Identification of a TPA-responsive element mediating preferential transactivation of the galanin gene promoter in chromaffin cells.
The gene encoding the neuropeptide galanin is upregulated by second messenger signal transduction pathways in bovine chromaffin cells. To identify its transcriptional regulatory elements, 5'-flanking sequences of the galanin gene were transiently transfected into primary cultures of bovine chromaffin cells within reporter gene constructs. Multiple regions of the galanin 5' flank seem to be necessary for basal activity. The most promoter-proximal of these regions contains a sequence (TGACG) -66 to -62 nucleotides upstream from the transcriptional start site which mediates stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA), as demonstrated by site-directed mutagenesis and cis-activation experiments. This cis-regulatory element mediates preferential TPA stimulation of transcription from the galanin promoter in chromaffin cells compared with bovine endothelial or HeLa cells. DNA-protein binding assays indicate that an oligonucleotide that includes the galanin TPA-responsive element (GTRE) binds specifically to proteins from nuclear extracts of chromaffin cells. TPA treatment persistently increases this binding activity in chromaffin but not in endothelial cells. Mutation of the galanin promoter within the -66 to -62 region renders it unresponsive to transcriptional stimulation by TPA, and a correspondingly mutated oligonucleotide fails to bind chromaffin cell nuclear proteins in a gel-shift assay. Chromaffin cell nuclear extracts also contain proteins that bind consensus TPA-responsive (TRE) and cyclic AMP-responsive (CRE) elements. GTRE, TRE, and CRE oligonucleotides all compete more efficiently for protein binding to their labeled congeners than for protein binding to either of the other labeled oligonucleotides, suggesting that the GTRE, TRE, and CRE oligonucleotides, suggesting that the GTRE, TRE, and CRE oligonucleotides each bind unique as well as common proteins, likely to be members of the Jun/Fos and cAMP-responsive element-binding protein/activating transcription factors (CREB/ATF) families of transcription factors, in chromaffin cells.
Anouar
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Section of Molecular Neuroscience, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892.
MacArthur
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Cohen
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Iacangelo
A L
AL
Eiden
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LE
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D019004:Galanin; D005786:Gene Expression Regulation / Q000187:drug effects*; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides / Q000096:biosynthesis / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D015290:Second Messenger Systems; D017384:Sequence Deletion; D015398:Signal Transduction; D016385:TATA Box; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects
D017931:DNA Primers; D009479:Neuropeptides; D009687:Nuclear Proteins; D010455:Peptides; D012333:RNA, Messenger; D005576:Colforsin; D019004:Galanin; D000242:Cyclic AMP; D008156:Luciferases; D013755:Tetradecanoylphorbol Acetate