Expression of Human Frataxin Is Regulated by Transcription Factors SRF and TFAP2 Kuanyu Li1*, Anamika Singh 2 , Daniel R. Crooks2,3, Xiaoman Dai1, Zhuangzhuang Cong1, Liang Pan1, Dung Ha2, Tracey A. Rouault2* 1 Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, Nanjing, China, 2 Molecular Medicine Program, National Institute of Child Health and Human Development, Bethesda, Maryland, United States of America, 3 Department of Biochemistry, Molecular and Cellular Biology, Georgetown University Medical Center, Washington, D. C., United States of America Abstract Background: Friedreich ataxia is an autosomal recessive neurodegenerative disease caused by reduced expression levels of the frataxin gene (FXN) due to expansion of triplet nucleotide GAA repeats in the first intron of FXN. Augmentation of frataxin expression levels in affected Friedreich ataxia patient tissues might substantially slow disease progression. Methodology/Principal Findings: We utilized bioinformatic tools in conjunction with chromatin immunoprecipitation and electrophoretic mobility shift assays to identify transcription factors that influence transcription of the FXN gene. We found that the transcription factors SRF and TFAP2 bind directly to FXN promoter sequences. SRF and TFAP2 binding sequences in the FXN promoter enhanced transcription from luciferase constructs, while mutagenesis of the predicted SRF or TFAP2 binding sites significantly decreased FXN promoter activity. Further analysis demonstrated that robust SRF- and TFAP2- mediated transcriptional activity was dependent on a regulatory element, located immediately downstream of the first FXN exon. Finally, over-expression of either SRF or TFAP2 significantly increased frataxin mRNA and protein levels in HEK293 cells, and frataxin mRNA levels were also elevated in SH-SY5Y cells and in Friedreich ataxia patient lymphoblasts transfected with SRF or TFAP2. Conclusions/Significance: We identified two transcription factors, SRF and TFAP2, as well as an intronic element encompassing EGR3-like sequence, that work together to regulate expression of the FXN gene. By providing new mechanistic insights into the molecular factors influencing frataxin expression, our results should aid in the discovery of new therapeutic targets for the treatment of Friedreich ataxia. Citation: Li K, Singh A, Crooks DR, Dai X, Cong Z, et al. (2010) Expression of Human Frataxin Is Regulated by Transcription Factors SRF and TFAP2. PLoS ONE 5(8): e12286. doi:10.1371/journal.pone.0012286 Editor: Sumitra Deb, Virginia Commonwealth University, United States of America Received March 27, 2010; Accepted July 24, 2010; Published August 20, 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by the intramural program of the NICHD, National Institutes of Health, and in part by a research grant from Friedreich Ataxia Research Alliance (FARA): http://www.curefa.org/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: likuanyu@nju.edu.cn (KL); trou@helix.nih.gov (TAR) Introduction Friedreich’s ataxia, the most common inherited ataxia, is an autosomal recessive neurodegenerative disease caused by expan- sion of triplet nucleotide GAA repeats in the first intron of the FXN gene. Expansion of the GAA region from fewer than 200 to as many as 1500 repeats results in significant reduction of frataxin protein levels in affected patient tissues. The exact physiological function of frataxin continues to be a subject of intense research. Early reports demonstrated robust mitochondrial iron accumula- tion in Friedreich ataxia patient cardiac tissue [1], as well as in a Saccharomyces cerevisiae strain lacking the yeast frataxin homologue Yfh1p [2]. Additionally, deficiency of iron-sulfur (Fe-S) cluster- containing mitochondrial respiratory chain enzymes is a feature found both in patient cardiac biopsies and in Yfh1p-deficient S. cerevisiae [3]. These seminal findings regarding frataxin function have led to further work suggesting potential roles for human frataxin (and its homologues in lower organisms) in cellular functions including as an iron donor for heme biosynthesis [4], as an iron storage protein [5], as an iron chaperone [6] or accessory protein [7] important for Fe-S cluster assembly. Although there is ongoing debate over the function(s) of frataxin, it seems clear that its absence in human cells results in impaired Fe-S protein activities as well as mitochondrial iron overload. The clinical manifestations of Friedreich ataxia involve neurodegeneration in the spinal cord and cerebellum, causing gait disturbances, speech impairment, and increased incidence of diabetes. Mitochondrial iron deposition in the heart is known to accompany the hypertrophic cardiomyopathy and eventual heart failure observed in Friedreich ataxia patients, which commonly leads to mortality in the third or fourth decade of life (reviewed elsewhere [8]). Since oxidative tissue damage is thought to result from mitochondrial iron overload, drug screening studies have focused on ameliorating cardiac iron accumulation using iron chelators [9,10], and enhancing respiratory chain function using coenzyme Q10 and/or reducing oxidative damage with antiox- PLoS ONE | www.plosone.org 1 August 2010 | Volume 5 | Issue 8 | e12286