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片段聚集在大片段上 (Mb)小鼠IgH基因座的区域。它们通过V（D）J重组的组装在多种情况下受到调节， 包括在VH至DJH连接之前有序地D至JH连接，调节后一步骤以促进利用 近端和远端VH以促进不同的库。IGCR 1控制区位于VH至D基因间区 通过两个CTCF结合元件（“CBE”）介导V（D）J重组控制，提出了介导 调节染色质环。为了启动V（D）J重组，RAG 1/2内切核酸酶（RAG）切割V（D）J 基因片段侧翼的互补重组信号序列（RSS）对，并介导其 随后加入。RAG可以定向跟踪Mb染色体环，这可以使它定位 用于切割活动的适当RSS对。RAG跟踪是用我们新的LAM-HTGTS技术发现的， 它是如此敏感，它揭示了大量的低频率RAG引发的切割/连接事件不可见 之前的检测我们已经将LAM-HTGTS用于V（D）J库测序（“HTGTS-Rep-seq”）。我们 提出四个具体目标，旨在利用这些发现和新技术阐明机制 其促进跨越Mb距离的IgH Vs、Ds和J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IG基因座和人抗原受体基因座。许多 建议的研究将使用G1停滞的、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谱系细胞的研究来证实或扩展。目标1建议阐明 通过使用大的c-Myc基因座环结构域作为RAG跟踪的机制和功能后果， 一种测试系统，用于排除许多RSS和其他调控因素的潜在混杂效应， 抗原受体基因座拟议的研究将评估RAG跟踪与其他机制相比对以下方面的贡献： 捕获遥远的RSS对，并阐明推动RAG跟踪的因素。目标2建议阐明 调节IgH D到JH重组的机制。在IgH中，Ds位于IGCR 1 - 1中JH的上游。 在这个结构域中，D到JH的重组起始于一个 包含JH和最近端D（DQ 52）的RAG结合的重组中心（“RC”）。我们将测试 假设RAG在RC中通过DQ 52和JH RSS配对而被激活，其他RSS的会聚12 RSS 然后通过其他机制捕获Ds，可能包括跟踪。我们还建议定义顺式元件 需要RC的格式。目的3提出阐明将远距离VH带入V（D）J的机制 重组结构域，包括跨IgH基因座的IgH CBE组织的独特方面和潜在的 Vh相关CBE的作用。该目的还建议将研究扩展到IGκ轻链位点。目标4 建议将目标2和3的研究扩展到人类抗原受体位点，这很重要，因为V（D）J 人IG或TCR基因座的重组控制机制尚未直接研究。

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.