Analysis of enhancer/silencer regions of TCR gene

TCR基因增强子/沉默子区域分析

• 批准号: 05670293
• 负责人: KANNO Masamoto
• 金额: $ 1.34万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670293/
• 关键词: TCR; ets; enhancer; T細胞抗原受容体; 転写因子; ets遺伝子

It has been known that TCR-alpha enhancer is subdivided to Talpha1-4. In our recent study we demonstrate several major developments : (1) Talpha2 region is most critical to exert enhancer function. This Talpha2 region has reported to have a binding sites for TCF-1/LEF-1, Talpha2B and Ets-1. (2) By our in vivo analysis, 5'-CATCCTC-3' sequence is very important for enhancer function, which is overlapped with reported Ets-1 binding site. Moreover, by in vitro analysis, we identified at least three different Ets-1 related proteins are interacting with this Talpha2 region and, especially, one of them seems to have Ets-1 like binding specificity to 5'-ATCC-3' sequence. However none of these proteins seems to have a character of reported Ets-1 nor reactivity to anti-Est-1 and anti-Ets-2 monoclonal antibodies by gel-shift assay. Our present dats is the first case to demonstrate the binding of several endogenous (natural) Ets-1 family proteins to the Talpha2 region.To determin the molecular weigth of the proteins, we performed UV-cross link experoment. It indicates novel 10kDa protein is binding to Talpha2 ets site with weakly associated 31kDa protein. By 3'-RACE RT-PCR,we islated a partial cDNA fragment with degenerated ets domain oligonucleotides. This cDNA frament sequence reveal that conserved ets domain with novel c-terminal half of new protein. We are currently trying to isolate full-length cDNA.

众所周知，TCR-Alpha增强子被细分为TALPHA1-4。在我们最近的研究中，我们证明了几个主要发展：（1）TALPHA2区域对于发挥增强子功能最为重要。据报道，该TALPHA2区具有用于TCF-1/LEF-1，TALPHA2B和ETS-1的结合位点。 （2）通过我们的体内分析，5'-catcctc-3'序列对于增强子函数非常重要，增强子功能与报告的ETS-1结合位点重叠。此外，通过体外分析，我们发现至少三种不同的ETS-1相关蛋白与该TALPHA2区域相互作用，尤其是其中一个似乎具有与5'-ATCC-3'序列的结合特异性一样。然而，这些蛋白质似乎都没有通过凝胶偏移测定法报告的ETS-1和反应性对抗1和抗EST-1和抗ETS-2单克隆抗体的反应。我们目前的DAT是第一个证明几种内源性（天然）ETS-1家族蛋白与TALPHA2区域的结合。为了确定蛋白质的分子元素，我们进行了UV-Cross Link经验。它表明新型10KDA蛋白与具有弱相关的31KDA蛋白的TALPHA2 ETS位点结合。通过3'-RACE RT-PCR，我们将部分cDNA片段与退化的ETS结构域寡核苷酸相解相关。该cDNA框序列表明，新蛋白质的新型C末端一半保守的ETS结构域。我们目前正在尝试隔离全长cDNA。

项目成果

Asano,H.: "The mel-18 RING-Filnger gene,genomic organization,promote analysis and Chromosome assignment." DNA Sequence.3. 369-377 (1993)

Asano, H.：“mel-18 RING-Filnger 基因、基因组组织、促进分析和染色体分配。”

Akasaka, T., Yamada, T., Koseki, H., Taniguchi, M.and Kanno, M.: "A novel ets-related 10kDa protein is responsible for the TCR alpha-chain enhancer activity but not ets-1 and ets-2." Int.Immunol.(Submitted.).

Akasaka, T.、Yamada, T.、Koseki, H.、Taniguchi, M. 和 Kanno, M.：“一种新型 ets 相关 10kDa 蛋白负责 TCR α 链增强子活性，但不是 ets-1 和 ets

Knno.M.: "mel-18,a Polycomb-related mammalian gene,encoded transcriptional negative regulator with tumor susppressive activity." ENBO J.(in press).

Knno.M.：“mel-18，一种与 Polycomb 相关的哺乳动物基因，编码具有肿瘤抑制活性的转录负调节因子。”

Akasaka,T.: "A novel ets-releted 10KDa protein is responsible for the TCRα chain enhancer activity but not ets-1 and ets-2" Int.Immunol.(in press).

Akasaka, T.：“一种新型 ets 相关 10KDa 蛋白负责 TCRα 链增强子活性，但 ets-1 和 ets-2 不负责”Int.Immunol。

Akasaka,T.: "A novel ets-related 10KDa protein is resposble for the TCRαchain enhuncer activity but not ets-1 and ets/2" Int.Immunol.(In press).

Akasaka, T.：“一种新型 ets 相关 10KDa 蛋白负责 TCRα 链增强子活性，但不负责 ets-1 和 ets/2”Int.Immunol。（正在出版）。