Somatic Activation of Testis-Specific PDHA2 Gene Figure 2. Functional deletion analysis of the human PDHA2 gene promoter. Progressive 59 deletion constructs were transiently transfected into HeLa, NT2 and SH-SY5Y cell lines. Transfections were carried out using 0.5 mg of the PDHA2 reporter constructs or the empty pGL2 vector. Normalized luciferase activities were expressed as mean values 6 SEM of duplicates for a minimum of three experiments (*p,0.001). doi:10.1371/journal.pone.0038076.g002 In order to corroborate the fact that DAC treatment induces a derepression of PDHA2 gene transcription, we analyzed the recruitment of RNA polymerase II (RNA pol II) to this gene by chromatin immunoprecipitation. We have designed three sets of primers, one targeting the promoter region (+1 bp), another targeting a distal upstream region (210 kb) and a third one targeting the CpG island II (+385 pb). Our results showed that we could only detect RNA pol II binding to the +1 bp region after DAC treatment (Student’s t-test, p,0.001), which most likely triggers the observed increase in PDHA2 gene transcription (Fig. 5). Interestingly, we could not detect the occupancy of the putative Sp binding site, by the Sp1 transcription factor (Fig. 5), as previously suggested by Datta and co-workers [5]. Furthermore, we investigated the possible recruitment of the elongation marker H3K36me3 to the gene body, but the results showed no occupancy of the targeted region. These data demonstrate that PDHA2 gene transcription was reactivated by DAC treatment in SH-SY5Y cells, suggesting that DNA demethylation may play a pivotal role in PDHA2 tissue- specific expression. DAC induces demethylation of PDHA2 coding region In order to confirm that DAC can induce the reactivation of PDHA2 expression in SH-SY5Y cells, in a demethylation dependent manner, we analyzed the methylation status of PDHA2 gene body by sodium bisulfite sequencing analysis. Since we have previously shown that the PDHA2 gene contains two CpG islands, one located in the promoter region (nucleotides 2128 to +73) and the other located in the coding region (nucleotides +197 to + 460) [14], we have analyzed the methylation patterns of both CpG islands. In accordance with our previous results, we observed that the CpG island I, located in the promoter region, is fully methylated in untreated SH-SY5Y cells, and seems to be insensitive to DAC, since it remains methylated after treatment with this drug (Fig. 6A). Effectively, its CpG sites remained fully methylated not only after DAC treatment, but after TSA and DAC plus TSA treatment, as well. Interestingly, the analysis of the CpG island II, located in the coding region, showed significant demethylation after treatment with DAC (Table S1) (Fig. 6A and 6B); TSA alone had no effect, but after pre-treatment with DAC, the demethylation level of this exonic CpG island seems to be slightly increased, which is particularly significant in the CpG sites +410 and +492 (Tukey for unequal N, p,0.01) (Fig. 6B). Recently, several potential alternative transcription start sites were identified in PDHA2 gene [15]. These are located in the coding region encompassing the CpG island II. Therefore, one could hypothesize that demethylation of the PDHA2 CpG island II PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38076