CTCF couples long-range loop extrusion and diffusion to mediate a diverse Igk repertoire

Dynamic genome folding is important for V(D)J recombination at the immunoglobulin kappa (Igk) locus, which recombines Jk and Vk gene segments across a 3.2 Mb region in both deletional and inversional orientations. Chromatin loop extrusion and diffusion are considered two key mechanisms underlying Igk folding, but how they coordinate remains unclear. Here we show that CTCF is a key regulator coupling loop extrusion and diffusion during Igk V-J rearrangement, promoting recombination in both orientations across long genomic distances. Mechanistically, the CTCF N-terminus promotes long-range loop extrusion that facilitates distal Vk usage by stabilizing cohesin against WAPL release, and also forms loop barriers enabling chromatin diffusion for inversional Vk joining. In CTCF N-terminal-deficient B cells, defects in inversional Vk joining are not restored by WAPL depletion but are instead largely rescued by a dCas9-blockade targeted to the Vk-Jk intergenic region, mimicking the CTCF barrier. Our findings thus highlight how CTCF coordinates distinct genome-folding mechanisms through its dual roles in cohesin stabilization and extrusion barrier formation to ensure the generation of a diverse Igk repertoire.

动态基因组折叠对于免疫球蛋白κ（immunoglobulin kappa, Igk）基因座的V(D)J重组至关重要。该基因座可在3.2 Mb的基因组区域内，以缺失型和倒位型两种重组方向，完成Jk与Vk基因片段的重组。染色质环挤出（chromatin loop extrusion）与染色质扩散（chromatin diffusion）被认为是调控Igk基因组折叠的两大核心机制，但二者的协同调控方式仍不明确。本研究发现，CTCF是Igk V-J重排过程中偶联环挤出与染色质扩散的关键调控因子，可促进长基因组距离范围内两种重组方向的重组事件。从机制上看，CTCF的N末端可通过稳定黏连蛋白（cohesin）以抵抗WAPL介导的释放，从而促进长距离环挤出，便于远端Vk基因的使用；同时还可形成环屏障，介导染色质扩散以支持倒位型Vk基因的重组连接。在CTCF N末端缺陷的B细胞中，倒位型Vk基因重组连接的缺陷无法通过敲低WAPL得到恢复，但通过靶向Vk-Jk基因间区的失活Cas9（dCas9）阻断来模拟CTCF屏障功能，可在很大程度上挽救该缺陷。综上，本研究揭示了CTCF如何通过黏连蛋白稳定与环挤出屏障形成的双重功能，协调不同的基因组折叠机制，从而确保多样化的Igk repertoire得以生成。