Somatic Activation of Testis-Specific PDHA2 Gene Figure 6. DNA methylation status of the human PDHA2 gene after sodium bisulfite PCR sequencing. A) Sample chromatograms obtained for both PDHA2 CpG Islands, before and after treatment with 5 mM DAC; B) DNA methylation status of CpG sites in the CpG island II located in the coding region of PDHA2 gene, before and after treatment with 5 mM DAC for 96 h and/or 0.25 mM TSA for 24 h. Results represent means of at least three independent experiments 6 SEM (* p,0.05; { p,0.01; 1 p,0.001 by Tukey HSD for unequal N test). doi:10.1371/journal.pone.0038076.g006 detection of several enhancer and repressor elements, because no significant differences in luciferase reporter activities were detected between the various deletion constructs. Additionally, the same authors suggested the existence of a putative Sp responsive element that would be important for Sp1-dependent transcription initiation [5]. However, our chromatin immunoprecipitation assays did not demonstrate any binding of Sp1 transcription factors to the proximal promoter of the PDHA2 gene. Additionally, and more importantly, our observation that PDHA2 promoter reporter constructs presented high luciferase activity in all somatic cell lines, where the PDHA2 mRNA cannot be detected, reinforces the idea that PDHA2 tissue-specific expression may be under strict control of epigenetic mechanisms of regulation. Furthermore, in vitro methylation of PDHA2 promoter constructs with SssI methylase resulted in a complete abrogation of luciferase activity. Our next approach was to further explore the involvement of epigenetics, namely DNA methylation and/or histone modifica- tions, on the regulation of PDHA2 gene expression. The results showed that treatment of SH-SY5Y cell cultures with DAC induced PDHA2 derepression with a concomitant accumulation of PDHA2 transcript. On the other hand, inhibition of histone deacetylation did not elicit any induction of PDHA2 expression, nor did it potentiate the DAC effect. Moreover, the accumulation of the PDHA2 mRNA after DAC treatment was correlated with an enrichment of RNA pol II at the PDHA2 proximal promoter (+1 bp region), which likely triggers the observed increase in PDHA2 mRNA levels. The fact that treatment with DAC alone elicited a significant effect upon PDHA2 gene expression suggests that changes associated with methylation are sufficient to drive transcription initiation of this testis-specific gene in somatic cells. Once proved that DAC was able to induce PDHA2 gene expression in cultured somatic cells, it would be important to investigate the methylation status of this gene, before and after treatment with the demethylating drug, in order to assure that demethylation was underlying PDHA2 transcription. Our results demonstrated that DAC promoted a relevant demethylation of PDHA2 coding region, which was fully methylated before treatment. However, the promoter CpG island I remained fully methylated, suggesting its insensibility to demethylation, at least by DAC treatment in the tested conditions. These findings corrob- orate our previous reported results, where we observed that PDHA2 expressing tissues (i.e spermatogenic cells) presented the PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38076