Differential protein-DNA binding analysis identifies a novel enhancer element, US-1, involved in the upregulation of the oxytocin receptor gene in human myometrium at term

In order to investigate the regulatory mechanisms involved in the transcrip tion of the human oxytocin receptor (OTR) gene in the human myometrium at t erm of pregnancy, we subjected the 5' flanking region of the gene to a diff erential EMSA (electrophoretic mobility shift assay) procedure. Comparing n uclear proteins from term myometrium, in which OTR gene transcription is ma ssively up-regulated, with those from the non-pregnant myometrium, indicate d a prominent DNA-protein complex using the former extract. The sequence of the protein binding site was determined within 20 bp (TCTGC-CTTCATCCAGCC) and designated as uterine stimulator motif-1 (US-1). The concatemerized US- 1 sequence exhibited enhancer activity using a minimal thymidine kinase pro moter (tk-81) in transfected SKN cells. We partially purified US-1 binding protein from SKN cells using a resin bead affinity procedure. Binding activ ity could be concentrated, although the protein eluate still comprised more than 20 component polypeptides. The molecular weight of the principal prot ein-DNA complex was determined following UV crosslinking to be 70 kDa. In c ircumstances where a cell-line mimicking the pregnant uterus at term is not available, the differential EMSA strategy, comparing OTR DNA-protein bindi ng in up- and down-regulated tissues, provides a powerful tool to investiga te OTR regulation in the uterus. However, the precise characterization and identity of the specific DNA-binding protein(s) and consequent experimental verification of regulatory mechansims still require elucidation. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.