Müller J R, Giese T, Henry D L, Mushinski J F, Marcu K B
Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Immunol. 1998 Aug 1;161(3):1354-62.
Ig heavy chain isotype switching in B lymphocytes is known to be preceded by transcription of a portion of the particular heavy chain gene segment that is targeted for recombination. Here, we describe an active role for these transcripts in the switch recombination process. Using an in vitro assay that exposes an artificial switch-mu (Smu) minisubstrate to switch region transcripts in the presence of nuclear extracts from switching cells, we demonstrate that free 3' ends of the Smu sequence are extended onto switch region transcripts by reverse transcription. The activity was induced in splenic B lymphocytes upon activation with LPS or CD40 ligand. This in vitro process is thought to be relevant to in vivo class switching for two reasons: 1) although only one-third of the Smu minisubstrate actually contains Smu sequence, all crossovers between switch regions occurred in the Smu portion; and 2) treatment of B lymphocytes with IL-4, which enriches for switching to S gamma 1, increases the ratio of Smu-S gamma 1 to Smu-S gamma 3 hybrids by 16% after LPS treatment and by 37% after CD40 ligand activation, implicating this S mu-primed reverse transcription of switch region transcripts as a novel mechanism of regulating the specificity of isotype switching. Further evidence for an active role of switch region transcripts was obtained by expressing S alpha RNA in trans in the Bcl1B1 B lymphoma line. Endogenous S mu-S alpha switch circles were detected in Bcl1B1 cells expressing exogenous S alpha RNA but not in mock-transfected cells.
已知B淋巴细胞中的Ig重链同种型转换之前，特定重链基因片段的一部分会进行转录，该片段是重组的目标。在此，我们描述了这些转录本在转换重组过程中的积极作用。使用一种体外测定法，在存在来自转换细胞的核提取物的情况下，将人工开关-μ（Smu）微型底物暴露于转换区域转录本，我们证明Smu序列的游离3'末端通过逆转录延伸到转换区域转录本上。用LPS或CD40配体激活后，脾B淋巴细胞中诱导了这种活性。该体外过程被认为与体内类别转换相关，原因有两个：1）尽管只有三分之一的Smu微型底物实际包含Smu序列，但转换区域之间的所有交叉都发生在Smu部分；2）用IL-4处理B淋巴细胞，其有利于转换为Sγ1，在LPS处理后，Smu-Sγ1与Smu-Sγ3杂种的比例增加16%，在CD40配体激活后增加37%，这表明这种由Sμ引发的转换区域转录本的逆转录是调节同种型转换特异性的一种新机制。通过在Bcl1B1 B淋巴瘤细胞系中反式表达SαRNA，获得了转换区域转录本发挥积极作用的进一步证据。在表达外源性SαRNA的Bcl1B1细胞中检测到内源性Sμ-Sα转换环，但在mock转染的细胞中未检测到。
