Figure 7. DAC activates a full-length PDHA2 transcript. Conventional RT-PCR analysis, using primers hybridizing to the 59 region, was used for detection of PDHA2 transcripts. (1) SH-SY5Y cells before treatment. (2) SH-SY5Y cells after treatment with 5 mM DAC. (3) Negative control – no biological sample. (4) Positive control – mature spermatozoa sample. (M) HyperLadder II (Bioline, London, UK). doi:10.1371/journal.pone.0038076.g007 coding region completely demethylated, while non-expressing tissues displayed it fully methylated [14]. Moreover, these results also correlate with our recent finding concerning a family displaying PDHA2 gene expression in somatic tissues [17], an interesting case that may configure the previously referred long dreamed therapy for PDC deficiency, i.e. the somatic activation of PDHA2 expression. The results obtained either then by in-vivo or now by ex-vivo experiments are overlapping; actually, both methylation analyses revealed a correlation between demethylation of the coding region and PDHA2 derepression. Moreover, and interestingly, the level of demethylation in the coding region is very similar in samples derived from the cultured cells treated with DAC and from the family individuals. However, Yamashita and colleagues [15] recently identified multiple transcriptional start sites downstream of the canonical one (+248, +253 and +269), which are located precisely within the CpG island II region (nucleotides +197 to +460). Accordingly, this region could eventually harbor alternative promoter that would trigger the transcription of truncated PDHA2 mRNAs. However, our RT-PCR results clearly show that DAC induces transcription of full-length transcripts. These data are corroborated by the significant enrichment of RNA pol II at the proximal promoter. Taken together, our results confirm that methylation of the coding region is a key point in somatic cell silencing of PDHA2 gene. Actually, DNA methylation appears to be particularly suitable for the regulation of germ line-specific genes, and this is probably related to the global demethylation process that occurs during the development of spermatogenic cells, which may provide the mechanism by which these germ line-specific genes Somatic Activation of Testis-Specific PDHA2 Gene are demethylated [18,19]. And, despite the fact that DNA methylation does not seem to be the primary control mechanism regulating the programmed expression of most tissue-specific genes resulting in tissue differentiation, there are several examples that indicate that DNA methylation can serve as the primary control mechanism for the expression of a number of germ line- specific genes [20,21,22]. Although the expression of previously referred genes proceeds via a promoter methylation-dependent mechanism, we also can find in the literature some references to human genes that are regulated by methylation of the coding region, namely mono- carboxylate transporter MCT3 [23]. Another particular interest- ing example is the mouse Tact1/Actl7 gene [24], which is also intronless and testis-specific, like the PDHA2 gene. Indeed, it has already been described that methylation of the coding region, per se, can control gene expression by preventing promoter activity at the level of the chromatin structure. Indeed, CpG methylation induces a local repressive chromatin structure, mediated by the binding of methyl-CpG binding domain (MBD) proteins which recruit other proteins including sin3A and histone deacetylase; when a sufficient amount of CpGs is methylated, this repression is transmissible in cis, spreading for several hundred base pairs [25,26,27]. Accordingly, more or less distant methylated sequences, like the PDHA2 coding region, can promote gene repression. Interestingly, and as stated by Nan and co-workers, this type of repression is greater if the promoter itself is methylated [28], which is the case of PDHA2 promoter. Furthermore, it has also been postulated that methylation of the coding region can inhibit gene expression by interfering with the elongation step rather than with transcription initiation, by causing RNA pol II to pause or to prematurely terminate [29,30]. Moreover, the inhibitory effect upon elongation is prominent when methylation occurs near the start codon [31]. Based on this hypothesis, we attempted to explore elongation of PDHA2 transcription by chromatin immunoprecipitation assays. The results did not show any particular differences in the chromatin recovered before and after DAC treatment, namely when using the elongation marker H3K36me3. A recent report has shown a clear correlation between H3K36me3 marking and transcriptional activity in intron-containing genes; however, in intronless genes H3K36me3 is detected at much lower levels irrespective of expression levels [32]. Indeed, there are other examples showing that transcriptional elongation is differently controlled in intronless genes when compared to longer intron- containing genes [33]. Accordingly, because PDHA2 gene lacks introns, different approaches need to be designed. In summary, we hypothesize that PDHA2 gene belongs not only to a restricted group of germ line-specific genes that use DNA methylation as a primary silencing mechanism, but to a unique subset of those genes whose expression is regulated by the methylation status of the coding region. Furthermore, these new insights on the regulatory mechanism underlying PDHA2 tissue- specific expression may open potential therapeutic avenues for PDC deficiency caused by PDHA1 mutations. Materials and Methods Cloning the human PDHA2 gene Two different fragments of the human PDHA2 gene (GenBank ID: M86808) were cloned in pCRH4Blunt-TOPOH vector (Invitrogen Corporation, Carlsbad, CA, USA) after PCR ampli- fication with PlatinumH Pfx Polymerase (Invitrogen) of genomic DNA isolated from circulating lymphocytes of a healthy individ- ual. The first fragment harboring the PDHA2 promoter region PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e38076