Roles of chromatin structure in gene conversion at chicken immunoglobulin locus

染色质结构在鸡免疫球蛋白位点基因转化中的作用

• 批准号: 14580689
• 负责人: OHTA Kunihiro
• 金额: $ 2.3万
• 依托单位: RIKEN Discovery Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580689/
• 关键词: recombination; transcription; DT40; chromatin; histone; acetylation; antibody

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.

在鸡B细胞系DT 40中，基因转换（一种同源重组）主要有助于免疫球蛋白（IG）基因的多样化。我们研究了鸡DT 40细胞系中染色质结构在调控该基因转换中的作用。我们发现重组活性的IG基因座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DT 40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，但在重组活性和非重组活性等位基因中均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。完整的CFP序列结合诱导型泰特启动子、3'增强子和基质附着区序列，我们进一步研究了转录活性和基因转换频率之间的关系。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DT 40细胞中检测到产生GFP型荧光。此外，利用TSA对基因转化的影响，我们建立了一种快速、灵活的系统，利用活化的基因转化细胞生产特异性单克隆抗体。选择系统（ADLib：自主多样化文库）能够快速建立（从多样化文库开始一周）产生具有足够的特异性和亲和力用于免疫测定的单克隆IgM的各种克隆，并且适用于包括合理蛋白质设计的各种生物技术。

项目成果

Yamada T. et al.: "Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot"EMBO J.. 印刷中. (2004)

Yamada T. 等人：“组蛋白乙酰化和染色质重塑因子在减数分裂重组热点中的作用”EMBO J.. 已出版（2004 年）。

Rapid generation of specific antibodies by enhanced homologous recombination

• DOI: 10.1038/nbt1092
• 发表时间: 2005-06-01
• 期刊: NATURE BIOTECHNOLOGY
• 影响因子: 46.9
• 作者: Seo, H;Masuoka, M;Ohta, K
• 通讯作者: Ohta, K

Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot.

组蛋白乙酰化和染色质重塑因子在减数分裂重组热点中的作用。

• DOI: 10.1038/sj.emboj.7600138
• 发表时间: 2004
• 期刊: The EMBO journal
• 作者: Yamada,Takatomi;Mizuno,Ken-ichi;Hirota,Kouji;Kon,Ning;Wahls,WayneP;Hartsuiker,Edgar;Murofushi,Hiromu;Shibata,Takehiko;Ohta,Kunihiro
• 通讯作者: Ohta,Kunihiro

Fission yeast global repressors regulate the specificity of chromatin alteration in response to distinct environment stresses

裂殖酵母全局抑制因子调节染色质改变的特异性以响应不同的环境压力

• 发表时间: 2004
• 期刊: Nucleic Acids Research 32
• 作者: Seo H.et al.;Ymada T. et al.;Hirota K. et al.
• 通讯作者: Hirota K. et al.

Tomita K. et al.: "Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double-strand break, but not telomeres"Mol.Cell.Biol. 23. 5186-5197 (2003)

Tomita K. 等人：“Rad50 复合体和 Ku 异二聚体之间的竞争揭示了 Exo1 在处理双链断裂中的作用，但不是端粒”Mol.Cell.Biol。