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Regulation and Molecular Mechanisms of Antigen Receptor Gene Rearrangements

抗原受体基因重排的调控及分子机制

基本信息

批准号：
07101001
负责人：
SAKANO Hitoshi
金额：
$ 159.36万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Specially Promoted Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1999
项目状态：
已结题

项目摘要

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the recombination-activating genes (RAGs). Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin homeodomain of the RAG1 protein and adjacent major and minor grooves of the 9mer DNA.When two RSSs are cleaved in V(D)J joining, the signal-end (SE) complex is formed with RAG1 and RAG2 proteins in the presence of DNA bending protein HMG1. Using magnetic beads, we have purified the SE complex and analyzed the SE DNA by DNaseI footprinting. It was found that the 7mer rather than the 9mer plays a primary role in the SE complex formation. It was also found that the 3'-OH of the 7mer is phosphorylated in the SE complex in both 12- and 23-RSS's after the cleavage. The 3'-phosphate is transferred from carrier DNA or RNA by trans-esterification after nicking of the carrier. In vitro assays demonstrated that the 3'-phosphorylation prevents the transposition of the SE complex. These results indicate that the 3'-phosphorylation may have an active role in preventing harmful transposition of an excision product of V(D)J joining.We have shown that the PU.1 binding motif (GAGGAA) in the 3'-enhancer of the IgィイD2κィエD2 gene is responsible for the negative regulation of VィイD2κィエD2-JィイD2κィエD2 joining. In the transcriptional activation, the PU.1 motif acts in a positive manner cooperatively with the PIP and BSAP motifs, which are located in the same enhancer core region. Interestingly, base substitutions in either the PIP or the BSAP motif did not affect the B/T or pro-B/pre-B specificity of VィイD2κィエD2-JィイD2κィエD2 joining. Thus, the PU.1 motif alone appears to regulate both temporal and tissue-specific rearrangements of the IgィイD2κィエD2 gene.

我们研究了重组信号序列（RSSs）与重组激活基因（rag）蛋白产物之间的相互作用。将RAG-RSS足迹数据与已发表的Hin模型进行比较，证实了序列特异性RSS-RAG相互作用主要发生在RAG1蛋白的Hin同源结构域和相邻的9mer DNA的主凹槽和小凹槽之间。当两个rss在V(D)J连接中被切割时，在DNA弯曲蛋白HMG1的存在下，RAG1和RAG2蛋白形成信号端（SE）复合物。我们利用磁珠纯化了SE复合物，并通过DNaseI足迹分析了SE DNA。结果表明，7mer而不是9mer在SE复合物的形成中起主要作用。还发现7mer的3'- oh在12-和23-RSS的SE复合物中被磷酸化。3'-磷酸通过反式酯化反应从载体DNA或RNA上转移。体外实验表明，3'-磷酸化可阻止SE复合物的转位。这些结果表明，3'-磷酸化可能在防止V(D)J连接的切除产物的有害转位中起积极作用。我们已经发现Ig γ γ - κ γ - D2基因3'-增强子中的PU.1结合基序（GAGGAA）负责负向调控V γ - κ γ - j γ - κ γ - D2连接。在转录激活中，PU.1基序与位于同一增强子核心区的PIP和BSAP基序积极合作。有趣的是，无论是PIP基序还是BSAP基序的碱基替换，都不会影响到V′′κ′- D2- j′′κ′- D2连接的B/T或pro-B/pre-B特异性。因此，单独的PU.1基序似乎可以调节Ig γ γ κ γ γ D2基因的时间和组织特异性重排。