PARENT SESSION
GENE REGULATION AND FUNCTION - B

Wednesday, August 4, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(691) TRANSCRIPTIONAL REGULATION OF THE OVINE PLACENTAL LACTOGEN GENE PROXIMAL PROMOTER.

Jeckel, Kimberly¹, Limesand, Sean¹, Anthony, Russell¹, ¹ Colorado State University, Fort Collins, CO

ABSTRACT- Placental lactogen (PL), a hormone expressed solely during gestation, may act to alter maternal metabolism through re-direction of nutrients to the fetus, and may be necessary for optimal fetal growth. Thus, understanding the cell-specific regulation of this gene could provide insight into the factors controlling fetal development. The proximal promoter (-383/+16) of the ovine (o) PL 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. Our current focus was to identify and functionally characterize the trans-acting factors interacting with FP6. Two base-pair transversion mutations were created spanning the length of FP6, and transiently transfected into BeWo cells. Significant decreases (p ≤ 0.05) in activity were identified within regions of FP6 that encompassed potential binding sites for CCAAT-enhancer binding protein (CEBP) and Specificity Proteins (Sp1 or Sp3). Transfection of CEBP dominant negative constructs resulted in a significant inhibition of transactivation of the wild-type -383/+16 promoter. Co-transfection of CEBP expression constructs (CEBP, CEBP and CEPB) with the wild-type promoter revealed that CEBP over-expression resulted in increased (p ≤ 0.003) transactivation, whereas CEBP and CEBP did not. Competitive electrophoretic mobility shift assays (EMSA) provided evidence that oligonucleotide sequences derived from FP6 were effective in competing for complex formation with consensus CEBP and Sp binding sites. Furthermore, antibodies raised against CEBP and Sp3 inhibited EMSA complex formation. These results indicate that CEBP and Sp proteins are capable of interacting with the proximal promoter region of the ovine placental lactogen gene, and may function to enhance its transactivation. Supported in part by USDA-NRI grant 99-35203-7817.

KEY WORDS: sp1, cebp, placental lactogen, sp3