Identification of the response region on the oxytocin receptor (OTR) promoter which is important for the synergistic transactivation of OTR by C/EBPβ and NF-κB transcription factors

Abstract

The OTR promoter contains response elements for several transcription factors often associated with inflammation/immune response. We have demonstrated previously that the OTR promoter is activated synergistically by overexpression of C/EBPβ and NF-κB. Our object is to identify the DNA region which mediates this synergy. Electromobility shift assays (EMSAs) showed specific DNA binding to nine OTR-specific C/EBP and 2 NF-κB sites. Single mutants of each site did not affect activation (fivefold) of OTR promoter with overexpressed C/EBPβ or NF-κB and the synergistic transactivation (30–50-fold). Transfections allowed analysis of the functional consequences of various OTR promoter lengths (350 bp, 656 bp and 1061 bp from the transcription start site). The 350 bp promoter did not demonstrate any transactivation with expression vectors for C/EBPβ and NF-κB, whereas the 656 bp promoter showed induction with C/EBPb and NF-kB but no synergy. EMSAs showed that an overlapping C/EBPβ NF-κB site (−961 to −945 bp) binds both transcription factors but the integrity of the C/EBPβ site is required for NF-κB binding. Mutation of the overlapping area or of the two NF-κB sites did not eliminate the synergistic transactivation.