Loop Extrusion Mediates Physiological Locus Contraction for V(D)J Recombination (ChIP-Seq)

Immunoglobulin heavy chain locus (Igh) VH, D, and JH gene segments are developmentally assembled into V(D)J exons. RAG endonuclease initiates V(D)J recombination by binding a JH-recombination signal sequence (RSS) within a chromatin-based recombination center (RC) and then, in an orientation-dependent process, scans upstream D-containing chromatin presented by cohesin-mediated loop extrusion for convergent D-RSSs to initiate DJH-RC formation. In primary pro-B cells, 100s of upstream VH-associated RSSs, embedded in convergent orientation to the DJH-RC-RSS, gain proximity to the DJH-RC for VH-to-DJH joining via a mechanistically-undefined VH-locus contraction process. Here, we report that a 2.4 mega-base VH locus inversion in primary pro-B cells nearly abrogates rearrangements of normally convergent VH-RSSs and cryptic RSSs, even though locus contraction per se is maintained. Moreover, this inversion activated rearrangement of both cryptic VH-locus RSSs normally in the opposite orientation and, unexpectedly, of normally-oriented cryptic RSSs within multiple, sequential upstream convergent-CBE domains. Primary pro-B cells had significantly reduced transcription of Wapl, a cohesin-unloading factor, versus levels in v-Abl pro-B lines that lack marked locus contraction or distal VH rearrangements. Correspondingly, Wapl depletion in v-Abl lines activated VH-locus contraction and orientation-specific RAG-scanning across the VH-locus. Our findings indicate that locus contraction and physiological VH-to-DJH joining both are regulated via circumvention of CBE scanning impediments. Overall design: We performed ChIP-Seq in BM pro-B cells and v- Abl transformed pro-B cells and its various mutant derivatives to study orientation-dependent linear RAG scanning and locus contraction in long-range cohesin-driven V(D)J recombination.

免疫球蛋白重链基因座（Immunoglobulin heavy chain locus, Igh）的VH、D及JH基因片段会在发育进程中组装为V(D)J外显子。重组激活基因（Recombination Activating Gene, RAG）核酸内切酶通过结合基于染色质的重组中心（recombination center, RC）内的JH重组信号序列（recombination signal sequence, RSS）启动V(D)J重组，随后以依赖方向的机制，扫描由黏连蛋白（cohesin）介导的环挤压（loop extrusion）所呈递的上游含D染色质，搜寻会聚型D-RSS以启动DJH-RC的形成。在原代前体B（pro-B）细胞中，数以百计的上游VH相关RSS嵌入与DJH-RC-RSS呈会聚方向的区域，通过机制尚未明确的VH基因座收缩过程，得以靠近DJH-RC以完成VH-to-DJH重排。本研究报道，原代pro-B细胞中2.4兆碱基的VH基因座倒位几乎完全阻断了正常会聚型VH-RSS及隐蔽RSS（cryptic RSS）的重排，即便基因座收缩过程本身仍可维持。此外，该倒位激活了两类原本方向相反的隐蔽VH基因座RSS的重排，且出乎意料地激活了多个连续上游会聚型CBE结构域内、方向正常的隐蔽RSS的重排。相较于缺乏显著基因座收缩或远端VH重排的v-Abl转化pro-B细胞系，原代pro-B细胞中黏连蛋白卸载因子Wapl的转录水平显著降低。相应地，在v-Abl细胞系中敲低Wapl可激活VH基因座收缩，并实现全VH基因座上依赖方向的RAG扫描。我们的研究结果表明，基因座收缩与生理性VH-to-DJH重排均通过规避CBE扫描障碍得以调控。实验整体设计：我们在骨髓pro-B细胞、v-Abl转化的pro-B细胞及其多种突变衍生细胞系中开展了染色质免疫沉淀测序（ChIP-Seq），以研究长程黏连蛋白驱动的V(D)J重组中依赖方向的线性RAG扫描与基因座收缩过程。