Regulation of transcription of the human prepronociceptin gene by Sp1

Abstract 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.