(nucleotides 2980 to 26) was amplified using the primers pPDHA2-975 bp and pPDHA2-R listed in Table 1. The second fragment designed to harbor both CpG islands of PDHA2 gene (2274 to +487) was amplified with pPDHA2-269 bp and PDHA2.CpGs-R primers (Table 1). The recombinant plasmids were sequenced by primer walking and named pPDHA2-TOPO and PDHA2.CpGs-TOPO, respectively. For sequence number- ing, nucleotide +1 was assigned to the adenosine of the initiation translation codon ATG. PDHA2 promoter reporter constructs Several different fragments derived from the human PDHA2 promoter region were subcloned into the luciferase expression vector pGL2-Basic vector (Promega Corporation, Madison, WI USA). We used the pPDHA2-TOPO plasmid as a template to amplify fragments of different lengths, using different forward primers and a reverse primer, which contains a XhoI overhang site, listed in Table 1. The PCR products, amplified with the PlatinumH Pfx Polymerase (Invitrogen), were subcloned in the pGL2 reporter plasmid into SmaI/XhoI sites generating plasmids 975.pGL2 (2980 to 26), 833.pGL2 (2838 to 26), 695.pGL2 (2700 to 26), 591.pGL2 (2596 to 26), 506.pGL2 (2511 to 26), 436.pGL2 (2441 to 26), 349.pGL2 (2354 to 26), 269.pGL2 (2274 to 26), and 196.pGL2 (2201 to 26). The pPDHA2- TOPO recombinant was also digested with enzymes MslI/XhoI and the 117 bp fragment was subcloned in the pGL2 reporter plasmid generating the 117.pGL2 recombinant (2122 to 26). An ultimate PDHA2 reporter construct was subcloned into the pGL2-Basic vector, using as template the PDHA2.CpGs-TOPO plasmid. This reporter construct was named CpGs.pGL2 and harbored both CpGs islands present in PDHA2 gene. In vitro methylation of PDHA2 constructs M.SssI was used to methylate the CpG dinucleotides of the PDHA2 gene/luciferase reporter constructs or the pGL2 basic vector, based on the protocol of the manufacturer (New England Biolabs, Beverly, MA). Mock reactions were carried out in parallel without adding the methylase. The samples were then incubated with methylation-sensitive restriction enzyme HpaII, followed by agarose gel electrophoresis. Complete methylation of the CpG sites of the constructs was verified by protection of the methylated DNA from digestion by this enzyme. Methylated and mock- Table 1. Sequence of oligonucleotides used in the human PDHA2 gene cloning. Oligonucleotides Sequence (59R39) pPDHA2 – R pPDHA2 – 975 bp pPDHA2 – 833 bp pPDHA2 – 695 bp pPDHA2 – 591 bp pPDHA2 – 506 bp pPDHA2 – 436 bp pPDHA2 – 349 bp pPDHA2 – 269 bp pPDHA2 – 196 bp PDHA2.CpGs – R TCACGGAGTGCTGTAGATGGCTCGAGCCG TGGAAACCTGCTGAAGACATT AAGGAAAAGTGGAATGTCACAAA ATACATTTTCCCTCCCCACT GTTAACGTGCGTGTGCTTGT GGCACATTATGGAGCAGGAT TGGTAGGAAGAAATACCTTTGGA TTGTCGGGAAAGCTTGAGAT GCGATTAGGATGCCCTGTAG GGCAGGCACTGTACAAATCA CGGCTCGAGCCATTGCCCCCATAGAAGT doi:10.1371/journal.pone.0038076.t001 Somatic Activation of Testis-Specific PDHA2 Gene treated plasmids were purified with a QIAquick gel extraction kit (Qiagen, Hilden, Germany) and used for reporter gene analyses after transient transfection in somatic cell lines. Transactivation studies The basal promoter activity of the different reporter plasmids was assayed by measuring the luciferase activity after transient transfection in HeLa, SH-SY5Y and Ntera2/clone D1 (NT2) cell lines. To minimize variations in transfection efficiency, replicates were transfected in single batch suspension with FuGENEH HD (Roche Diagnostics GmbH, Penzberg, Germany) according to the manufacturer’s instructions. Plates containing 200,000 cells were co-transfected with 0.5 mg of the reporter plasmid together with an expression plasmid containing the b-galactosidase gene-coding region (pSV40-bGAL). Cells were inoculated in 24-well plates and maintained for 48 h. These cells were harvested and lysed in reporter lysis buffer (Promega). Cell extracts were assayed for luciferase and b-galactosidase activity (b-Gal Reporter Gene Assay, Roche), which was used to normalize the results. All experiments were performed at least three times in duplicate well. Cell cultures and treatments SH-SY5Y (human neuroblastoma, ATCC CRL-2266) cell line was maintained in low glucose Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Inc., St. Louis, MO, USA), while HeLa (human cervix adenocarcinoma, ATCC CCL-2) and NT2 (human teratocarcinoma, ATCC CRL-1973) cell lines were cultured in high glucose Dulbecco’s modified Eagle’s medium (Sigma). All media were supplemented with 10% heat inactivated fetal bovine serum (Biochrom AG, Berlin, Germany), 2 mM L-glutamine (Sigma), 100 units/mL penicillin and 100 mg/mL streptomycin (Sigma), with the exception of NT2 culture medium that was supplemented with 4 mM L-glutamine. All cell cultures were carried out at 37uC in humidified 5% CO2. Dose response assays of 5-Aza-29-deoxycytidine (DAC) and Trichostatin A (TSA) were performed as stated in Milagre and collaborators (2008) [34]. Subsequently, cells were treated with 5 mM DAC and 0.25 mM TSA for the indicated periods (or vehicle as control) and every 24 hours the medium was changed. RNA isolation and qPCR for PDHA2 expression analysis Total RNA was prepared from all cells, treated and untreated, using the RNeasyH Mini Kit (Qiagen) and contamination by genomic DNA was eliminated from each RNA sample by pre- treatment with DNase I using RNase-free DNase Set (Qiagen). Total RNA was reverse transcribed using the Reverse Transcription System A3500 Kit (Promega) with random primers following the manufacturer’s instructions. First strand DNA (1 mg) was used as template for quantitative real-time PCR with an ABI PRISM 7300 sequence detection system (Applied Biosystems). The cycling conditions were 95uC/ 10 min followed by 40 cycles of 95uC/15 sec and 60uC/1 min. It was used the TaqManH Gene Expression Assay - ID: Hs01043024_s1 (Applied Biosystems) for specific detection of PDHA2 mRNA, and the TaqManH b-actin Control Reagents as endogenous control (Applied Biosystems). In every reaction we used the TaqManH Gene Expression Master Mix (Applied Biosystems) following enclosed instructions. Each sample was assayed in triplicate and results show a minimum of three independent experiments. Transcript levels were normalized to b- actin and expressed in pg PDHA2 mRNA per ng of b-actin mRNA. PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38076