Regulation of Frataxin Figure 1. Transcription factors SRF and TFAP2 bind to the FXN gene promoter region in vivo. (A) Sequence of 59-end of the human FXN gene. Sequences are color-coded: intronic sequences in green, exon 1 and exon 2 in black. Putative transcription factor binding sites identified by Genomatix analysis are annotated in bold black for SRF, in bold blue for TFAP2, in bold red for SP1, and in bold pink for EGR3. Start codon (AUG) in exon 1 is in bold black. (B) Schematic diagram of human FXN promoter region showing the location of the putative regulatory sequences. Black-filled square: SRF binding site; blue-filled square: TFAP2 binding site; red-filled square: SP1 binding site; pink-filled square: EGR3 binding site. Primers used in the ChIP assays are designated as: P189, P195, P204, P205, P206, P364, and P365. (C) Binding of SRF and TFAP2 to the FXN promoter in vivo was quantified by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR). The upper panel is the quantification of FXN promoter chromatin, immunoprecipitated using antibodies raised against SRF, TFAP2, and SP1. Three pairs of primers flanking the putative binding sites of these transcription factors were used for the qPCR analysis. The lower panel shows the end-products of the qPCR reactions separated by agarose gel electrophoresis. Primers specific for human FXN intron 4 were included as an internal negative control (data not shown). doi:10.1371/journal.pone.0012286.g001 SRF or TFAP2 binding sites in construct IV resulted in dramatically decreased luciferase activity in three distinct cell lines (Figure 3B). In contrast, minimal decreases in luciferase activity were observed following mutagenesis of the SRF or TFAP2 binding sites in constructs I and II (not shown), which lack the intronic sequence downstream of exon 1 that is included in construct IV. Interestingly, mutagenesis of the EGR3 binding site in the FXN intronic sequence in construct IV had dramatically different effects on luciferase expression depending on the cell lines tested, causing severe, mild, or no significant effect on the promoter activities in K562, SH SY5Y, and HEK293 cells, respectively. Taken together, these results further suggest that SRF and TFAP2 are transcription factors important for frataxin expression, and the intronic sequence downstream of exon 1 which contains a putative EGR3 binding site is required for full expression of frataxin. Previously, we demonstrated that frataxin protein and mRNA transcript levels are altered by perturbations in cellular iron status brought on by treatment with the iron chelator DFO [21]. To test whether iron-mediated changes in frataxin transcript levels might be related to altered expression of either SRF or TFAP2, we treated HEK293 and SH SY5Y cells with DFO and measured mRNA levels of SRF and TFAP2 by qRT-PCR. While no significant change in SRF mRNA levels was observed in iron- depleted (+DFO) versus iron-replete (-DFO) cells, TFAP2 mRNA levels were significantly decreased by iron depletion in both HEK293 and SH SY5Y cells (Figure 4A). Frataxin mRNA levels were also decreased by DFO treatment in HEK293 cells (as reported previously [21]), but not in SH SY5Y cells (Figure 4A). These data suggest that iron-mediated alterations in TFAP2 expression levels might influence frataxin mRNA expression during cellular iron deficiency. We also checked the mRNA expression levels of SRF, TFAP2, and EGR3 in lymphoblasts derived from an Friedreich ataxia patient (GM16214) and from a healthy control (GM16215). We found that EGR3 expression was very low, while TFAP2 expression was undetectable (not shown). As expected, frataxin mRNA levels were reduced in the Friedreich ataxia patient lymphoblasts as compared to the control lymphoblasts, while SRF mRNA expression levels were only moderately different between the two cell lines (Figure 4B). Finally, a ChIP experiment revealed that SRF occupancy of the FXN promoter was reduced in the Friedreich ataxia patient lymphoblasts as compared to the control lymphoblasts (Figure 4C). The above data suggest that frataxin expression could be enhanced by heterologous expression of SRF or TFAP2. To test this hypothesis we over-expressed SRF or TFAP2 in SH SY5Y cells, HEK293 cells, and in lymphoblasts obtained from an PLoS ONE | www.plosone.org 3 August 2010 | Volume 5 | Issue 8 | e12286