Transcriptional regulation of ovine placental lactogen

2005 2005

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Abstract (summary)

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.

Indexing (details)

Molecular biology
0307: Molecular biology
Identifier / keyword
Biological sciences; Ovine; Placental lactogen; Sp3; Transcriptional regulation
Transcriptional regulation of ovine placental lactogen
Jeckel, Kimberly M.
Number of pages
Publication year
Degree date
School code
DAI-B 66/08, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
0542268485, 9780542268489
Anthony, Russell
Colorado State University
University location
United States -- Colorado
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
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