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FIGURE 7. The N-terminal transactivation domains but not the DNA binding capacity of the zinc fingers of BTEB1 are required for TrβA autoinduction. A, N-terminal truncated forms of xBTEB1 fail to enhance TrβA autoinduction in XTC-2 cells. XTC-2 cells were transfected with the indicated expression vectors, and 48 h later cells were treated with 5 nM T3 for 6 h. Gene expression analysis was done by RTqPCR. Data shown are the means ± S.E. for the T3--treated cells only; n = 6/treatment. Letters indicate significant differences among treatments (i.e. means with the same letter are not significantly different; p < 0.05; Bonferroni's multiple comparison test). B, mutations in the three zinc fingers of BTEB1 do not affect activity on TrβA autoinduction. The first histidine residue in each of the Cys2-His2 zinc finger DNA binding domain of BTEB1 was mutated to alanine to generate pCS2-xBTEB C2AH. XTC-2 cells were transfected with the indicated expression vectors, and 48 h later cells were treated with 5 nM T3 for 6 h. Data shown are the means ± S.E. from one transfection experiment (n = 6/treatment) and the experiment was repeated twice with similar results. Letters indicate significant differences among treatments (i.e. means with the same letter are not significantly different; p < 0.05; Bonferroni's multiple comparison test). C, electrophoretic mobility shift assay showed that the BTEB1 C2AH mutant does not bind to DNA. Recombinant wild type BTEB1 and BTEB1 C2AH mutant proteins were generated by coupled in vitro transcription/translation, and varying amounts were tested for their ability to bind to the 32P-BTE probe in vitro. Radioinert BTE oligonucleotide was added to some reactions as a competitor, and antibody supershift was used to verify the presence of BTEB1 protein in the protein-DNA complexes formed. Western blot analysis confirmed that equal amounts of wild type and mutant BTEB1 proteins were used in the EMSA (data not shown). Image published in: Bagamasbad P et al. (2008) Copyright © 2008. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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