Mechanisms That Control Antigen Receptor Variable Region Exon Assembly

免疫球蛋白(Ig)重链基因座(IGH)VH、D和JH片段聚集在兆数据库上 (MB)小鼠免疫球蛋白轨迹的区域。它们通过V(D)J重组的组装在多个上下文中受到调控， 包括在VH至DJH加入之前按顺序D至JH加入，并对后一步骤进行调整以促进对 近端和远端VHS以推广多样化的曲目。VH-D基因间隔区的IGCR1控制区 通过两个CTCF结合元件(CBE)介导V(D)J重组调控 调节性染色质环。为了启动V(D)J重组，RAG1/2内切酶(RAG)裂解V(D)J 以互补重组信号序列(RSS)对为侧翼的基因片段 随后加入。RAG可以在Mb染色体环上定向追踪，这可能使它能够定位 适合切割活动的RSS对。RAG跟踪是通过我们的新LAM-HTGTS技术发现的， 它是如此敏感，以至于它揭示了大量看不见的低频RAG启动的切割/连接事件 之前的化验结果。我们已经将LAM-HTGTS改编为V(D)J谱系测序(“HTGTS-Rep-seq”)。我们 提出四个具体目标，旨在以这些发现和新技术为基础来阐明机制 这促进了免疫球蛋白V、D和J跨越Mb距离的连接，以产生不同的抗体库。我们 还建议将这些研究扩展到其他小鼠免疫球蛋白基因座和人类抗原受体基因座。许多 拟议的研究将使用G1抑制、RAG诱导的v-Abl转化的Pro-B细胞系作为测试系统， 但关键结果将通过对正常B系细胞的研究来证实或推广。目标1建议澄清 利用大的c-Myc基因座环结构域进行RAG示踪的机制和功能后果 测试系统以消除大量RSS和其他监管元素在 抗原受体基因座。拟议的研究将评估RAG跟踪与其他机制对 捕获距离较远的RSS对并阐明推动RAG跟踪的因素。目标2建议澄清 调节IgH D到JH重组的机制。在IGCR1中，D位于JHS的上游- 将它们与上游V隔离的从属区域。在这个结构域中，D到JH的重组始于一个 RAG结合的重组中心(RC)，包括JHS和最近端的D(DQ52)。我们将测试 假设RAG是通过DQ52和JH RSS与其他RSS的12个RSS配对而激活的 DS然后被其他机制捕获，可能包括跟踪。我们还建议定义独联体元素 RC队形所必需的。目标3建议阐明将远距离VHS带入V(D)J的机制 重组结构域，包括IGH CBE组织跨IGH基因座和潜力的独特方面 与VH相关的CBE的作用。这一目标还建议将研究扩展到Igκ轻链基因座。目标4 建议将目标2和目标3扩展到人类抗原受体基因座，这一点很重要，因为V(D)J 人类Ig或TCR基因座的重组调控机制尚未被直接研究。

Immunoglobulin (Ig) heavy chain locus (IgH) VH, D, and JH segments are clustered over megabase (Mb) regions of the mouse IgH locus. Their assembly via V(D)J recombination is regulated in multiple contexts, including ordered D to JH joining prior to VH to DJH joining, with the latter step regulated to promote utilization of proximal and distal VHs to promote diverse repertoires. The IGCR1 control region in VH to D inter-genic region mediates V(D)J recombination control via two CTCF-binding elements ("CBEs") proposed to mediate regulatory chromatin loops. To initiate V(D)J recombination, the RAG1/2 endonuclease (RAG) cleaves V(D)J gene segments flanked by complementary recombination signal sequence (RSS) pairs and mediates their subsequent joining. RAG can directionally track across Mb chromosomal loops, which could allow it to locate proper RSS pairs for cleavage activities. RAG tracking was discovered with our new LAM-HTGTS technique, which is so sensitive that it reveals multitudes of low frequency RAG-initiated cleavage/joining events invisible to prior assays. We have adapted the LAM-HTGTS for V(D)J repertoire sequencing (“HTGTS-Rep-seq”). We propose four specific aims designed to build on these findings and new technologies to elucidate mechanisms that promote joining of IgH Vs, Ds, and Js across Mb distances to generate diverse antibody repertoires. We also propose to extend these studies to other mouse Ig loci and to human antigen receptor loci. Many proposed studies will employ G1-arrested, RAG-inducible v-Abl transformed pro-B cell lines as a test system, but key results will be confirmed or extended by studies of normal B lineage cells. Aim 1 proposes to elucidate the mechanism and functional consequences of RAG tracking by using a large c-Myc locus loop domain as a test system to obviate potential confounding effects of numerous RSSs and other regulatory elements in antigen receptor loci. Proposed studies will evaluate contribution of RAG tracking versus other mechanisms for capture of distant RSS pairs and elucidate factors that propel RAG tracking. Aim 2 proposes to elucidate mechanisms that regulate IgH D to JH recombination. In IgH, Ds are located upstream of JHs within an IGCR1- dependent domain that isolates them from upstream Vs. In this domain, D to JH recombination initiates from a RAG-bound recombination center ("RC") comprising JHs and the most proximal D (DQ52). We will test the hypothesis that RAG is activated in the RC by pairing of DQ52 and JH RSSs, with convergent 12RSSs of other Ds then captured by other mechanisms, potentially including tracking. We also propose to define cis elements required for RC formation. Aim 3 proposes to elucidate mechanisms that bring distant VHs into V(D)J recombination domains, including unique aspects of IgH CBE organization across the IgH locus and potential roles of VH-associated CBEs. This aim also proposes to extend studies to the Igκ light chain locus. Aim 4 proposes to extend Aim 2 and 3 studies to human antigen receptor loci, which is important because V(D)J recombination control mechanisms of human Ig or TCR loci have not been studied directly.