Two Progesterone-Dependent Endometrial Nuclear Factors Bind to an E-Box in the Rabbit Uteroglobin Gene Promoter: Involvement in Tissue-Specific Transcription

Abstract

We studied the implications of progesterone-dependent transcription factors in the hormonal and tissue-specific induction of the uteroglobin gene (ug) in the rabbit endometrium. Previously, we have observed the interaction of two progesterone-dependent endometrial nuclear proteins (TRBPs) with sequences downstream from theugTATA box. Using electrophoretic mobility shift assays (EMSA) we show here that TRBPs specifically interacted with an E-box localized almost immediately downstream from theugTATA box. UV crosslinking of affinity-purified TRBPs to the radiolabeled oligonucleotide probe confirmed that these factors were proteins with molecular mass of about 40–50 kDa. Ferguson's analysis of theMrof the DNA–TRBP complexes suggested that TRBPs interacted with the E-box either as homo- or heterodimers. This interaction did not result in detectable bending of the DNA. EMSA analysis with nuclear extracts from different rabbit tissues suggested that TRBPs might be endometrium-specific nuclear factors. Involvement of the E-box in the tissue-specific transcription from theugpromoter was assessed by transient expression experiments using different cell lines transfected with a reporter gene driven by theugpromoter which contained either the intact E-box or a mutated version that completely abolished its interaction with TRBPs. These experiments indicated that, in all cell lines of nonendometrial lineage, destruction of the E-box increased transcription from theugpromoter, whereas in two cell lines of endometrial origin this mutation either had no appreciable effect or slightly reduced the transcription from the promoter. Thus, this E-box and endometrial helix–loop–helix proteins might be involved in the hormonal and tissue-specific regulation ofugtranscription.