Analysis of enhancer/silencer regions of TCR gene

已知TCR-α增强子被细分为Talpha1-4。在我们最近的研究中，我们展示了几个主要的进展：(1)Talpha2区域是发挥增强子功能最关键的区域。据报道，该Talpha2区域具有TCF-1/Lef-1、Talpha2B和ETS-1的结合位点。(2)经体内分析，5‘-CATCCTC-3’序列与已报道的Ets-1结合位点重叠，对增强子功能具有重要作用。此外，通过体外分析，我们发现至少有三个不同的Ets-1相关蛋白与该Talpha2区域相互作用，其中一个似乎与5‘-ATCC-3’序列具有类似Ets-1的结合特异性。然而，这些蛋白似乎都不具有已报道的Ets-1的特性，也不能与抗Est-1和抗Ets-2的单抗发生凝胶偏移反应。我们目前的数据是第一个证明了几个内源性(天然的)ETS-1家族蛋白与Talpha2区域结合的案例。为了确定这些蛋白的分子质量，我们进行了紫外光交联实验。这表明新的10 kDa蛋白与Talpha2 Ets结合，与弱结合的31 kDa蛋白结合。通过3‘-RACE RT-PCR，分离出含有简并Ets结构域寡核苷酸的部分c DNA片段。该序列揭示了Ets结构域与新蛋白的新的c-末端部分的保守性。我们目前正在尝试分离全长的cDNA.

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.