Mechanisms regulating the target specificity of gene rearrangements in immune system

主要进行了Ig H链类开关重组实验，其中每个基因的区域染色质状态和特异性调控能够在体外重现，以及Ig类开关重组所必需的AID基因表达调控。结果表明，靶染色质区域为重组打开，使用该区域组蛋白乙酰化水平作为染色质状态的一般指标。通过加入HDAC抑制剂和种系转录抑制信号，可以维持靶区染色质的超乙酰化状态。即使目标染色质在这样的距离上保持开放，也不能使其成为重组的目标。这一结果表明，在重组之前发生的种系转录不仅仅是染色质打开的结果，而是直接参与了这一过程，因此对使基因成为靶基因至关重要。此外，还获得了B淋巴细胞在AID基因表达激活过程中释放特异性转录抑制的结果。此外，发现含有CpG的寡核苷酸阵列通过抑制种系转录来抑制IgG1和IgE的类开关重组。通过敲除可能参与这种抑制的基因的小鼠的B淋巴细胞来分析其分子机制。结果表明，分别抑制E2A和STAT6的Id2和Bcl6不参与这一过程，而是通过NFκB和IRF4信号介导的。

Experiments on the class switch recombination of Ig H chain, in which the regional chromatin status and specific regulation of each gene are able to be reproduced in vitro, and on the regulation of AID gene expression, which is essential for Ig class switch recombination were mainly done. It is shown that the chromatin region of target is opened for the recombination using the level of histone acetylation at the region as the general indicator of chromatin status.By adding HDAC inhibitor together with the repression signal for the germline transcription, the hyper-acetylation status of the target region chromatin could be maintained. Even though the target chromatin was kept open in such away, it was not able to make it the target for the recombination. This result indicates that the germline transcription occurring prior to the recombination is not simply the result of the chromatin opening but it directly involved in the process and therefore essential to make the gene the target. Moreover, results suggesting that the release from specific transcriptional repression occurs in B lymphocyte during the activation of AID gene expression were also obtained.In addition, the oligonucleotide containing CpG array was found to repress the class switch recombination to IgG1 and IgE through the repression of germline transcription. The molecular mechanism for this was analyzed by using B lymphocytes from knockout mice of the genes that might be involved in this repression. The results indicate that Id2 and Bcl6 that repress E2A and STAT6 respectively are not involved in this process, but it is mediated by the signals through NFκB and IRF4.