Demethylation of the Coding Region Triggers the Activation of the Human Testis-Specific PDHA2 Gene in Somatic Tissues Ana Pinheiro1, Maria Joa˜ o Nunes1, Ineˆ s Milagre1, Elsa Rodrigues1,2, Maria Joa˜ o Silva1,2, Isabel Tavares de Almeida1,2, Isabel Rivera1,2* 1 Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Lisbon, Portugal, 2 Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal Abstract Human PDHA2 is a testis-specific gene that codes for the E1a subunit of Pyruvate Dehydrogenase Complex (PDC), a crucial enzyme system in cell energy metabolism. Since activation of the PDHA2 gene in somatic cells could be a new therapeutic approach for PDC deficiency, we aimed to identify the regulatory mechanisms underlying the human PDHA2 gene expression. Functional deletion studies revealed that the 2122 to 26 promoter region is indispensable for basal expression of this TATA-less promoter, and suggested a role of an epigenetic program in the control of PDHA2 gene expression. Indeed, treatment of SH-SY5Y cells with the hypomethylating agent 5-Aza-29-deoxycytidine (DAC) promoted the reactivation of the PDHA2 gene, by inducing the recruitment of the RNA polymerase II to the proximal promoter region and the consequent increase in PDHA2 mRNA levels. Bisulfite sequencing analysis revealed that DAC treatment induced a significant demethylation of the CpG island II (nucleotides +197 to +460) in PDHA2 coding region, while the promoter region remained highly methylated. Taken together with our previous results that show an in vivo correlation between PDHA2 expression and the demethylation of the CpG island II in testis germ cells, the present results show that internal methylation of the PDHA2 gene plays a part in its repression in somatic cells. In conclusion, our data support the novel finding that methylation of the PDHA2 coding region can inhibit gene transcription. This represents a key mechanism for absence of PDHA2 expression in somatic cells and a target for PDC therapy. Citation: Pinheiro A, Nunes MJ, Milagre I, Rodrigues E, Silva MJ, et al. (2012) Demethylation of the Coding Region Triggers the Activation of the Human Testis- Specific PDHA2 Gene in Somatic Tissues. PLoS ONE 7(6): e38076. doi:10.1371/journal.pone.0038076 Editor: Wei-Guo Zhu, Peking University Health Science Center, China Received January 25, 2012; Accepted April 30, 2012; Published June 1, 2012 Copyright: 2012 Pinheiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the PhD grant SFRH/BD/31264/2006, from the Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT), Lisboa, Portugal, awarded to Ana Pinheiro. 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: iarivera@ff.ul.pt Introduction Pyruvate dehydrogenase complex (PDC) is a mitochondrial matrix enzyme system that catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA, a key metabolite for energy metabo- lism. The rate-limiting component is the E1 enzyme, which is a heterotetramer (a2b2). The a subunit, besides forming with the b subunit the active and the cofactor binding sites, is also the target for regulatory mechanisms governing global activity of PDC. The E1a subunit can be encoded by two different genes: PDHA1 located on X chromosome and expressed in somatic tissues; and PDHA2 an intronless gene located on chromosome 4 (Fig. 1). This autosomal gene is repressed in all somatic tissues but actively transcribed in post-meiotic germ cells where the X chromosome is absent or inactive [1,2]. It was suggested that the translocation of PDHA to the eutherian X chromosome, which is inactivated during spermatogenesis, led to the evolution of this second testis- specific locus by retroposition to an autosome [3]. Besides the potential of PDHA2 as a model for unraveling the mechanisms that govern gene expression during spermatogenesis [4], some authors also postulated that PDHA2 gene activation in somatic cells could be an effective therapy for PDC deficiency [5], an inborn error of metabolism mainly caused by mutations in PDHA1 gene [6]. The amount of published work on PDHA2 gene regulation is scarce and the studies performed relied on the mouse orthologue, Pdha2 [4,7,8,9,10]. However, it is believed that the regulatory mechanisms that control these orthologous genes are different, once there is no gross homology between their promoters, which seem to have evolved from different retroposons [11]. Datta and collaborators publish the first and, to our knowledge, unique study on human PDHA2 gene regulation [5]. They isolated and characterized approximately 800 nucleotides of the PDHA2 promoter region, identified the location of the transcription start site and performed functional studies that suggested the existence of multiple regulatory elements. More importantly, these authors proposed that the PDHA2 tissue-specific expression could be modulated by mechanisms, such as DNA methylation, that would limit PDHA2 expression to spermatogenic cells. Indeed, DNA methylation is a widely known epigenetic mechanism that plays a central role in the selective expression of particular genes in different tissues. The methylation of cytosine residues acts as a negative regulator of transcription by three potential mechanisms: direct interference with the binding of PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38076