Regulation of Frataxin Figure 5. Overexpression of SRF or TFAP2 enhances frataxin expression in HEK293 cells and Friedreich ataxia patient lymphoblasts. (A) In order to assess the effect of over-expression of SRF or TFAP2 on frataxin expression, plasmids pcDNA-SRF and pcDNA-AP2 were constructed and the correct sizes of the translation products of the cloned SRF and TFAP2 cDNA were verified by in vitro translation with [35S]- labeled methionine. The plasmid constructs were then transfected into HEK293 or SH SY5Y cells (B), or lymphoblasts (C) derived from healthy individuals (GM15799, GM16215) or Friedreich ataxia patients (GM16179, GM16214), and frataxin mRNA levels were measured by qRT-PCR (see MATERIALS and METHODS). Empty plasmid pcDNA3.1(-) was used as a control for comparison. The results from two different experiments were similar; the data presented here are average values from the two experiments. Levels of mature frataxin protein were determined by western blot (D). A representative western blot is shown here for HEK293 cells. Three separate experiments were carried out, and for each experiment two transfections per sample were performed. Statistical analysis was performed using the Student’s t-test: *: r,0.05; **: r,0.001. doi:10.1371/journal.pone.0012286.g005 cellular frataxin mRNA levels were decreased after experimental induction of iron deficiency [21]. Here we have demonstrated that mRNA levels of the transcription factor TFAP2, but not SRF, are also regulated by iron in HEK293 cells. In affected tissues of Friedreich ataxia patients, cytosolic iron depletion brought on by decreased frataxin expression due to expansion of the GAA repeat might result in decreased expression of TFAP2, resulting in an even further reduction in frataxin expression. Unexpectedly, overexpression of TFAP2 did not significantly increase mRNA levels of frataxin in SH-SY5Y, HEK293, or lymphoblasts derived from healthy control individuals. However, frataxin expression levels were significantly enhanced by TFAP2 over-expression in the Friedreich ataxia patient lymphoblast cell line. These effects may be a result of different levels of occupancy of endogenous TFAP2 on the putative TFAP2 binding site in the frataxin promoter. Additionally, the TFAP2 family of transcription factors is composed of five distinct gene products in humans (AP-2a, AP- 2b, AP-2x, AP-2d, and AP-2e), which can heterodimerize and elicit tissue-specific transcriptional regulatory effects [24]. There- fore, heterologous expression of additional TFAP2 family members may be needed in order to see positive effects on frataxin expression in all cell types utilized in this study. Many genes contain intronic regulatory sequences that are important for gene expression [34,35]. Bisulfite sequence mapping of the region immediately upstream of GAA repeats within FXN intron 1 (724 bp) identified sequences that enhance FXN promoter activity [15]. However, this prior study did not address the function of upstream sequences found immediately following exon 1 (530 bp away from the 724 bp fragment). We observed diminished luciferase activity in FXN promoter constructs that lack this intronic region (Figure 3A; constructs I and II), which contains a predicted consensus binding site for the EGR3 transcriptional regulator. Since distinct band shift patterns were observed in further EMSA experiments performed with nuclear extracts from K562 and SH SY5Y cells (data not shown), we may speculate that SRF and TFAP2 interact with other regulatory elements to modulate frataxin expression in a cell-type dependent manner. EGR3 is a good candidate co-regulator, since mutagenesis of the predicted EGR3 binding site in our luciferase experiments resulted in variable enhancer effects in the different cell lines that were tested (Figure 3B). Interestingly, EGR3- deficient mice exhibit several neuromuscular defects including gait ataxia and scoliosis [36], symptoms which resemble those exhibited by Friedreich ataxia patients. Moreover, EGR1 and EGR3 double knockout mice displayed decreased frataxin mRNA levels (57.3% of control) in thymocytes [37], indi- cating that loss of EGR1/3 can transcriptionally alter frataxin expression in vivo. PLoS ONE | www.plosone.org 6 August 2010 | Volume 5 | Issue 8 | e12286