Additionally, we analyzed PDHA2 mRNA by conventional RT- PCR analysis with a gene specific set of primers, designed to amplify the 59 region of the transcript: the forward primer 59- TGCCATCTACAGCACTCCGT-39 hybridizing to nucleotides 227 to 28 and the reverse primer 59-AGCA- CAACCTCCTCTTCTTCC-39 hybridizing to nucleotides +412 to +432. The 459 pb product was amplified with SYBR green Master Mix in an ABI 7300 sequence detection system (Applied Biosystems) and visualized by agarose gel electrophoresis. Genomic DNA isolation and bisulfite sequencing for PDHA2 methylation analysis Genomic DNA was isolated from treated and untreated cells using a salting-out method (CitogeneH DNA Blood Kit – Citomed, Lisbon, Portugal). The number and distribution of scattered CpG sites and CpG islands of PDHA2 gene has been previously analyzed [14] using the online program MethPrimer, available at www.urogene.org/ methprimer, which defines CpG islands as sequences longer than 200 bp, with a calculated CG composition .50% and an observed to expected CpG ratio of .0.6 [35]. Bisulfite PCR sequencing was carried out using the EpiTectH Bisulfite Kit (Qiagen) and CpG islands were amplified by PCR using the specific primers and the conditions previously described [14]. The reaction products were purified by MinEluteH PCR Purification Kit (Qiagen) and sequenced in both directions by primer walking with ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kits, in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Chromatin immunoprecipitation assay Chromatin immunoprecipitation assays were performed as described previously [36]. Briefly, chromatin isolated from cell cultures was immunoprecipitated using the following antibodies: anti RNA pol II clone CTD4H8 (#05-623, Millipore, Bedford, References 1. Brown RM, Dahl HH, Brown GK (1989) X-chromosome localization of the functional gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex. Genomics 4: 174–181. 2. Dahl HH, Brown RM, Hutchison WM, Maragos C, Brown GK (1990) A testis- specific form of the human pyruvate dehydrogenase E1 alpha subunit is coded for by an intronless gene on chromosome 4. Genomics 8: 225–232. 3. Fitzgerald J, Wilcox SA, Graves JA, Dahl HH (1993) A eutherian X-linked gene, PDHA1, is autosomal in marsupials: a model for the evolution of a second, testis- specific variant in eutherian mammals. Genomics 18: 636–642. 4. Iannello RC, Young J, Sumarsono S, Tymms M, Kola I (1995) A model for understanding gene regulation during spermatogenesis: the mouse testis Pdha-2 promoter. Reprod Fertil Dev 7: 705–712. 5. Datta U, Wexler ID, Kerr DS, Raz I, Patel MS (1999) Characterization of the regulatory region of the human testis-specific form of the pyruvate dehydroge- nase alpha-subunit (PDHA-2) gene. Biochim Biophys Acta 1447: 236–243. 6. Brown GK, Brown RM, Scholem RD, Kirby DM, Dahl HH (1989) The clinical and biochemical spectrum of human pyruvate dehydrogenase complex deficiency. Ann N Y Acad Sci 573: 360–368. 7. Iannello RC, Dahl HH (1992) Transcriptional expression of a testis-specific variant of the mouse pyruvate dehydrogenase E1 alpha subunit. Biol Reprod 47: 48–58. 8. Iannello RC, Gould JA, Young JC, Giudice A, Medcalf R, et al. (2000) Methylation-dependent silencing of the testis-specific Pdha-2 basal promoter occurs through selective targeting of an activating transcription factor/cAMP- responsive element-binding site. J Biol Chem 275: 19603–19608. 9. Iannello RC, Young J, Sumarsono S, Tymms MJ, Dahl HH, et al. (1997) Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation. Mol Cell Biol 17: 612–619. 10. RC Iannello, JC Young, S Sumarsono, MJ Tymms, Kola I (1994) Mouse testis Pdha-2 promoter upstream sequences confer tissue-and temporal-specific activity in transgenic mice. Reprod Fertil Dev 6: 599–604. Somatic Activation of Testis-Specific PDHA2 Gene MA, USA), H3K36me3 (#ab9050, Abcam, Cambridge, UK), Sp1 (PEP 2) X (#SC-59X, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and normal rabbit immunoglobulin (#X0903, DakoCytomation, Denmark). The recovered DNA was analyzed by quantitative real-time PCR with SYBR green Master Mix in an ABI 7300 sequence detection system (Applied Biosystems). The qPCRs were performed using primers designed to cover three different regions of the PDHA2 gene: the proximal promoter region (+1 bp) 59-GGCAGGCACTGTACAAATCA-39 (forward) and 59-CAGTGCACACGGGTGATAGA-39 (reverse); the CpG island II region (+385 bp) 59-GGGCTCATGGTGTGTGCTAT- 39 (forward) and 59-AGCACAACCTCCTCTTCTTCC-39(re- verse); and a distal upstream region (210 Kb) 59-GCATGG- CAGGACTTCTCTC-39 (forward) and 59-TTACAGG- CAATGCTTG ACCA-39 (reverse). Statistical analysis Statistical analysis was performed using the Student’s t-test and the ANOVA one-way test with the Tukey Honestly Significant Differences (HSD) post-hoc test, the Tukey HSD for unequal N (Spjotvoll/Stoline test). All analyses were performed using the STATISTICA (data analysis software system), version 7.1 StatSoft, Inc. (Tulsa, OK, USA; 2006). A value of p,0.05 was considered significant. Supporting Information Table S1 Statistical analysis of PDHA2 gene methyla- tion results by the ANOVA one-way test. (DOCX) Author Contributions Conceived and designed the experiments: IR ER. Performed the experiments: AP MJN IM MJS. Analyzed the data: IR ER ITA. Contributed reagents/materials/analysis tools: MJS ITA. Wrote the paper: IR ER AP. 11. Fitzgerald J, Dahl HH, Jakobsen IB, Easteal S (1996) Evolution of mammalian X-linked and autosomal Pgk and Pdh E1 alpha subunit genes. Mol Biol Evol 13: 1023–1031. 12. Miranda TB, Jones PA (2007) DNA methylation: the nuts and bolts of repression. J Cell Physiol 213: 384–390. 13. Christman JK (2002) 5-Azacytidine and 5-aza-29-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21: 5483–5495. 14. Pinheiro A, Faustino I, Silva MJ, Silva J, Sa R, et al. (2010) Human testis-specific PDHA2 gene: methylation status of a CpG island in the open reading frame correlates with transcriptional activity. Mol Genet Metab 99: 425–430. 15. Yamashita R, Sathira NP, Kanai A, Tanimoto K, Arauchi T, et al. (2011) Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis. Genome Res 21: 775–789. 16. Robinson BH, MacKay N, Chun K, Ling M (1996) Disorders of pyruvate carboxylase and the pyruvate dehydrogenase complex. J Inherit Metab Dis 19: 452–462. 17. Pinheiro A, Silva MJ, Florindo C, Barroso M, Afonso A, et al. (2012) The unique case of a family expressing the testis-specific PDHA2 gene in somatic tissues. Manuscript under submission. 18. del Mazo J, Prantera G, Torres M, Ferraro M (1994) DNA methylation changes during mouse spermatogenesis. Chromosome Res 2: 147–152. 19. Rocamora N, Mezquita C (1989) Chicken spermatogenesis is accompanied by a genomic-wide loss of DNA methylation. FEBS Lett 247: 415–418. 20. Choi YC, Chae CB (1991) DNA hypomethylation and germ cell-specific expression of testis-specific H2B histone gene. J Biol Chem 266: 20504–20511. 21. De Smet C, Lurquin C, Lethe B, Martelange V, Boon T (1999) DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter. Mol Cell Biol 19: 7327–7335. 22. Kroft TL, Jethanandani P, McLean DJ, Goldberg E (2001) Methylation of CpG dinucleotides alters binding and silences testis-specific transcription directed by the mouse lactate dehydrogenase C promoter. Biol Reprod 65: 1522–1527. PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e38076