Regulation of Frataxin Figure 2. Transcription factors SRF and TFAP2 bind to the FXN promoter region in vitro. EMSA analysis was performed to investigate binding of TFAP2 (A) and SRF (B) to the promoter region of FXN in vitro. Nuclear extracts from HEK293 cells were incubated with [c-32P]-ATP labeled oligonucleotides coding the predicted TFAP2 or SRF binding site on the FXN promoter region of interest for 1 hour at 4uC. The binding products were resolved in native polyacrylamide gels (see MATERIALS AND METHODS). Specific competitor (non-radioactive oligonucleotide, so called cold probe, 10 mM) or non-specific competitor (poly(dI-dC), 0.5 mU/mL) was added to assess the specificity of the binding of SRF or TFAP2. Antibodies of TFAP2 (2 mg/ml) and SRF (0.5 mg/ml) were added for supershift, respectively. I: SRF antibody purchased from Santa Cruz Biotech.; II: SRF antibody purchased from Active Motif. A: HEK293 cell nuclear extracts, prepared by the authors, B: Jurkat cell nuclear extracts, purchased from Active Motif (Carlsbad, CA). doi:10.1371/journal.pone.0012286.g002 Figure 3. The SRF and TFAP2 binding sites in the FXN promoter are important for frataxin expression. (A) Luciferase analysis of a novel intronic regulatory region of the FXN gene. The upper panel portrays the upstream region of the FXN gene including the exon 1 and 2 and intron 1. Bottom panel: luciferase activity was measured in cells transfected with luciferase constructs containing truncated FXN promoter fragments containing the SRF and TFAP2 binding sites. Filled square: SRF binding site; open square: TFAP2 binding site; dotted square: EGR3 binding site. The four luciferase constructs are designated as I, II, III, IV (see MATERIALS AND METHODS). (B) Mutation of the SRF and TFAP2 binding sites in the FXN promoter dramatically decreased luciferase activity driven from FXN promoter fragment IV. Mutation of the predicted EGR3 transcription factor binding site in intronic sequence of the FXN gene showed cell line-specific effects on transcriptional activity. Three separate experiments were carried out. For each experiment, duplicate transfections were performed. Error bars represent the standard deviation. doi:10.1371/journal.pone.0012286.g003 PLoS ONE | www.plosone.org 4 August 2010 | Volume 5 | Issue 8 | e12286