idants [11,12]. The effectiveness of these treatments in improving cardiac and neurological outcomes in Friedreich ataxia patients is under continued evaluation. A recent study demonstrated an association between the GAA repeats within the FXN gene and aberrant frataxin pre-mRNA processing [13], and the authors proposed that binding of transcribed GAA repeats to nuclear splicing factors can interfere with turnover of intronic RNA and lead to decreased abundance of mature mRNA [13]. However, accumulating evidence indicates that epigenetic changes caused by heterochromatin formation in the promoter region and/or the first intron of the FXN gene also contribute to the dramatic reduction of frataxin protein levels in Friedreich ataxia patients. Decreased histone acetylation and extensive methylation of CpG regions upstream of the GAA repeat are observed in Friedreich ataxia patient cell lines and tissues [14,15], suggesting that enhanced heterochromatin formation might impede the transcription of frataxin, leading to lower frataxin protein levels [14,16,17]. Recently, a study employing an experimental histone deacetylase (HDAC) inhibitor in a mouse model of Friedreich ataxia revealed that this drug can substantially increase frataxin mRNA and protein levels [16]. Reduction of frataxin transcription very likely results from reduced accessibility of transcriptional regulatory factors to the promoter region and/or trinucleotide repeat region [15,18,19,20]. However, the identity and number of the regulatory factors influencing frataxin expression are largely unknown. Thus, in-depth investigation of the transcriptional regulatory machinery involved in frataxin expression would aid in the identification of drugs or therapies directed at restoring frataxin protein levels in Friedreich ataxia patient tissues. In this study, we used bioinformatic and molecular techniques to identify two transcription factors, SRF and TFAP2, which directly bind to the promoter region of the FXN gene. TFAP2 up- regulated frataxin mRNA expression in several cell lines, whereas SRF showed cell-line specific influences on frataxin expression. Finally, over-expression of either transcription factor in Friedreich ataxia patient-derived lymphoblasts or cell lines significantly increased frataxin mRNA levels. Identification and further characterization of these two new factors involved in frataxin expression may aid in the development of new therapeutic avenues for the treatment of Friedreich ataxia. Results In previous work we observed significant decreases in frataxin mRNA levels in multiple human cell lines as well as primary human fibroblasts and lymphoblasts derived from Friedreich ataxia patients and controls, when treated with the iron chelator desferrioxamine (DFO) [21]. These data suggested that one or more regulatory elements might modulate transcription of frataxin under varying metabolic conditions such as iron starvation. We initiated the current study by using Genomatix software (www. genomatix.de) to identify putative transcription factor binding sites within the promoter that might serve as transcriptional regulatory elements of the FXN gene. Although the FXN promoter region was reported to extend at least 1255 bp upstream of the translation start site (AUG) [22], in vitro experiments suggested that more than 60% of FXN promoter activity is conferred by the first 221 bp of this upstream sequence [22]. We chose to pursue putative SRF (serum response factor), TFAP2 (transcription factor AP2), and SP1 binding sites within this 221 bp region due to a high matrix similarity score of greater than 0.95 for these sequences (Figure 1A). SRF binds to sequences known as serum response elements, and is a member of the MADS (MCM1, Agamous, Regulation of Frataxin Deficiens, and ARF) box superfamily of transcription factors that can stimulate cell proliferation and differentiation [23]. TFAP2 is a developmentally regulated, retinoic-acid inducible transcriptional activator [24], while SP1 is a ubiquitously expressed factor that interacts with numerous other transcription regulators involved in many pathways (e.g., HIV-1 Tat, P53, RNAPII, EGR1, TFAP2) [25,26]. Notably, the SRF and TFAP2 binding sites were found within-, and immediately downstream of one of the few regions of the FXN promoter that shows significant sequence similarity between rodents and primates due to the presence of an L2 retrotransposon-like sequence [22]. To test whether SRF, TFAP2 or SP1 bind to the predicted FXN promoter sequence elements defined above, chromatin immuno- precipitation (ChIP) was performed using anti-SRF, TFAP2, and SP1 primary antibodies, and qRT-PCR was used to quantify the degree of transcription factor binding in the FXN promoter region. Robust SRF and TFAP2 binding was observed in HEK293 cells using several qRT-PCR primer sets (Figure 1B) encompassing the promoter region of FXN, while binding of SP1 was found to be far weaker than that of SRF or TFAP2 (Figure 1C). Similar results were obtained for each primer set (Figure 1C, lower panel) and comparable results were also obtained using K562 erythroleuke- mia cells (not shown). Next, in vitro electrophoretic mobility shift assays (EMSA) were carried out to assess the binding of nuclear proteins to synthetic dsDNA oligonucleotides encoding the putative TFAP2 and SRF sequences found in the FXN promoter. In vitro binding of nuclear proteins to the TFAP2 and SRF sequences was observed (Figure 2A, 2B). Moreover, when antibodies raised against either TFAP2 or SRF were included in the reactions, a ‘supershift’ was observed (Figure 2A lane 8, 9, 10 and 2B lane 9, 10, 12), suggesting formation of specific complexes between SRF or TFAP2 and the respective antibodies, resulting in altered mobility within the gel. These results strongly suggest that both SRF and TFAP2 can bind to specific sequences within the promoter region of the FXN gene. Transcriptional regulatory elements have previously been identified within the first intron of several genes [27,28,29,30]. Recently a region immediately upstream of the intronic GAA expansion which was reported to be important for maximal frataxin expression [15]. Further experiments showed that expansion of the GAA repeats leads to changes in DNA methylation and chromatin structure in this region of the FXN gene [15]. Accordingly, we analyzed the region immediately downstream of exon 1 using Genomatix software, and found a putative EGR3 transcription factor binding site in this intronic region (Figure 3A). To investigate whether this putative EGR3 binding site and the identified SRF and TFAP2-binding sites are involved in transcription of frataxin, we generated four luciferase reporter constructs. The first two constructs contained promoter fragments that extended 558-bp (construct I) or 228-bp (construct II) 59 of the start codon AUG linked to the luciferase gene; the latter two (constructs III and IV) contained the same promoter regions as construct I and II, respectively, but they also contained part of the first intron including the proposed EGR3 binding site (GCGCCGCGCACGCCGGG) (Figure 3A, dotted square). The insert sizes were 1030-bp (III) and 650-bp (IV), separately. Luciferase activity from constructs III and IV was substantially higher than activity measured from constructs I and II (Figure 3A, bottom panel), suggesting that the intronic region downstream of exon 1 is important for transcription of the FXN gene. We next used the luciferase assay to probe promoter activity of variants of the above constructs in which the putative binding sites for SRF, TFAP2, or EGR3 were altered. Mutation of either the PLoS ONE | www.plosone.org 2 August 2010 | Volume 5 | Issue 8 | e12286