FIG. 8.
TLF inhibits c-fos reporter gene activity by sequestering TFIIA. (A) Asparagine 37 of TLF-GFP was replaced with tyrosine in the construct TLFN37Y-GFP to disrupt the TFIIA-binding site of TLF. (B) TLF-GFP and TLFN37Y-GFP localizations in COS-7 cells were similar. Scale bars, 10 μm. (C) Western blot analysis showing that TLF-GFP and TLFN37Y-GFP are expressed at similar levels. BCA protein assays were used to ensure equal amounts of total protein were loaded in the lanes. (D and E) Western blots of TLF-GFP and TLFN37Y-GFP immunoprecipitated with an anti-GFP antibody from transfected HEK-293 cells. Staining with the anti-GFP antibody indicates that TLF-GFP and TLFN37Y-GFP were immunoprecipitated with similar efficiencies (D). Analysis with an anti-TFIIA antibody shows that TFIIA did not coimmunoprecipitate withTLFN37Y-GFP, indicating that the mutation disrupted TFIIA-TLF interactions (E). Interestingly, the uncleaved p55 product (TFIIA α and β) was also efficiently immunoprecipitated with TLF. (F) Repression of the c-fos reporter by TLF-GFP, TLFN37Y-GFP, GFP plus TFIIA, and TLF-GFP plus TFIIA. The addition of excess TFIIA or mutation of the TFIIA-binding site of TLF alleviated the TLF-mediated inhibition of c-fos reporter gene activity. Error bars indicate the standard error of the mean (n = 6, 5, 3, and 5, respectively, from left to right). (G) Model for TLF function. TLF and TBP bind to the NF1 and c-fos promoters, respectively. In the model, TLF competes with TBP for TFIIA, thus inhibiting TBP-responsive promoters. TBP competes with TLF for an undetermined general transcription factor (GTF) to inhibit TLF-responsive promoters.