Western Blotting Analysis Proteins were resolved in 12% NUPAGE gels (Invitrogen, cat# NP0342box) and transferred onto nitrocellulose membranes (Invitrogen, cat# IB3010-01). Primary antibodies used were rabbit anti-TFAP2 and anti-SP1 (Sant Cruz Biotech, Inc, Santa Cruz, CA), mouse anti-SRF and anti-tubulin (Abcam, Cambridge, MA), and mouse anti-frataxin (MitoScience, Eugene, Oregon). The mature frataxin form (,14 kDa) was quantified for comparison when needed. Western blot band intensities were quantified using program ImageJ. Any change of the intensities was compared with the controls, which value was set as 1. Luciferase assay The indicated plasmids derived from pGL4.10 [luc2] (Promega) were co-transfected with pGL4.75 [hRluc/CMV] into HEK293, K562, or SH SY5Y cells. Luciferase activities were measured with Dual-LuciferaseH Reporter Assay System (Promega) by using References 1. Sanchez-Casis G, Cote M, Barbeau A (1976) Pathology of the heart in Friedreich’s ataxia: review of the literature and report of one case. Can J Neurol Sci 3: 349–354. 2. Babcock M, de Silva D, Oaks R, Davis-Kaplan S, Jiralerspong S, et al. (1997) Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin. Science 276: 1709–1712. 3. Rotig A, de Lonlay P, Chretien D, Foury F, Koenig M, et al. (1997) Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia. Nat Genet 17: 215–217. 4. Lesuisse E, Santos R, Matzanke BF, Knight SA, Camadro JM, et al. (2003) Iron use for haeme synthesis is under control of the yeast frataxin homologue (Yfh1). Hum Mol Genet 12: 879–889. 5. Gakh O, Adamec J, Gacy AM, Twesten RD, Owen WG, et al. (2002) Physical evidence that yeast frataxin is an iron storage protein. Biochemistry 41: 6798–6804. 6. Yoon T, Cowan JA (2003) Iron-sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe-2S] clusters in ISU-type proteins. J Am Chem Soc 125: 6078–6084. 7. Ramazzotti A, Vanmansart V, Foury F (2004) Mitochondrial functional interactions between frataxin and Isu1p, the iron-sulfur cluster scaffold protein, in Saccharomyces cerevisiae. FEBS Lett 557: 215–220. 8. Schulz JB, Boesch S, Burk K, Durr A, Giunti P, et al. (2009) Diagnosis and treatment of Friedreich ataxia: a European perspective. Nat Rev Neurol 5: 222–234. 9. Kakhlon O, Manning H, Breuer W, Melamed-Book N, Lu C, et al. (2008) Cell functions impaired by frataxin deficiency are restored by drug-mediated iron relocation. Blood 112: 5219–5227. 10. Richardson DR, Mouralian C, Ponka P, Becker E (2001) Development of potential iron chelators for the treatment of Friedreich’s ataxia: ligands that mobilize mitochondrial iron. Biochim Biophys Acta 1536: 133–140. 11. Hart PE, Lodi R, Rajagopalan B, Bradley JL, Crilley JG, et al. (2005) Antioxidant treatment of patients with Friedreich ataxia: four-year follow-up. Arch Neurol 62: 621–626. 12. Rustin P (2003) The use of antioxidants in Friedreich’s ataxia treatment. Expert Opin Investig Drugs 12: 569–575. 13. Baralle M, Pastor T, Bussani E, Pagani F (2008) Influence of Friedreich ataxia GAA noncoding repeat expansions on pre-mRNA processing. Am J Hum Genet 83: 77–88. 14. Al-Mahdawi S, Pinto RM, Ismail O, Varshney D, Lymperi S, et al. (2008) The Friedreich ataxia GAA repeat expansion mutation induces comparable epigenetic changes in human and transgenic mouse brain and heart tissues. Hum Mol Genet 17: 735–746. 15. Greene E, Mahishi L, Entezam A, Kumari D, Usdin K (2007) Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia. Nucleic Acids Res 35: 3383–3390. 16. Rai M, Soragni E, Jenssen K, Burnett R, Herman D, et al. (2008) HDAC inhibitors correct frataxin deficiency in a Friedreich ataxia mouse model. PLoS One 3: e1958. 17. Soragni E, Herman D, Dent SY, Gottesfeld JM, Wells RD, et al. (2008) Long intronic GAA*TTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia. Nucleic Acids Res 36: 6056–6065. 18. Grabczyk E, Usdin K (2000) The GAA*TTC triplet repeat expanded in Friedreich’s ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner. Nucleic Acids Res 28: 2815–2822. Regulation of Frataxin Veritas microplate luminometer (Turner Biosystems, Sunnyville, CA). For control of transfection efficiency in each well, firefly luciferase activity was normalized to Rellina luciferase activity. Bioinformatic and statistical analysis Online software Genomatix (http://www.genomatix.de) was used to search the binding sites of putative transcription factors in the promoter region of the human FXN gene. Other statistical analyses in this study were performed using the Student’s t-test (http://www.danielsoper.com/statcalc/). Author Contributions Conceived and designed the experiments: KL DRC TR. Performed the experiments: KL AS XD ZC LP DH. Analyzed the data: KL AS DRC TR. Contributed reagents/materials/analysis tools: KL AS XD ZC LP DH TR. Wrote the paper: KL DRC TR. 19. Grabczyk E, Usdin K (2000) Alleviating transcript insufficiency caused by Friedreich’s ataxia triplet repeats. Nucleic Acids Res 28: 4930–4937. 20. Krasilnikova MM, Kireeva ML, Petrovic V, Knijnikova N, Kashlev M, et al. (2007) Effects of Friedreich’s ataxia (GAA)n*(TTC)n repeats on RNA synthesis and stability. Nucleic Acids Res 35: 1075–1084. 21. Li K, Besse EK, Ha D, Kovtunovych G, Rouault TA (2008) Iron-dependent regulation of frataxin expression: implications for treatment of Friedreich ataxia. Hum Mol Genet 17: 2265–2273. 22. Greene E, Entezam A, Kumari D, Usdin K (2005) Ancient repeated DNA elements and the regulation of the human frataxin promoter. Genomics 85: 221–230. 23. Knoll B, Nordheim A (2009) Functional versatility of transcription factors in the nervous system: the SRF paradigm. Trends Neurosci 32: 432–442. 24. Eckert D, Buhl S, Weber S, Jager R, Schorle H (2005) The AP-2 family of transcription factors. Genome Biol 6: 246. 25. Mao XR, Moerman-Herzog AM, Chen Y, Barger SW (2009) Unique aspects of transcriptional regulation in neurons–nuances in NFkappaB and Sp1-related factors. J Neuroinflammation 6: 16. 26. Sysa P, Potter JJ, Liu X, Mezey E (2009) Transforming growth factor-beta1 up- regulation of human alpha(1)(I) collagen is mediated by Sp1 and Smad2 transacting factors. DNA Cell Biol 28: 425–434. 27. Reid LH, Gregg RG, Smithies O, Koller BH (1990) Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells. Proc Natl Acad Sci U S A 87: 4299–4303. 28. Smith AN, Barth ML, McDowell TL, Moulin DS, Nuthall HN, et al. (1996) A regulatory element in intron 1 of the cystic fibrosis transmembrane conductance regulator gene. J Biol Chem 271: 9947–9954. 29. Antoine M, Kiefer P (1998) Functional characterization of transcriptional regulatory elements in the upstream region and intron 1 of the human S6 ribosomal protein gene. Biochem J 336(Pt 2): 327–335. 30. Brudno M, Gelfand MS, Spengler S, Zorn M, Dubchak I, et al. (2001) Computational analysis of candidate intron regulatory elements for tissue- specific alternative pre-mRNA splicing. Nucleic Acids Res 29: 2338–2348. 31. Campuzano V, Montermini L, Lutz Y, Cova L, Hindelang C, et al. (1997) Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes. Hum Mol Genet 6: 1771–1780. 32. Campuzano V, Montermini L, Molto MD, Pianese L, Cossee M, et al. (1996) Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271: 1423–1427. 33. Montermini L, Richter A, Morgan K, Justice CM, Julien D, et al. (1997) Phenotypic variability in Friedreich ataxia: role of the associated GAA triplet repeat expansion. Ann Neurol 41: 675–682. 34. Lee JG, Dahi S, Mahimkar R, Tulloch NL, Alfonso-Jaume MA, et al. (2005) Intronic regulation of matrix metalloproteinase-2 revealed by in vivo transcriptional analysis in ischemia. Proc Natl Acad Sci U S A 102: 16345–16350. 35. van Helden J, Rios AF, Collado-Vides J (2000) Discovering regulatory elements in non-coding sequences by analysis of spaced dyads. Nucleic Acids Res 28: 1808–1818. 36. Tourtellotte WG, Milbrandt J (1998) Sensory ataxia and muscle spindle agenesis in mice lacking the transcription factor Egr3. Nat Genet 20: 87–91. 37. Carter JH, Lefebvre JM, Wiest DL, Tourtellotte WG (2007) Redundant role for early growth response transcriptional regulators in thymocyte differentiation and survival. J Immunol 178: 6796–6805. PLoS ONE | www.plosone.org 8 August 2010 | Volume 5 | Issue 8 | e12286