Hempel W M, Stanhope-Baker P, Mathieu N, Huang F, Schlissel M S, Ferrier P
Centre d'Immunologie Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Marseille-Luminy, Marseille Cedex 9, France.
Genes Dev. 1998 Aug 1;12(15):2305-17. doi: 10.1101/gad.12.15.2305.
Deletion of the TCRbeta transcriptional enhancer (Ebeta) results in nearly complete inhibition of V(D)J recombination at the TCRbeta 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 Ebeta-deleted (Ebeta-/-) mice using assays that detect various reaction intermediates and products. We found double-strand DNA breaks at recombination signal sequences flanking Dbeta and Jbeta gene segments in Ebeta-/- thymuses at about one-third to one-thirtieth the level found in thymuses with an unaltered TCRbeta locus. These sites are also subject to in vitro cleavage by the V(D)J recombinase in both Ebeta-/- and Ebeta+/+ thymocyte nuclei. However, the corresponding Dbeta-to-Jbeta coding joints are further reduced (by 100- to 300-fold) in Ebeta-/- thymuses. Formation of extrachromosomal Dbeta-to-Jbeta signal joints appears to be intermediately affected and nonstandard Dbeta-to-Dbeta joining occurs at the Ebeta-deleted alleles. These data indicate that, unexpectedly, loss of accessibility alone cannot explain the loss of TCRbeta recombination in the absence of the Ebeta element and suggest an additional function for Ebeta in the process of DNA repair at specific TCRbeta sites during the late phase of the recombination reaction.
TCRβ转录增强子（Eβ）的缺失导致TCRβ基因座处的V（D）J重组几乎完全受到抑制，并阻碍αβT细胞的发育。这一结果，连同许多实验室之前的研究工作，引发了这样一种假说，即转录增强子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，我们通过使用检测各种反应中间体和产物的实验，对Eβ缺失（Eβ-/-）小鼠的重组缺陷进行详细分析，来检验这一假说。我们发现，在Eβ-/-胸腺中，Dβ和Jβ基因片段侧翼的重组信号序列处的双链DNA断裂，其水平约为TCRβ基因座未改变的胸腺中的三分之一至三十分之一。在Eβ-/-和Eβ+/+胸腺细胞核中，这些位点也会受到V（D）J重组酶的体外切割。然而，在Eβ-/-胸腺中，相应的Dβ到Jβ编码接头进一步减少（减少100至300倍）。染色体外Dβ到Jβ信号接头的形成似乎受到中等程度的影响，并且在Eβ缺失的等位基因处发生非标准的Dβ到Dβ连接。这些数据表明，出乎意料的是，仅可及性的丧失并不能解释在没有Eβ元件的情况下TCRβ重组的丧失，并暗示Eβ在重组反应后期特定TCRβ位点的DNA修复过程中具有额外功能。
