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，主要是导致免疫球蛋白(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。我们发现具有重组活性的Ig基因座表现出更高的组蛋白乙酰化水平。用组蛋白去乙酰酶抑制剂Tricostatin A(TSA)处理DT40细胞，显著诱导基因转换频率和重组活性等位基因的转录活性，但在重组活性等位基因和非活性等位基因中观察到组蛋白乙酰化水平的升高，提示重组激活和转录活性之间存在联系。在转录激活后，部分GFP和CFP序列之间的基因转换事件被诱导，从而在DT40细胞的一小部分中检测到GFP类型的荧光。此外，利用TSA对基因转换的影响，我们开发了一种快速而灵活的系统，利用经历了激活的基因转换的DT40细胞来制备特异性的单抗。该选择系统(adlib：自主多样化文库)可以快速(从多样化文库到一周)建立产生具有足够特异性和亲和力的单抗IgM的各种克隆，并适用于包括合理蛋白质设计在内的各种生物技术。

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.