Abstract
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 nucleotides -54 and -62 upstream of the major transcriptional start site. Targeting of the CpG dinucleotide within this cis-element significantly disrupts the ability of this basal promoter to activate gene expression in vitro and completely abolishes promoter activity in vivo. DNase I footprinting experiments indicated that availability of the nuclear factor(s) binding this element is limiting in sexually immature mouse testis, and as such, these factors may play an important role in the coordinate activation of early spermatogenic gene expression. Interestingly, CpG dinucleotides associated with the hypersensitive region flanking the activating transcription factor/cAMP-responsive element-binding site appear to confer some conformational structure on the promoter since mutations at these specific CpG dinucleotides result in elevated basal levels of transcription. This raises the possibility of a potential bifunctional role for CpG dinucleotides in either methylation-dependent or -independent processes. Our data support the notion that hypomethylation and transcription factor recruitment are necessary events that precede gene activation at the early stages of spermatogenesis.
Highlights
The molecular processes that lead to gene activation at the onset of spermatogenesis are not well defined
We investigated the relative importance of specific CpG dinucleotides residing within the Pdha-2 core promoter and to what extent the ATF/ CRE-binding site and -binding factor(s) may be involved in this process
CpG Methylation Abrogates Pdha-2 Promoter Activity through an ATF/CRE cis-Element—In a previous study, we had established an in vivo correlation between transcriptional activation and demethylation of 5-methylcytosine nucleotides along the length of the Pdha-2 core promoter [9]
Summary
The molecular processes that lead to gene activation at the onset of spermatogenesis are not well defined Those involved in (i) the activation of early cell-specific gene expression coincident with the appearance of primary spermatocytes and (ii) the coordination of the expression of these genes during this period are poorly understood. We demonstrated that this constitutive activity in somatic cells can be significantly reduced following in vitro methylation and showed that an outcome of methylation is the specific abolition of factor binding to a consensus ATF1/CRE-binding site within the Pdha-2 core promoter [9]. We investigated the relative importance of specific CpG dinucleotides residing within the Pdha-2 core promoter and to what extent the ATF/ CRE-binding site and -binding factor(s) may be involved in this process. Activation of the promoter in vivo is dependent upon the binding of an ATF/CRE-binding factor that appears to be in limiting quantities early in spermatogenesis
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