Methylation-dependent silencing of the testis-specific Pdha-2 basal promoter occurs through selective targeting of an activating transcription factor/cAMP-responsive element-binding site

In this study, we demonstrate that methylation-dependent repression of the Pdha-2 core promoter is mediated regionally through a consensus activating transcription factor/cAMP-responsive element-binding site located between n ucleotides -54 and -62 upstream of the major transcriptional start site. Ta rgeting of the CpG dinucleotide within this cis-element significantly disru pts the ability of this basal promoter to activate gene expression in vitro and completely abolishes promoter activity in vivo, DNase I footprinting e xperiments indicated that availability of the nuclear factor(s) binding thi s element is limiting in sexually immature mouse testis, and as such, these factors may play an important role in the coordinate activation of early s permatogenic gene expression. Interestingly, CpG dinucleotides associated w ith the hypersensitive region flanking the activating transcription factor/ cAMP-responsive element-binding site appear to confer some conformational s tructure on the promoter since mutations at these specific CpG dinucleotide s result in elevated basal levels of transcription. This raises the possibi lity of a potential bifunctional role for CpG dinucleotides in either methy lation-dependent or -independent processes. Our data support the notion tha t hypomethylation and transcription factor recruitment are necessary events that precede gene activation at the early stages of spermatogenesis.