A functional composite cis-element for NFκB and RBPJκ in the rat pregnancy-specific glycoprotein gene

The rat pregnancy-specific glycoprotein gene rnCGM3 is primarily expressed in the placenta. Previously, three DNase I footprinting sites (FPI, FPII, and FPIII) were identified in the rnCGM3 promoter region, a yeast one-hybrid screen was performed to identify the nuclear factors binding to the FPIII (5'-GCCTGGGAAAAAACTC-3') element, and RBPJ kappa, a downstream effector of the Notch signaling pathway, was identified as one of the FPIII-binding factors. In the present study, the NF kappaB member p65 was identified as another FPIII-binding factor. Electrophoretic mobility shift assays showed that NF kappaB members, including p50 and p65, bound to the FPIII site. The core binding sequence in the FPIII element for p50 and p65 is GGGAAA, which overlaps with that for RBPJ kappa. Competition exists between p50 and RBPJ kappa for binding to the FPIII element. Transient expression analyses revealed that p65 significantly stimulated the expression of a reporter gene directed by the NF kappaB core sequence in the FPIII element. However, RBPJ kappa could block this stimulation. These results suggest that the regulation of rnCGM3 expression involves both NF kappaB and RBPJ kappa, and they are mutually exclusive in the FPIII element.