Contribution of IGCR1 and 3' CBE Super Anchor to Developmental Regulation of Igh V(D)J Recombination [HTGTS_VDJ_Seq]. Contribution of IGCR1 and 3' CBE Super Anchor to Developmental Regulation of Igh V(D)J Recombination [HTGTS_VDJ_Seq]

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease orchestrates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJHRC, from which RAG scans upstream for VHs to assemble VHDJH exons in an ordered recombination process. Cohesin-mediated chromatin loop extrusion is impeded by CTCF-bound elements (CBEs), with extrusion between two CBEs, particularly if convergently-oriented, forming chromatin-contact loops genome-wide. Igh has provocative CBE organization, with two divergently-oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, dozens of VH domain CBEs convergent to CBE1, and 10 3'Igh-CBEs convergent to CBE2. IGCR1 CBEs segregate D/JH and VH domains by impeding RAG-scanning. After DJH formation, down-regulation of WAPL, a cohesin unloader, neutralizes IGCR1 and VH-domain CBEs, allowing RAG to scan the VH domain. To elucidate potential mechanisms underlying the developmental transition from D-to-JH to VH-to-DJH recombination and potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning, we tested effects of deleting or inverting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments its RAG-scanning impediment activity and that the 3'Igh-CBEs reinforce RC activity as a dynamic loop extrusion impediment, thereby, increasing its ability to support V(D)J recombination. Finally, our findings implicate a mechanism by which developmental WAPL down-regulation contributes to ordered V(D)J recombination. Overall design: We performed HTGTS-VDJ-Seq to determine the roles of IGCR1 and 3'Igh CBEs in v-abl cells, pro-B cells.

免疫球蛋白重链可变区外显子在祖B细胞（progenitor-B cells）中组装，其组装模板为分布于Igh基因座不同簇内的VH、D及JH基因片段。RAG核酸内切酶（RAG endonuclease）依托基于JH的重组中心（RC）调控V(D)J重组进程。黏连蛋白（Cohesin）介导的上游染色质环挤出过程，可使结合于重组中心的RAG接触到D片段，使其与JH片段结合形成DJH重组中心（DJHRC）；随后RAG可通过该中心向上游扫描VH片段，以有序重组的方式组装VHDJH外显子。
黏连蛋白介导的染色质环挤出会被CTCF结合元件（CBEs）阻碍；当两个CTCF结合元件之间发生环挤出时，若二者呈收敛定向，更易在全基因组范围内形成染色质接触环。Igh基因座的CTCF结合元件排布颇具特点：在VH结构域与D/JH结构域之间的IGCR1元件内，存在两个呈发散定向的CTCF结合元件（CBE1与CBE2）；VH结构域内存在大量与CBE1呈收敛定向的CTCF结合元件；另有10个3'端Igh-CTCF结合元件（3'Igh-CBEs）与CBE2呈收敛定向。IGCR1的CTCF结合元件可通过阻碍RAG扫描，实现D/JH结构域与VH结构域的物理分隔。
在DJH结构形成后，黏连蛋白卸载因子WAPL的表达下调会中和IGCR1及VH结构域CTCF结合元件的阻碍活性，使RAG得以扫描VH结构域。为阐明从D-JH重组向VH-DJH重组发育转变的潜在机制，以及基于IGCR1的CTCF结合元件与3'Igh-CBEs在调控RAG扫描中的潜在作用，我们在小鼠及/或祖B细胞系中对IGCR1或3'Igh-CBEs进行了敲除或倒置操作，并检测其相关效应。
本研究发现，正常的IGCR1 CBE定向能够增强其对RAG扫描的阻碍活性；3'Igh-CBEs可作为动态的环挤出阻碍物强化重组中心的活性，进而提升其支持V(D)J重组的能力。最后，我们的研究结果揭示了发育过程中WAPL表达下调促进有序V(D)J重组的潜在机制。
整体实验设计：我们在v-abl转化细胞与前B细胞（pro-B cells）中开展了HTGTS-VDJ测序（HTGTS-VDJ-Seq），以此明确IGCR1与3'Igh CBEs的功能作用。