Transcriptional regulation of ovine placental lactogen
The proximal promoter (−380/+16) of the ovine placental lactogen (oPL) gene provides trophoblast-specific expression in vitro. Footprint 6 (FP6; −319/−349) lies within this region, and block mutations spanning FP6 inhibit transactivation in BeWo (human choriocarcinoma) cells. Two-base pair mutations were created spanning the length of FP6 and transiently transfected into forskolin treated BeWo cells. Decreases (p ≤ 0.05) in activity were identified with those mutations that encompassed potential binding sites for CCAAT-enhancer binding protein (CEBP) and Specificity Proteins (Sp). Co-transfections with dominant negative CEBP constructs did not decrease transactivation. Additionally, over-expression of CEBP-α, CEBP-β and CEPB-δ did not result in increased activation. Furthermore, electroporetic mobility shift analysis (EMSA), using binucleate cell (BNC), HeLa and Juarkat cell nuclear extracts, the FP6 sequence and CEBPα antisera did not result in a supershift, indicating that the CEBP proteins may not interact with the FP6 domain.
In contrast, co-transfection assays with Sp1 and Sp3 over-expression constructs and the −380 oPL promoter construct significantly (p ≤ 0.05) increased transactivation in BeWo cells. Co-transfections with these Sp1 and Sp3 over-expression constructs and a FP6/minimal prolactin promoter construct also resulted in significant (p ≤ 0.01) increases in transactivation. Additionally, co-transfections with Sp1 and Sp3 short-hairpin RNA constructs caused reduced (p ≤ 0.01) transactivation. In EMSA supershift assays, Sp1 and Sp3 antibodies were able to inhibit migration of the complexes formed from labeled oligonucleotide sequences derived from FP6, and nuclear extracts from BNC and BeWo cells. Western analysis showed that Sp3, but not Sp1, is present in nuclear extracts of HeLa, Jurkat and BNC cells. Furthermore, Southwestern analysis with a FP6 oligonucleotide identified a nuclear protein specifically binding the FP6 region which corresponds with the Sp3 protein identified via Western analysis. In conclusion, these results indicate that Sp3 is capable of interacting with the FP6 region of the ovine placental lactogen gene proximal promoter and may function to enhance its transactivation.