重组激活基因蛋白造成B细胞非抗原受体基因位点发生转位的机制研究

The lymphocytes can produce a highly diverse repertoire of antigen receptors through V(D)J recombination by using the lymphocyte specific proteins（RAGs）encoded by the recombination activating gene. RAGs bind and cleave the DNA at the recombination signal sequences (RSSs) that flank each antigen receptor gene segment. The mouse and human genomes contain millions of cryptic RSS（cRSS） sequences， which might cause chromosomal deletions and translocations by RAGs potential binding and cleaveage. Based on this issue, We will purify pre-B cells from the mice which express RAGs, establish the genome-wide binding for RAG targeting by using RAG ChIP-seq assay, and select the nonantigen receptor genes which could be bound by RAGs. We will clone the vectors which include the nonantigen receptor genes targeted by RAGs, and will show whether the RAGs have the abilities to cleave them or not. We will figure out which regions in RAGs are important for RAG binding to nonantigen receptor loci. We will indicate that abnormal V(D)J recombination by RAG overexpression and so on will cause the chromosomal translocations between antigen receptor loci and nonantigen receptor loci (oncogenes).All these results not only will elucidate the mechanisms for RAG targeting to the nonantigen receptor loci, but also will provide the reasons why lymphoid neoplasms occur.

淋巴细胞通过重组激活基因蛋白（recombination activating gene，RAG）特异性结合并断裂抗原受体基因片段旁的重组信号序列（recombination signal sequences, RSSs）实现V(D)J的重组，产生多样性的抗原受体基因。基因组含有大量类似RSS（cRSS）序列，RAG对cRSS序列的结合和断裂可能造成了基因的丢失和转位。为此，本研究从小鼠分离出表达RAG的pre-B细胞，运用RAG ChIP-seq技术筛选出基因组中RAG结合的非抗原受体基因，构建含有上述基因载体并检测RAG对这些基因的断裂作用。研究不同结构的RAG结合非抗原受体基因位点的机制，阐明RAG过度表达等异常V(D)J重组是造成免疫球蛋白基因与非抗原受体基因（原癌基因）转位的原因。该研究不仅发现了RAG诱导非抗原受体基因位点发生转位的机制，而且为淋巴细胞肿瘤的发生提供了理论依据。

早期发育的T或B淋巴细胞通过重组激活基因蛋白（recombination activating gene，RAG）特异性结合并断裂抗原受体基因片段旁的重组信号序列（recombination signal sequences, RSSs）实现抗原受体基因重排，产生多样性的抗原受体基因。但是基因组含有大量类似RSS（cRSS）序列，RAG对cRSS序列的结合和断裂可能造成了基因的丢失和转位。为此，本研究从小鼠分离出表达RAG的pre-B细胞，运用RAG ChIP-seq技术筛选出基因组中RAG结合的1500个非抗原受体基因，分析这些基因的cRss序列发现，它们具有RAG的结合位点但没有断裂位点。我们提出基因组cRss的结构对基因组的稳定性起着保护作用。IgH基因重组是一个连续的过程，先发生DH到JH的重组，再发生VH到DJH的重组。由于VH基因数量众多、VH基因片段与DJH连接体距离较远以及缺乏合适的研究模型，VH到DJH重组的机理尚未完全阐明。我们发现RAG对IgH的VH基因的结合机制为在DH到JH重组后，DJH连接体这种结构有助于VH基因发生高水平转录，基因活化的H3k4me3和H3K27ac组蛋白修饰，RAG方有机会结合到VH。完成VH到DJH重组。DNA甲基化作为表观遗传学的重要标志，在Igk重组和Igk等位基因排斥中起作用。我们的研究成果提示了RAG2的350-383可以调节pre-B细胞阶段的Igk基因位点发生DNA去甲基化，但突变了RAG2的350-383区域维持Igk基因位点DNA甲基化但并不影响Igk重组。提示DNA去甲基化可能不影响基因重排但对Igk等位基因排斥中起作用，从而保持免疫球蛋白分子的特异性和基因组的稳定性。