Abstract
Human PDHA2 is a testis-specific gene that codes for the E1α subunit of Pyruvate Dehydrogenase Complex (PDC), a crucial enzyme system in cell energy metabolism. Since activation of the PDHA2 gene in somatic cells could be a new therapeutic approach for PDC deficiency, we aimed to identify the regulatory mechanisms underlying the human PDHA2 gene expression. Functional deletion studies revealed that the −122 to −6 promoter region is indispensable for basal expression of this TATA-less promoter, and suggested a role of an epigenetic program in the control of PDHA2 gene expression. Indeed, treatment of SH-SY5Y cells with the hypomethylating agent 5-Aza-2′-deoxycytidine (DAC) promoted the reactivation of the PDHA2 gene, by inducing the recruitment of the RNA polymerase II to the proximal promoter region and the consequent increase in PDHA2 mRNA levels. Bisulfite sequencing analysis revealed that DAC treatment induced a significant demethylation of the CpG island II (nucleotides +197 to +460) in PDHA2 coding region, while the promoter region remained highly methylated. Taken together with our previous results that show an in vivo correlation between PDHA2 expression and the demethylation of the CpG island II in testis germ cells, the present results show that internal methylation of the PDHA2 gene plays a part in its repression in somatic cells. In conclusion, our data support the novel finding that methylation of the PDHA2 coding region can inhibit gene transcription. This represents a key mechanism for absence of PDHA2 expression in somatic cells and a target for PDC therapy.
Highlights
Pyruvate dehydrogenase complex (PDC) is a mitochondrial matrix enzyme system that catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA, a key metabolite for energy metabolism
The E1a subunit can be encoded by two different genes: PDHA1 located on X chromosome and expressed in somatic tissues; and PDHA2 an intronless gene located on chromosome 4 (Fig. 1)
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
Summary
Pyruvate dehydrogenase complex (PDC) is a mitochondrial matrix enzyme system that catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA, a key metabolite for energy metabolism. The a subunit, besides forming with the b subunit the active and the cofactor binding sites, is the target for regulatory mechanisms governing global activity of PDC. The E1a subunit can be encoded by two different genes: PDHA1 located on X chromosome and expressed in somatic tissues; and PDHA2 an intronless gene located on chromosome 4 (Fig. 1). This autosomal gene is repressed in all somatic tissues but actively transcribed in post-meiotic germ cells where the X chromosome is absent or inactive [1,2]. It was suggested that the translocation of PDHA to the eutherian X chromosome, which is inactivated during spermatogenesis, led to the evolution of this second testisspecific locus by retroposition to an autosome [3]
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