Mechanisms That Control Antigen Receptor Variable Region Exon Assembly

7.项目摘要/摘要。 在过去的资助期间，我们发现粘附素介导的环状挤出过程 参与染色体构型的全基因组调控在V(D)J中起基础作用 重组和抗体多样性的产生。在发育祖细胞B细胞的过程中，我们发现 粘附素介导的环挤出将免疫球蛋白重链基因(IgH)VH、D和JH片段线性呈现给 V(D)J重组的RAG内切酶。我们的初步数据表明，长程V(D)J 至少部分地，免疫球蛋白轻链基因座中的重组可能是通过与 那就是伊格的。我们提出了两个特定的目标来阐明IgH和IgK的潜在差异机制。 长程V(D)J复合。指导目标1和AIM2研究的一个主要假设是 范围V(D)J通过线性RAG染色质扫描重组，导致主要缺失 重组事件。目标2需要检验的一个主要假设是，Igk在结构上进行了优化，可以使用 一种相关的基于环挤出的机制，同时适应健壮的删除和倒置的VK-to-VK JK加盟。这些假设得到了大量已发表的初步数据的支持，这些数据大部分来自于 来自我们在当前资助期内开发的强大的新技术。尤其是我们 建立了检测V(D)J重组的LAM-HTGTS-V(D)J-Seq，具有前所未有的灵敏度和LAM-3C- HTGTS以比以前的分析高得多的分辨率映射染色质结构域之间的序列相互作用。我们 进一步发展了G1期滞留、RAG可诱导的v-Abl转化的前B细胞(“v-Abl细胞”)检测方法 通过介绍特定顺式元件或反式作用因子在远程RAG染色质扫描中的作用 Ig H或Ig k基因座修饰和/或靶向蛋白缺失。AIMS 1和2一起实验将会 深入比较和对比IGH和IGK用于将VS合并到 V(D)J复合反应。虽然大多数最初的目标1和2研究将使用v-Abl细胞；但所有关键结果都将 通过对正常祖细胞和前体B细胞群体的研究来证实/扩展这些研究可以 揭示理解体内V(D)J重组的新范式并阐明一系列机制 用于抗原受体基因座上的长距离V(D)J重组。解决目标1和目标2应 通过提供对基本机制的重大新见解，极大地影响了免疫学领域 建立高度多样化的一级抗体库。目标1和目标2中提议的研究也将进一步 阐明扫描路径中的障碍如何聚焦染色质受阻区域内的隐蔽RSS靶点 用于并入RAG复合体和随后的重排。因此，这些研究应该提供 了解RAG如何针对其他基因中的隐蔽RSS并促进共同的关键信息 进展期B、T淋巴癌中常见的移位或间质缺失 人体内的淋巴细胞。

7. Project Summary/Abstract. During the past funding period, we discovered that the cohesin-mediated loop extrusion process involved in genome-wide modulation of chromosome architecture plays fundamental roles in V(D)J recombination and the generation of antibody diversity. In developing progenitor B cells, we found that cohesin-mediated loop extrusion linearly presents Ig heavy chain locus (Igh) VH, D, and JH gene segments to the RAG endonuclease for V(D)J recombination. Our preliminary data indicate that long-range V(D)J recombination in the Igk light chain locus may occur, at least in part, by a mechanistically distinct process from that of Igh. We propose 2 specific aims to elucidate the potentially differential mechanisms of Igh versus Igk long-range V(D)J recombination. A major hypothesis guiding Aim 1 and Aim2 studies is that Igh achieves long range V(D)J recombination via linear RAG chromatin scanning that leads to predominantly deletional recombination events. A major hypothesis to be tested for Aim 2 is that Igk is structurally-optimized to employ a related loop extrusion-based mechanism that accommodates both robust deletional and inverted Vk-to- Jk joining. These hypotheses are supported by a wealth of published and preliminary data derived in large part from powerful new technologies that we developed during the current funding period. In particular we developed LAM-HTGTS-V(D)J-Seq to assay V(D)J recombination with unprecedented sensitivity and LAM-3C- HTGTS to map sequence interactions across chromatin domains at far higher resolution than prior assays. We further developed G1-arrested, RAG inducible v-Abl transformed pro-B cell cell ("v-Abl cell") approaches to test roles specific cis elements or trans-acting factors in long-range RAG chromatin-scanning through introduced Igh or Igk locus modifications and/or targeted protein depletion. Aims 1 and 2 experiments together will compare and contrast, in depth, the long-range mechanisms used by Igh and Igk to incorporate Vs into the V(D)J recombination reaction. While most initial Aim 1 and 2 studies will employ v-Abl cells; all key results will be confirmed/extended by studies of normal progenitor and precursor B cell populations These studies may reveal new paradigms for understanding V(D)J recombination in vivo and illuminate the range of mechanisms employed for long-range V(D)J recombination in antigen receptor loci. Addressing Aim 1 and 2 goals should greatly impact the immunology field by providing major new insights into fundamental mechanisms that establish highly diverse primary antibody repertoires. The studies proposed in Aim 1 and 2 will also further elucidate how impediments in the scanning path focus cryptic RSS targets within impeded areas of chromatin for incorporation into the RAG complex and subsequent rearrangement. Thus, these studies should provide critical information for understanding how RAG targets cryptic RSSs in other genes and promotes common translocations or interstitial deletions frequently found in B and T lymphocyte cancers of developing lymphocytes in humans.