Temporal and tissue-specific interactions involving novel transcription factors and the proximal promoter of the mouse Pdha-2 gene.
We have determined by deletion analysis that the most proximal region of the Pdha-2 promoter between nucleotide position -187 to +22 harbors a transcriptionally active core. This "core" promoter directs high levels of CAT (chloramphenicol acetyltransferase) reporter gene transcription in HeLa cells. DNase I footprinting of the proximal promoter revealed four regions of protection. One of these contains the consensus sequence for the Sp1 binding site and another the ATF/CREB binding site. The cis-sequences of the remaining two protected regions (designated MEP-2 and MEP-3; Mouse E1 alpha Promoter site) show no apparent consensus homology with cis-elements of other known transcription factors. Results of electrophoretic mobility shift assays confirm that the ATF/CREB and MEP binding sites interact in a characteristic and specific manner with factors present in nuclei of both testis and somatic tissue. The factor which recognizes the MEP-3 motif appears to be ubiquitous, whereas the MEP-2-protein complexes were tissue-specific. Interestingly, formation of a complex involving MEP-2 and a putative testis-specific binding factor (tau-MEP-2BF) is first observed in the testis of 2-week-old mice, this correlates with the expression of Pdha-2. In contrast, the formation of complexes between the MEP-2 binding site and a somatic variant of MEP-2BF (sigma-MEP-2BF) decreases in the testis as spermatogenesis proceeds. Our results suggest that 1) the MEP-2 binding factors are temporally regulated during spermatogenesis, and 2) interactions involving these factors with the MEP-2 cis-element may be important for modulating Pdha-2 expression.
Iannello
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RC
Murdoch Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.
Kola
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Dahl
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D000375:Aging / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D013737:Testis / Q000201:enzymology*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D014157:Transcription Factors; D012313:RNA; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase