Enhancer control of V(D)J recombination at the TCRβ locus:: differential effects on DNA cleavage and joining
Enhancer control of V(D)J recombination at the TCRβ locus:: differential effects on DNA cleavage and joining
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DOI：
10.1101/gad.12.15.2305
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发表时间：
1998-08-01
影响因子：
10.5
通讯作者：
Ferrier, P
中科院分区：
文献类型：
--
作者：
Hempel, WM;Stanhope-Baker, P;Ferrier, P
TCRβ转录增强子(EP)的缺失几乎完全抑制了TCRβ基因上的V(D)J重组，阻碍了αβT细胞的发育。这一结果与许多实验室以前的工作一起，导致了一种假设，即转录增强剂通过调节基因座对重组酶的可及性来影响V(D)J重组。在这里，我们通过对Eβ缺失(E beta(-/-))小鼠的重组缺陷进行详细分析来检验这一假设，方法是检测各种反应中间产物和产物。我们在Eβ(-/-)胸腺的Dβ和Jβ基因片段两侧的重组信号序列上发现了双链DNA断裂，其水平大约是胸腺的三分之一到三十分之一，而TCRβ基因座没有改变。在Eβ(-/-)和Eβ(+/+)胸腺细胞核中，这些位点也可被V(D)J重组酶体外切割。然而，在Eβ(-/-)胸腺中，相应的Dβ-Jβ编码关节进一步减少(100-300倍)。染色体外Dβ到Jβ信号关节的形成似乎受到了中间影响，EP缺失的等位基因发生了非标准的DP到DP连接。这些数据表明，意想不到的是，仅凭可及性的丧失不能解释缺乏EP元件时TCRβ重组的丧失，并表明EP在重组反应后期特定TCRβ位点的DNA修复过程中具有额外的功能。
Deletion of the TCR beta transcriptional enhancer (EP) results in nearly complete inhibition of V(D)J recombination at the TCR beta locus and a block in alpha beta T cell development. This result, along with previous work from many laboratories, has led to the hypothesis that transcriptional enhancers affect V(D)J recombination by regulating the accessibility of the locus to the recombinase. Here we test this hypothesis by performing a detailed analysis of the recombination defect in E beta-deleted (E beta(-/-)) mice using assays that detect various reaction intermediates and products. We found double-strand DNA breaks at recombination signal sequences flanking D beta and J beta gene segments in E beta(-/-) thymuses at about one-third to one-thirtieth the level found in thymuses with an unaltered TCR beta locus. These sites are also subject to in vitro cleavage by the v(D)J recombinase in both E beta(-/-) and E beta(+/+) thymocyte nuclei. However, the corresponding D beta-to-J beta coding joints are further reduced (by 100- to 300-fold) in E beta(-/-) thymuses. Formation of extrachromosomal D beta-to-J beta signal joints appears to be intermediately affected and nonstandard DP-to-DP joining occurs at the Ep-deleted alleles. These data indicate that, unexpectedly, loss of accessibility alone cannot explain the loss of TCR beta recombination in the absence of the EP element and suggest anadditional function for EP in the process of DNA repair at specific TCR beta sites during the late phase of the recombination reaction.