Regulation of Frataxin Figure 4. Cellular iron depletion decreases frataxin expression. (A) Iron depletion decreases frataxin and TFAP2 mRNA levels in tested cells. qRT-PCR was performed to quantify the mRNA levels of frataxin, SRF, and TFAP2 in SHSY5Y and HEK293 cells following treatment with 30 mM DFO for 48 hours. GAPDH was used as an internal control. Three separate experiments were carried out. For each experiment, duplicate iron treatment was performed. The error bars represent standard deviation. Statistical analysis was performed using the Student’s t-test: **: r,0.001. (B) mRNA levels of SRF and frataxin in lymphoblasts derived from patient (GM16214) and healthy control (GM16215). qRT-PCR was performed to quantify the mRNA levels of frataxin and SRF with the total RNA isolated from patient and control. (C) Binding of SRF to the FXN promoter is dramatically decreased in patient lymphoblasts. ChIP assays were conducted as described in Figure 1. The relative expression level in the patient cells is normalized to the healthy control cells, which are set at 1. doi:10.1371/journal.pone.0012286.g004 Friedreich ataxia patient or from a healthy control individual, and qRT-PCR was performed to assess changes in cellular frataxin mRNA levels following transfection (Figure 5). First, we performed in vitro translation to verify that our plasmid constructs expressed SRF and TFAP2 protein products of the correct size (Figure 5A). SRF and TFAP2 mRNA levels were found to be more than 100- fold higher in cells transfected with plasmids pcDNA-SRF or pcDNA-TFAP2 than in control cells transfected with an empty plasmid control, pcDNA3.1(-) (data not shown). Over-expression of SRF in HEK293, but not SH-SY5Y cells resulted in significantly elevated frataxin mRNA levels, while over-expression of TFAP2 in either cell line resulted in modest increases in frataxin mRNA levels (Figure 5B). Strikingly, over-expression of either TFAP2 or SRF in Friedreich ataxia patient lymphoblasts resulted in significant increases in frataxin mRNA levels, while over- expression of either transcription factor in control lymphoblasts had no significant effect on frataxin mRNA levels (Figure 5C). Finally, we measured frataxin protein levels in HEK293 cells after transfection with either SRF or TFAP2. Consistent with the changes in frataxin mRNA levels observed in these cells (Figure 5B), western blots demonstrated that frataxin protein levels were also increased (Figure 5D). Together, these results suggest that frataxin expression levels can be influenced by both SRF and TFAP2 expression in a variety of cell types. Discussion Deficiency of the frataxin protein is the primary molecular defect in Friedreich ataxia disease. Frataxin levels in Friedreich ataxia patients vary from between 5% and 30% of normal levels, while healthy heterozygous carriers typically express more than 50% of normal frataxin levels [31,32,33]. Thus, it has been suggested that restoration of frataxin gene expression levels in Friedreich ataxia patients to levels observed in heterozygotes may substantially slow disease progression. Characterization of the regulatory elements controlling frataxin expression is critical in the development of therapies directed at restoring frataxin expression levels in Friedreich ataxia patients. In this study we identified two transcription factors, SRF and TFAP2, which directly bind the promoter region of the human FXN gene and alter frataxin mRNA and protein levels. Over-expression of either of these two transcription factors led to a significant increase in frataxin mRNA levels in Friedreich ataxia patient lymphoblasts. Further- more, frataxin protein levels were increased following over- expression of SRF or TFAP2 in HEK293 cells. Thus, we conclude that transcription factors SRF and TFAP2 directly influence frataxin expression. We previously demonstrated that cells derived from Friedreich ataxia patients show signs of cytosolic iron deficiency, and that PLoS ONE | www.plosone.org 5 August 2010 | Volume 5 | Issue 8 | e12286