Roles for Transcription in Antibody Diversity

免疫球蛋白开关MU区的重复DNA序列形成含有RNA的二级结构，并通过激活诱导脱氨酶(AID)发生超突变。为了研究DNA结构如何影响转录和超突变，我们将RNA聚合酶II分子的位置和跨越内含子增强子的5kb区域的突变映射到Constant Mu基因。对于RNA聚合酶II，用核连续法和染色质免疫沉淀法测定其在体外刺激的B细胞中的分布。在形成开关区核心的1kb重复序列两侧的DNA中发现了高密度的RNA聚合酶。我们认为，开关区域DNA结构的改变会暂停RNA聚合酶II。我们目前正在研究可变区的二级结构和转录模式，使用的是在两个等位基因上都包含一个重排可变基因的敲入小鼠。

Repetitive DNA sequences in the immunoglobulin switch mu region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5 kb region spanning the intronic enhancer to the constant mu gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1 kb repetitive sequence that forms the core of the switch region. We propose that altered DNA structure in the switch region pauses RNA polymerase II. We are currently examining secondary structure and transcription patterns in the variable region, using knock-in mice containing one rearranged variable gene on both alleles.