从DJ _H -RC-RSS到上游收敛V _H -RSSs的RAG扫描受D-近端CTCF绑定元素（CBE）^2,3,4,5的阻碍。原代祖细胞B发生机械不确定的V _H收缩建议提供远端V _H进入DJ _H -RC ^6,7,8,9的轨迹。在这里我们报告说，小鼠原代祖细胞中整个2.4 Mb V _H基因座的倒置消除了V _H -RSSs和通常会聚的隐性RSS的重排，即使基因座收缩仍然发生。另外，这种反演激活了通常沿相反方向的隐秘V _H -RSS的利用和通过多个会聚CBE域到达端粒的RAG扫描超出V _H基因座。总之，这些发现暗示原代祖细胞中CBE障碍的广泛解除促进了V _H的RAG扫描环挤出介导的基因座。我们进一步发现，与缺乏收缩和RAG扫描的v-Abl转化的祖细胞B细胞系相比，原代祖细胞B中的鸡翅样蛋白同源物（WAPL）¹⁰（一种黏着素卸载因子）的表达较低。V _H轨迹。相应地，在v-Abl转化的细胞系中WAPL的耗尽激活了两个过程，进一步牵涉了在基因座收缩机制中的环挤出。