Transcriptional regulation of ovine placental lactogen

Abstract

The placenta is a transitory organ, produced only during gestation, with the sole purpose of maintaining and promoting fetal growth. During gestation the growth and development of the fetus is regulated by a variety of hormones and growth factors, many of which are produced by the placenta. The placental lactogens are among the hormones produced during this unique period and they function to maintain fetal growth through repartitioning of nutrients to the fetus. During gestation there are many abnormalities which can occur, many of which result from aberrations in the production of these hormones. Intrauterine growth restriction (IUGR) is one such gestational abnormality thought to result from placental insufficiency. With IUGR pregnancies the risk of death increases with gestational age. In addition, the long term consequences of IUGR include a predisposition to diseases such as cardiovascular disease, hypertension, diabetes and coronary artery disease. Placental lactogen is also decreased in these IUGR pregnancies, indicating its importance in fetal development. The sheep provides a good model by which to study the hormones produced during gestation. Therefore, understanding the regulation of ovine placental lactogen (oPL) may lead to potential genetic therapies for IUGR pregnancies. The ovine placental lactogen gene has been characterized and its transcriptional regulation examined to identify cis elements which bind nuclear proteins that function as transactivators. Within 1.1 kb of the oPL gene proximal promoter, 19 footprints were identified via DNase I protection analysis. Within -383 bp of the proximal promoter region, relative to the transcriptional start site, six footprints resided. Three of these, FP1, FP2 and FP3 were within -124/+16 bp, the minimal promoter region. This region was responsible for trophoblast-specific transactivation in human (BeWo) and rodent (Rcho-1) choricarcinoma cell lines. Various trans-acting factors were found to interact with these footprints. Footprint I contained an initiator element, FP2 contained a GATA site and an AP-2α site, FP3 contained another GATA element and a Purα element and FP4 contained an E-Box. Two footprints, FP5 and FP6, reside within the -383/-217 region responsible for full in vitro activation of the oPL promoter, containing a direct repeat of a GAGGAG sequence. This GAGGAG sequence was found to be functional through mutation analysis. The region between -383/-217 contains the FP6 sequence found to be necessary for trophoblast-specific transactivation through block mutation analysis. Potential binding sites for CEBP-α and the Sp proteins were identified within the FP6 sequence (-319/ -349) through a transcription factor analysis program. This coincided with the GAGGAG sequence within FP6 previously found to be functional through mutation analysis. Transfection analysis utilizing two-base pair transversion mutations along the length of FP6 showed significant (p ≤ 0.05) reductions in transactivation with mutations 2, 4-6, 810 and 12 in human (BeWo) choriocarcinoma cells compared to the wild type -380 promoter construct indicating these base-pairs were necessary for transactivation. Co transfection analysis with over-expression constructs for the CEBP-α, β and δ proteins did not significantly increase activation of the oPL promoter. Additionally, dominant negative co-transfections with a CEBP-α specific and a general CEBP construct did not result in significant reductions in transactivation. In conjunction with supershift analysis, in which a CEBP-α antibody did not produce a supershift in BeWo and binucleate cell nuclear extracts, these results suggest that the CEBP proteins are not interacting with FP6 to activate transcription. Co-transfection analysis with over-expression constructs for both Sp1 and Sp3 significantly (p ≤ 0.05) increased transactivation in BeWo cells compared to the wild type -380 promoter construct. Additionally, co-transfections with the FP6 sequence in front of a minimal prolactin promoter construct, and Sp1 and Sp3 expression constructs also significantly (p ≤ 0.01) increased transactivation. When these proteins were inhibited through siRNA co-transfections both resulted in significant (p ≤ 0.01) decreases in transcriptional activation, indicating that they are important for regulation of the oPL promoter. Sp1 increased transcription to a greater degree than Sp3 in the over-expression co-transfections, while Sp3 resulted in a greater inhibition in the siRNA co-transfections, indicating that perhaps Sp3 is more important and already being expressed at a higher concentration. Supershift analysis showed that both Sp1 and Sp3 were in binucleate cells (BNC) and were binding to the FP6 region. Western analysis showed that Sp3 was in BNC and further Southwestern analysis revealed that this protein was specifically binding to the FP6 region. However, Sp1 did not show the same migration pattern in Western or Southwestern analysis, indicating that Sp1 may not be binding to the FP6 region in order to stimulate transcription. Together, these data indicate that Sp3 may be the trans-acting factor binding to the FP6 region and functioning to stimulate transcription of the oPL gene.