NF-Y antagonizes renin enhancer function by blocking stimulatory transcription factors.
We previously reported that the promoter proximal portion of the mouse renin enhancer contains a binding site for NF-Y (Ea) that overlaps with a positive regulatory element (Eb). In the context of the renin enhancer, NF-Y acts to oppose enhancer activity. We tested the hypothesis that NF-Y acts as a negative regulator by physically blocking the binding of transcription factors to element-b (Eb). Increasing the spacing between the NF-Y binding site (Ea) and Eb by 2, 5, or 10 nucleotides increased activity of the enhancer to the same extent as mutations abolishing NF-Y binding. The increase in transcription caused by increasing the spacing between Ea and Eb was not due to a shift of NF-Y from a negative regulator to a positive regulator because there was no loss of activity when Ea was also mutated. Oligonucleotides containing the normal or increased spacing mutants still allowed the binding of both NF-Y to Ea and transcription factors to Eb. In fact, we present evidence that both NF-Y and the Eb-binding factor(s) can each bind together on the same oligonucleotide containing either a 5- or 10-bp spacing between Ea and Eb. Our data strongly suggest that the mechanism by which NF-Y opposes renin enhancer activity is to sterically block the binding of factors to Eb.
Shi
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Departments of Internal Medicine and Physiology and Biophysics, University of Iowa, College of Medicine, Iowa City 52242, USA.
Gross
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KW
Sigmund
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CD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics* / Q000502:physiology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012083:Renin / Q000235:genetics*; D014162:Transfection
D023081:CCAAT-Binding Factor; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D008156:Luciferases; D012083:Renin