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

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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.