Roles of chromatin structure in gene conversion at chicken immunoglobulin locus

在鸡b细胞系DT40中，基因转换是一种同源重组，主要有助于免疫球蛋白（Ig）基因的多样化。我们在鸡DT40细胞系中研究了染色质结构在该基因转化调控中的作用。我们发现重组活性Ig位点表现出更高水平的组蛋白乙酰化。用组蛋白去乙酰化酶抑制剂Tricostatin A （TSA）处理DT40细胞可显著诱导重组活性等位基因的基因转换频率和转录活性，但在重组活性和非活性等位基因中均观察到组蛋白乙酰化水平升高，提示重组激活与转录活性之间存在联系。利用含有人工染色体重组标记的DT40细胞，包括部分GFP序列、完整的CFP序列与可诱导的Tet启动子、3′增强子和基质附着区序列，我们进一步研究了转录活性与基因转换频率的关系。转录激活后，诱导部分GFP和CFP序列之间的基因转换事件；从而在一小部分DT40细胞中检测到gfp型荧光。因此，我们认为基因转换可能与一些转录相关的机制耦合而激活。此外，利用TSA对基因转化的影响，我们开发了一种快速灵活的系统，利用激活基因转化的DT40细胞产生特异性单克隆抗体。该选择系统（ADLib: autonomous diversity Library）能够快速建立（从多样化文库中提取约一周）产生具有足够特异性和亲和力的单克隆igm，用于免疫学试验，适用于各种生物技术，包括合理的蛋白质设计。

In the chicken B-cell line DT40, gene conversion, a type of homologous recombination, primarily contributes to diversification of the immunoglobulin (Ig) gene. We have studied roles of chromatin structure in the regulation of this gene conversion in the chicken DT40 cell lines. We found that recombination-active Ig locus exhibits much higher levels of histone acetylation. Treatment of DT40 cells with histone deacetylase inhibitor Tricostatin A (TSA) markedly induced gene conversion frequency and transcriptional activity at recombination-active allele, but observed elevation of histone acetylation levels in both recombination-active and inactive-alleles, suggesting links between recombination activation and transcriotional activity.Using DT40 cells harboring an artificial chromosomal recombination marker including partial GFP sequence, complete CFP sequence combined with inducible Tet promoter, 3' enhancer, and a matrix attachment region sequence, we further examined relationship between transcriptional activity and gene conversion frequency. After transcriptional activation, gene conversion events between partial GFP and CFP sequences were induced; thereby generating GFP-type fluorescence was detected in a small portion of the DT40 cells. Thus, it is suggested that gene conversion may be activated coupled to some transcription-related mechanisms.In addition, using the effects by TSA on gene conversion, we developed a rapid and flexible system to produce specific monoclonal antibodies using the DT40 cells undergoing activated gene conversion. The selection system (ADLib: Autonomously Diversifying Library) enables quick establishment (〜one week from a diversifying library) of various clones producing monoclonal IgMs with enough specificity and affinity for immunological assays, and is applicable to various biotechnologies including rational protein design.