Regulation of transcription of the human prepronociceptin gene by Sp1.
Nociceptin/orphanin FQ is a recently discovered neuropeptide and the endogenous ligand for opioid receptor-like-1. The promoter region of the precursor protein prepronociceptin (ppN/OFQ) has been cloned and sequenced. We have previously shown that a stretch of 110 bases immediately 5' to the first intron 23 bp upstream of the ATG start codon is responsible for significant enhancement of transcription of the human ppN/OFQ gene. We performed electromobility shift assays (EMSAs) using oligonucleotides spanning portions of the promoter region close to the intron to determine which DNA elements were important for transcriptional regulation. EMSAs using Sp1 antibody revealed a cis-acting regulatory element from bases 35-67 that appeared to bind Sp1 transcription factor and cause a shift to higher molecular weight. Deletion of this 30-bp region of DNA from the 1.2 kb promoter caused a significant loss of transcription as measured by luciferase reporter assays. Mutation of four bases at the Sp1 binding site also induced a significant loss of transcription compared to wildtype constructs. Finally, an Sp1- but not Etf-binding consensus oligonucleotide was able to compete with the interaction of the oligo with the NS20Y nuclear extract. Combined with the data from the supershift EMSAs, it appears that Sp1 is the transcription factor binding to the GC region close to the intron to regulate transcription of the human ppN/OFQ gene.
Zaveri
Naunihal T
NT
Neurobiology Program, SRI International, LA 214A, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA. naunihal.zaveri@sri.com
Green
Christopher J
CJ
Polgar
Willma E
WE
Huynh
Nhung
N
Toll
Lawrence
L
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011498:Protein Precursors / Q000235:genetics*; D011957:Receptors, Opioid / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D009841:Oligonucleotides; D011498:Protein Precursors; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; C121527:prepronociceptin; D004247:DNA; D008156:Luciferases