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

淋巴组织特异性高迁移组(HMG)蛋白Lef-1被激活 T细胞受体(阿尔法链)和HIV-1增强剂在上下文中- 在T细胞中的限制性方式。我们之前已经证明了Lef-1 包含一个有效的、模块化的反式激活结构域，它在 与HMG DNA结合结构域一致，不类似于酸性， 经典转录中富含谷氨酰胺或脯氨酸的激活结构域 各种因素。LEF-1已被提出用于组织立体特异性 增强子上的多蛋白复合体，便于组装或 附近其他增强子结合蛋白的作用。虽然有能力 Lef-1对DNA的强烈弯曲可能在这一过程中发挥重要作用， N-末端反式激活结构域也必须发挥重要作用， 要么通过提供额外的蛋白质表面来稳定 组装立体特异性增强子复合体，或通过发挥更多 转录中的直接作用及其与基础转录的相互作用 启动子上的因子或共激活蛋白。有趣的是，两者 的LEF-1反应增强子包含附近的结合位点 淋系特异性因子Ets-1与Ets结合域的突变 极大地减少体内Lef-1的激活。ETS-1与 TCRpha增强子与T细胞特异性的PEBP2/AML1合作， 与许多T细胞核心区结合的矮小相关蛋白 增强剂。我们最近已经证明，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。这种化验提供了一种有用的生化 进一步剖析LeF-1激活机制的途径 在这里，我们建议调整体外染色质组装转录 系统分析Lef-1，ETS-1，AML1(PEBP2)和 CREB/ATF激活核心TCRpha增强子。LEF-1细胞的功能 与ETS-1和AML1配合使用的功能将在 CREB的存在和缺失，以及LEF-1激活的特定模型 将会被检查。LEF-1和ETS-1的功能目标 将对激活结构域进行生化分析，并对 这些蛋白质的局部染色质结构将被检测。在……里面 此外，我们将测试嵌合融合蛋白AML1/MTG8/ETO， 它是由急性髓系白血病t(8；21)易位产生的 白血病是AML1/PEBP2活性的有效抑制因子 体外培养。这种方法将有助于阐明其作用机制。 Lef-1和其他T细胞特异性转录因子，以及 AML1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.