Functional Domains of Transcription Factor hGABP( 1/E4TF1-53 Required for Nuclear Localization and Transcription Activation

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Abstract
Transcription factor E4TF1 is the human homolog of GABP and has been renamed hGABP (human GABP). hGABP is composed of two types of subunits; hGABP-β1/E4TF1–53 and the ets-related protein hGABP-α/E4TF1–60. Both bind together to form an (α)2(β1)2 heterotetrameric complex on DNA and activate transcription at specific promoters in vitro. Tetramer formation depends on two regions of hGABPβ1; the N-terminal region containing the Notch/ankyrin-type repeats is necessary for binding to hGABPα and the C-terminal region is necessary for homodimerization. In this report, we constructed various deletion mutants of hGABPβ1 in order to delimit the functional regions required for nuclear localization and transcription activity. We found that hGABPβ1 localization in the nucleus is dependent on a region located between amino acids 243 and 330 and that the presence of hGABPβ1 influences the efficiency of hGABPα transport into the nucleus. Next, we demonstrated that the hGABP complex composed of α and β1 subunits activates transcription from the adenovirus early 4 promoter in vivo. This transcription activation needs the C-terminal region of hGABPβ1 and is consistent with results obtained with the in vitro assay. Furthermore, site-directed mutagenesis analysis of the C-terminal region reveals that the α-helix structure and the leucine residues are important for formation of a heterotetrameric complex with hGABPα in vitro and for transcription activation in vivo. These results suggest that hGABPβ1 stimulates transcription as part of a heterotetrameric complex with hGABPα in vivo.