Somatic Activation of Testis-Specific PDHA2 Gene Figure 1. Schematic representation of the PDHA2 gene showing the localization of the 2 CpG islands (CpG I and CpG II), the transcriptional start site and the putative Sp1 binding site. doi:10.1371/journal.pone.0038076.g001 specific transcription factors to promoters, direct binding of specific transcriptional repressors and alteration of the chromatin structure [12]. DNA methylation can be inhibited by 5-Aza-29- deoxycytidine (DAC), a potent inhibitor of DNA methyltransferase (DNMT) activity, through the irreversible binding of DNMTs to DAC substituted DNA [13]. Recently, we have demonstrated an in vivo correlation between an increase in PDHA2 mRNA levels and the demethylation of a CpG island in its coding region [14], which is a strong evidence for a role of DNA methylation in the epigenetic control of the human PDHA2 gene. In this study we show that inhibition of DNMTs with DAC reactivates PDHA2 gene expression in SH-SY5Y cells, in a demethylation-dependent manner. Results PDHA2 promoter constructs display basal activity in human somatic cell lines Human PDHA2 gene expression is restricted to post-meiotic germ cells [2] suggesting the absence of positive modulating factors and/or the presence of repressors in somatic tissues. However, the lack of suitable human spermatogenic germ cell lines makes it difficult to easily elucidate the regulatory mechanisms involved upon PDHA2 expression. In order to evaluate the regions important for transcriptional modulation and, more precisely, to define the regions putatively involved in the repression of PDHA2 gene in somatic cells, several deletion promoter constructs were generated (Fig. 2), and their ability to direct expression of the reporter luciferase gene was analyzed after transient transfection in different somatic cell lines. Accordingly, the various PDHA2 reporter plasmids and the parental pGL2-Basic vector were transfected into HeLa (cervix adenocarcinoma), NT2 (human teratocarcinoma) and SH-SY5Y (human neuroblastoma) cells, which do not normally express PDHA2 mRNA. Surprisingly, PDHA2 reporter constructs displayed high lucifer- ase activities in all three somatic cell lines (Fig. 2). Significant differences were found between the luciferase activities of the several constructs, in HeLa (ANOVA one-way test: F = 20.29, df = 10, p,0.001), NT2 (ANOVA one-way test: F = 26.01, df = 10, p,0.001), and SH-SY5Y (ANOVA one-way test: F = 48.89, df = 10, p,0.001) cells. However, post hoc comparisons only revealed significant differences between the luciferase activities of the empty pGL2-Basic and all the other reporter constructs (Tukey HSD p,0.001). These results firstly show that the region between nucleotides 2122 and 26 seems to contain all the sequences necessary to drive transcription initiation, and thus most probably correspond- ing to the basal proximal promoter. Secondly, the fact that PDHA2 promoter reporter constructs present high luciferase activity in somatic cell lines where the PDHA2 mRNA cannot be detected, and that there are no significant differences between the luciferase activities of the different deletion constructs, suggest an important role of epigenetic modifications in the regulation PDHA2 tissue- specific expression. In vitro methylation prevents PDHA2 promoter constructs activity To evaluate the role of DNA methylation on the activity of the PDHA2 proximal promoter, we performed an in vitro methylation assay using SssI methylase (Fig. 3A). The results revealed that the PDHA2 proximal promoter activity was completely abolished after in vitro methylation (Fig. 3B). Moreover, since we had previously demonstrated an in vivo inverted correlation between methylation of a CpG island localized in the coding region (see CpG II in Fig. 1) and the PDHA2 mRNA levels [14], we wanted to assess if the presence of a methylated CpG island II region could further decrease PDHA2 construct’s luciferase activity. Nevertheless, since there were already no significant differences between the luciferase activity of the 296.pGL2 construct and of the empty pGL2.Basic vector (Fig. 3B and 3C), the inclusion of the CpG II region did not result in a further decrease in promoter activity. These results indeed show that methylation plays an important repressive role upon PDHA2 gene expression. Activation of PDHA2 expression in somatic cells after treatment with epigenetic drugs To further investigate the role of epigenetic mechanisms upon PDHA2 gene regulation, we treated SH-SY5Y cells, where this gene is not normally expressed, with a potent inhibitor of the de novo methylation (DAC) and with a pharmacological inhibitor of histone deacetylases (TSA). PDHA2 expression was quantified by real-time PCR (qPCR) and the results revealed that after treatment with 5 mM DAC for 96 h or 120 h, there is a significant increase of PDHA2 mRNA, when compared with untreated cells (ANOVA one-way test: F = 27.84, df = 3, p,0.001; Tukey HSD for unequal N p,0.001) (Fig. 4A). Since it is generally accepted that DAC and TSA synergistically affect gene expression, by a mechanism that depends on promoter demethylation induced by DAC and subsequent reinforcement by histone acetylation induced by TSA, we also treated SH-SY5Y with TSA for 24 h, and with DAC for 96 h prior to a 24 h treatment with TSA. Incubation with 0.25 mM TSA for 24 h did not significantly affect PDHA2 mRNA levels. Moreover, pretreat- ment with DAC for 96 h, prior to the 24 h TSA treatment, did not further increase the observed PDHA2 mRNA accumulation with DAC treatment only (Fig. 4B). PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38076