ACTIVATION OF THE TCR ALPHA-CHAIN ENHANCER BY THE LEF-1 PROTEIN

淋巴特异性高迁移率族（HMG）蛋白LEF-1激活 T细胞受体（α链）和HIV-1增强剂在一个背景下- T细胞中的限制性方式。 我们之前已经证明，LEF-1 包含一个有效的模块化反式激活结构域， 与HMG DNA结合结构域一致并且不类似于酸性， 富含谷氨酰胺或脯氨酸的经典转录激活结构域 因素 LEF-1已被提议组织立体特异性 在促进组装的增强子处的多蛋白复合物，或 其他邻近增强子结合蛋白的作用。 虽然能力 LEF-1对DNA的强烈弯曲可能在这一过程中起重要作用， N-末端反式激活结构域也必须发挥重要作用， 或者通过提供额外的蛋白质表面以稳定 组装立体特异性增强子复合物，或通过发挥更大的 直接参与转录并与基础转录相互作用 因子或共激活蛋白。 有趣的是，两者 的LEF-1应答增强子含有邻近的结合位点， 淋巴特异性因子Ets-1及其结合域突变 大大降低了LEF-1在体内的激活。 Ets-1与 TCR α增强子与PEBP 2/AML 1协同作用，PEBP 2/AML 1是T细胞特异性的， 与许多T细胞核心结构域结合的Runt相关蛋白 增强剂。 我们最近发现，纯化的重组LEF-1和 Ets-1蛋白强烈激活染色质上的HIV-1增强子- 体外重组DNA。 该测定提供了有用的生物化学方法。 进一步剖析LEF-1激活机制的方法 在这里，我们建议调整体外染色质组装转录 系统来分析LEF-1、Ets-1、AML 1（PEBP 2）和 CREB/ATF激活核心TCR α增强子。 LEF-1的能力 与Ets-1和AML 1一致的功能将被检查，无论是在 CREB的存在和不存在，以及LEF-1激活的特定模型 将被审查。 LEF-1和ETS-1的功能目标 激活域将进行生物化学分析， 将检查这些蛋白质对局部染色质结构的影响。 在 此外，我们将测试嵌合融合蛋白，AML 1/MTG 8/ETO， 它是由急性髓细胞性白血病中的t（8;21）易位产生的。 白血病，作为AML 1/PEBP 2活性的有效阻遏物， 体外 这种方法将有助于阐明的作用机制， LEF-1和其他T细胞特异性转录因子，以及 AML 1在白血病发生中的作用

The lymphoid-specific high mobility group (HMG) protein, LEF-1, activates the T-cell receptor (alpha-chain) and HIV-1 enhancers in a context- restricted manner in T cells. We have previously shown that LEF-1 contains a potent, modular trans-activation domain that functions in concert with the HMG DNA-binding domain and does not resemble acidic, glutamine- or proline-rich activation domains of classical transcription factors. LEF-1 has been proposed to organize a stereospecific multiprotein complex at the enhancer that facilitates the assembly or action of other nearby enhancer-binding proteins. Although the ability of LEF-1 to bend DNA strongly may play an important role in this process, the N-terminal trans-activation domain must also play an essential role, either by providing an additional protein surface to stabilize the assembly of the stereospecific enhancer complex, or by playing a more direct role in transcription and interacting with basal transcription factors or co-activator proteins at the promoter. Interestingly, both of the LEF-1 responsive enhancers contain nearby binding sites for the lymphoid-specific factor, Ets-1, and mutation of the Ets-binding domain greatly reduces activation by LEF-1 in vivo. Binding of Ets-1 to the TCRalpha enhancer is co-operative with PEBP2/AML1, a T cell-specific, Runt-related protein that binds to the core domains of many T cell enhancers. We have recently shown that purified recombinant LEF-1 and Ets-1 proteins strongly activate the HIV-1 enhancer on chromatin- reconstituted DNA in vitro. This assay provides a useful biochemical approach to further dissect the mechanism of activation by LEF-1 Here we propose to adapt the in vitro chromatin-assembly transcription system to analyze the mechanism by which LEF-1, Ets-1, AML1 (PEBP2) and CREB/ATF activate the core TCRalpha enhancer. The ability of LEF-1 to function in concert with Ets-1 and AML1 will be examined, both in the presence and absence of CREB, and specific models for LEF-1 activation will be examined. The functional targets for the LEF-1 and ETS-1 activation domains will be analyzed biochemically, and the effects of these proteins on local chromatin structure will be examined. In addition, we will test whether a chimeric fusion protein, AML1/MTG8/ETO, which is generated by the t(8;21) translocation in acute myelogenous leukemia, functions as a potent repressor of AML1/PEBP2 activity in vitro. This approach will help to elucidate the mechanism of action of LEF-1 and other T cell-specific transcription factors, as well as the role of AML1 in leukemogenesis.