Somatic Activation of Testis-Specific PDHA2 Gene Figure 3. In vitro methylation with SssI methylase abrogates PDHA2 promoter constructs activity. Constructs, either methylated or mock- methylated, were transiently transfected into SH-SY5Y cell line. 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). (A) Control of the methylation reaction by digestion with the methylation-sensitive endonuclease HpaII. (B) Methylation of PDHA2 constructs prevents proximal promoter activity. (C) No significant decrease in the luciferase activity was observed in the PDHA2 construct including the CpG II region (CpGs.pGL2). doi:10.1371/journal.pone.0038076.g003 corresponds to the demethylation of alternative promoters that control transcription of truncated PDHA2 transcripts. Indeed, as the qPCR assay used a TaqMan probe hybridizing to the 39UTR of the PDHA2 mRNA, the presence of eventual shorter truncated transcripts could not be discarded. Accordingly, we further analyzed PDHA2 expression by conventional RT-PCR with a set of specific primers designed to amplify the 59 region of the full- length transcript (nucleotides 227 to +432). We detected the expected amplification product with 459 bp only in DAC-treated cells, which proves that DAC promotes the transcription of a full- length PDHA2 transcript (Fig. 7). These results are consistent with our previous work [14], and strongly suggest that PDHA2 reactivation in somatic cells proceeds via a mechanism independent of promoter demethylation, most probably dependent on demethylation of the coding region. Discussion PDHA2 gene activation in somatic tissues has been postulated as a conceptual therapy for PDC deficiencies caused by PDHA1 gene mutations [5,16]. Accordingly, it is crucial to understand the mechanisms controlling human PDHA2 tissue-specific expression, namely what factors are responsible for the silencing of this gene in somatic tissues, and its activation in spermatogenic ones (diploid and haploid germ cells). However, the lack of a suitable cell line to study the regulatory mechanisms underlying PDHA2 gene expression has hampered this elucidation for a long time. The first step towards understanding the regulation of a particular gene is the identification of regulatory elements and factors involved in basal expression. Accordingly, our studies began by the functional analysis of human PDHA2 promoter. The results showed that PDHA2 promoter-directed transcription of the luciferase reporter gene occurred in cultured somatic cells (HeLa, NT2 and SH-SY5Y), where PDHA2 mRNA is undetectable. Deletion analysis further revealed that the region spanning from 2122 to 26 is indispensable for basal expression of this TATA- less promoter. Moreover, and in each cell line, no significant differences were found among the reporter gene activities driven by all the other PDHA2 deletion promoter constructs. Addition- ally, each construct displayed comparable reporter activities in the three different somatic cell lines. These observations suggested that the mechanisms involved in the repression of PDHA2 expression in somatic cells are not operative when the core promoter or the 59 flanking region are transiently transfected into somatic cells. Moreover, these results corroborated the previous transactivation experiments described by Datta and colleagues [5], who also observed PDHA2 promoter- directed transcription in human hepatocellular carcinoma cells. However, we could not replicate their results concerning the PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38076