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 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.
Iannello
R C
RC
Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. Rocco.Iannello@med.monash.edu
Gould
J A
JA
Young
J C
JC
Giudice
A
A
Medcalf
R
R
Kola
I
I
D016475:3T3 Cells; D000367:Age Factors; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D018899:CpG Islands / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism*; D051786:Cyclic AMP Response Element Modulator; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D008297:Male; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D030481:Pyruvate Dehydrogenase (Lipoamide); D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins; D020218:Response Elements / Q000235:genetics; D013091:Spermatogenesis / Q000502:physiology; D013737:Testis / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D011768:Pyruvate Dehydrogenase Complex; D012097:Repressor Proteins; D014157:Transcription Factors; D051786:Cyclic AMP Response Element Modulator; D000242:Cyclic AMP; D030481:Pyruvate Dehydrogenase (Lipoamide); C065640:pyruvate dehydrogenase E1alpha subunit; D015500:Chloramphenicol O-Acetyltransferase