In conclusion, we have identified two nuclear transcription factors, SRF and TFAP2, which can directly bind sequences in the promoter of the human FXN gene, likely enhancing frataxin expression. Expression of recombinant transcription factors SRF or TFAP2 in two distinct human cell lines, as well as in Friedreich ataxia patient lymphoblasts, resulted in increased frataxin mRNA levels. These results demonstrate that frataxin expression can be enhanced by these two key transcription factors. Potential interaction partners of SRF and TFAP2, including EGR3, should be further explored to shed light on the regulatory network governing frataxin expression, as well as the tissue-specific pathology of Friedreich ataxia disease. Materials and Methods Cell culture and transfection Cell lines HEK293, K562, and SH SY5Y were purchased from ATCC (Manassas, VA). Lymphoblasts derived from healthy controls (GM15799, GM16215) and Friedreich ataxia patients (GM16197, GM16214) were obtained from the Coriell Cell Repository (Camden, NJ). HEK293 cells were grown in alpha- modified MEM medium (Sigma, St. Louis, MO), SH SY5Y in DMEM/F-12 medium (Invitrogen, Carlsbad, CA), and lympho- blasts in RPMI 1640 medium (Invitrogen), all supplemented with 10% fetal calf serum and 2 mM glutamine. For transfection of HEK293 and SH SY5Y cells, Fugene 6 (Roche, Indianapolis, IN) was used according to the supplier’s manuals. For transfection of lymphoblast cells, cell line Nucleofector Kit V (Lonza, cat# VCA- 1003, Gaithersburg, MD) was used. DNA constructs Plasmid constructs expressing the transcription factors were generated with primers 59- tttCTCGAGatgttaccgacccaagctgg-39 and 59-ggggAAGCTTtcattcactcttggtgctgt-39 for SRF, primers 59- ttCTCGAGccagactcttcgcagatgtt-39 and 59- ccAAGCTTtcac- tttctgtgcttctcctc-39 for TFAP2, and primers 59- ccCTCGAGat- gagcgaccaagatcactc-39 and 59-ccAAGCTTtcagaagccattgccactga - 39 for SP1. The PCR products were cloned into plasmid pcDNA3.1(-) with XhoI and HindIII restriction sites. The plasmids for luciferase assay were constructed by cloning PCR products (558 bp (construct I), 228 bp (II), 980 bp (III), and 650 bp (IV)), covering the direct upstream of human FXN gene coding sequence or lengthening into the first intron 257 bp of FXN gene, into pGL4.10 (Promega, Madison, WI). Site-directed mutagenesis was carried out by using primers 59- GCAGGCTCTGGAT- TCGCGTTAAATGCACG -39 and 59- CGTGCATTTAACG- CGAATCCAGAGCCTGC -39, or primers 59- CTGCTC- CCCCACAGAAGAGTAAATGCAACCAGTGGCCACCAGG -39 and 59- CCTGGTGGCCACTGGTTGCATTTACTCTT- CTGTGGGGGAGCAG-39, or primers 59- CGCAGCACCA- GCGCTGGAAAACAAAGCGGAGCGGGCGGCAGA-39 and 59- TCTGCCGCCCGCTCCGCTTTGTTTTCCAGCGCTG- GTGCTGCG-39, or primers 59- GAGGCGCCGCGCTAT- TCGGGGTCGCTC-39 and 59- GAGCGACCCCGAATAG- CGCGGCGCCTC -39, which contained the mutated predicted binding sites of SRF, TFAP2, SP1, or EGR3, respectively, following the supplier’s instructions (Stratagene, La Jolla, CA). Plasmid DNA was isolated with Qiagen miniprep or midiprep kits (Valencia, CA) as needed. Chromatin immunoprecipitation (ChIP) ChIP was conducted with Magna ChIPTM G Kit (Millipore, cat#MAGNA0002, Billerica, MA) following the manufacturer’s instruction. Briefly, cells were treated with formaldehyde (1%) for Regulation of Frataxin 10 min to crosslink the chromatin with the potential transcription factors. Harvested cells were sonicated to shear chromosomal DNA to an average length between 200 to 600 base pairs. Isolated chromatin was incubated with the proper primary antibody against human transcription factor SRF, TFAP2, SP1, or EGR3 (Santa Cruz Biotech. Inc., Santa Cruz, CA). After a series of wash steps, the chromatin was eluted for further real-time PCR analysis. Electrophoretic mobility shift assays (EMSAs) DNA gel-shifts were carried out with a double-stranded oligonucleotide 201 (oligo 201) 59- CGTGCATTTAACAA- AAATGGAGAGCCTGCTTT-39 for transcription factor SRF, oligo 202 59- CAGAAGAGTGCCTGCGGCCAGTGGCCA- CCA-39 for transcription factor TFAP2, oligo 203 59- CCA- GCGCTGGAGGGCGGAGCGGGCGGCAGA-39 for transcrip- tion factor SP1 in 20 ml of 25 mM Hepes (pH 7.5), 40 mM NaCl, 1 mM EDTA, 4 mM DTT, and 10% glycerol for 1 hour at 4uC, after labeling the double stranded oligonucleotide with [c-P32]- ATP (Perkin Elmer, cat#NEG035C, Waltham, MA). Nuclear extracts were prepared from HEK293 cells using NE-PER Nuclear Extraction Reagents (Pierce, Rockford, IL, USA) according to the supplier’s instructions. For assessment of binding specificity, poly(dI-dC) (Sigma, cat#P4925) was added to each reaction, from a stock concentration of 0.5 mU/ml. Antibody supershift reactions were performed following manufacturers instructions (Active Motif, Carlsbad, CA). Antibodies of SRF and TFAP2 and Jurkat nuclear extract for supershift were purchase from Active Motif. Following 5% native polyacrylamide gel electrophoresis (29:1, acrylamide:bisacrylamide) with Tris- Borate-EDTA (1X TBE) casting and running buffer, gels were dried and exposed to a phosphor screen, which was visualized using a Typhoon Imager (GE, Piscataway, NJ). Real-Time PCR The comparative Ct method with SYBR Green was conducted with the ABI 7000 Real-Time PCR System (Applied Biosystems, Foster City, CA). For human FXN gene detection, the following primers were used: 59-CCTTGCAGACAAGCCATACA-39 and 59-CCACTGGATGGAGAAGATAG-39 [21]; for SRF, primer 249 59-AGAAGGCCTATGAGCTGTCC-39 and primer 250 59- TTGCCGGTCTCACTGGTGAT-39; for TFAP2, primer 274 59-TCCAACAGCAATGCCGTCTC-39 and primer 275 59- GC- CACCGTGACCTTGTACTT-39; for EGR3, primer 265 59- GCCAGGACAACATCATTAGC-39 and primer 266 59-AG- GTCGCCGCAGTTGGAGTA-39. Endogenous GAPDH was used as an internal control with primers 59-TGCACCAC- CAACTGCTTAGC-39 and 59-GGCATGGACTGTGGTCAT- GAG-39 for normalization. Different primer combinations for transcription factor binding sites in ChIP assays were primer 204 59- TTACACAAGGCATCCGTCTC-39 and primer 205 59- GGAGCAGCTAGAGGTTAGAC-39; primer 206 59- GGTC- TAACCTCTAGCTGCTC-39 and primer 195 59- TTTTT- TAAGCTTCTGCTCCGGGTCTGCCGCCC-39; primer 189 59- ATTGGTACCACCAGGCTGCAGTCTCCCTT-39 and primer 195; primer 364 59-TAAGTATCCGCGCCGGGAAC- 39 and primer 365 59-AATGCAACCGGGAGAACCAG-39 with template DNA from isolated chromatin (see ChIP experiment) for quantitative real-time PCR. A part of sequence of Human FXN intron 4 was amplified as a ChIP negative internal control with primers 59-AGGCCAAGGCCTGTGGATCA-39 and 59- AGTGGCGCGATCTTGGCTCA-39. PLoS ONE | www.plosone.org 7 August 2010 | Volume 5 | Issue 8 | e12286