Biochemistry and Regulation of V(D)J Recombination

抗原受体是由其组成基因片段通过一系列高度调控的位点组装而成的。 特定的DNA重组反应称为V(D)J重组。这项研究的总体目标是 该计划的目的是详细了解V(D)J调节的分子机制 两种淋巴细胞限制性蛋白RAG1和RAG2相互形成一个复合体，并且 识别重组基因片段两侧的重组信号序列(RSS)对。先把破布拿出来 点击DNA，然后引入dsDNA中断。然后，适当的断端通过 DNA修复蛋白的作用导致编码和信号关节的形成。重组酶的靶向性研究 与未重排基因片段的转录相关，并在RSS可及性水平上受到调节 在染色质结构中。重组酶靶向错误与染色体易位有关 这会导致淋巴系恶性肿瘤和V(D)J重组缺陷导致免疫功能障碍。我们建议 1)验证由远端生殖系k启动子定位的H3K4me3修饰染色质的梯度 决定了Igk基因座重组的效率，对等位基因排除是重要的；2)执行 逆转录病毒cdna文库筛选影响重组的染色质修饰物；3)使用新高 通过DNA测序技术扩展我们对单链DNA划痕和RSS末端插入的研究 V(D)J体内重组；4)详细研究关键转录因子E2a如何改变染色质 结构以促进免疫球蛋白K基因的重组；5)使用基因打靶来删除CTGF结合位点 在IgHC V和D基因片段之间的DNase I敏感区，以测试其是否调节V-to-D 重排；以及6)检测h5T)融合，上游启动子和下游启动子之间的相互作用 增强剂是通过将断裂的编码末端结合在一起来促进编码关节形成所必需的。V(D)J 利用生化系统对重组进行了广泛的研究。我们的方法是将已经发生的事情 在体外学习，并使用它来框架和测试关于体内重组调控的假说。

Antigen receptors are assembled from their component gene segments by a series of highly regulated site- specific DNA recombination reactions known as V(D)J recombination. The over-arching goal ofthis research program is to understand in molecular detail the mechanisms underlying the regulation ofthe V(D)J recombinase.Two lymphocyte restricted proteins, RAGl and RAG2 form a complex with one another and recognize pairs ofrecombination signal sequences (RSSs) that flank rearranging gene segments. The RAGs first nick the DNA then introduce dsDNA breaks. Appropriate broken ends are then joined to each other through the action of DNA repair proteins resulting in the formation ofcoding and signal joints. Targeting ofthe recombinase correlates with transcription ofthe unrearranged gene segments and is regulated at the level of RSS accessibility within chromatin structure. Errors in recombinase targeting are associated with chromosomal translocations that result in lymphoid malignancy and defects in V(D)J recombination result in immunodefiency. We propose to 1) test the idea that a gradient of H3K4me3 modified chromatin positioned by the distal germline k promoter determines the efficiency of Igk locus recombination and is important for allelic exclusion; 2) perform a retroviral cDNA library screen for chromatin modifiers that influence recombination; 3) use new high throughput DNA sequencing technologies to extend our studies ofssDNA nicking and RSS end insertion during V(D)J recombination in vivo; 4) examine in detail how a critical transcription factor, E2A, alters chromatin structure to promote recombination in the Igk locus; 5) use gene targeting to delete a CTGF binding site in a DNAse I sensitive region in between the IgHC V and D gene segments in order to test whether it regulates V-to-D rearrangement; and 6) test the h5T)othesis that the interaction between upstream promoters and downstream enhancers is necessary to promote coding joint formation by holding broken coding ends together. V(D)J recombination has been extensively studied using biochemical systems. Our approach is to take what has been learned in vitro and use it to frame and test hypotheses regarding the regulation of recombination in vivo.