Transcriptional regulation of the ovine placental lactogen gene

Abstract

Prenatal growth and development influences the quality of life one has after birth. The placenta is an essential organ during prenatal development and has endocrine functions that regulate maternal intermediary metabolism to provide nutrients for fetal growth. A member of the growth hormone / prolactin gene family produced exclusively by the placenta, placental lactogen (PL) has been described as a key hormone in nutrient regulation in the mother and fetus. However, conclusive data on PL's biological action has not been forthcoming and a reduction/ablation of PL will be required for true analysis. Therefore, transcriptional regulation of ovine (o) PL was examined to identify important c/s-acting elements that interact with nuclear proteins to regulate its production, which in turn could lead to methodologies to reduce oPL expression in vivo. The oPL gene has been structurally characterized and the in vitro transcriptional regulation was localized to the proximal 383 bp of the oPL gene promoter. Trophoblastspecific transactivation in rat (Rcho-1) and human (BeWo) choriocarcinoma cell lines was demonstrated within the -124/+16 bp region (minimal promoter) of the oPL gene, but full in vitro activity of the promoter required a region within -383/-217 bp. DNase I footprinting assays with chorionic binucleate cell (BNC) nuclear proteins identified potential protein-DNA interactions that may function to enhance transcription of the oPL gene. An initiator element encompassing the transcriptional start site (-12/+7) of the oPL gene is thought to be required for transcription because a functional TATA box was not identified. Footprint 2 (FP2) was confirmed to interact with AP-2α. Immunohistochemical analysis of AP-2α expression in the ovine placentome localized AP-2α in the BNC, but expression was not restricted to BNC and was identified in other cells in the chorionic epithelium. Three AP-2α splice variants were purified from a placental cDNA library. Two of the AP-2α spike variants (AP-2αv6 and AP-2αv7) stimulated transactivation through the oPL gene AP-2 element in HepG2 cells, which lack endogenous AP-2 expression. A third AP-2α was specific to sheep, but was not capable of enhancing transcription. These data indicate that regulation of AP-2α expression during trophoblast differentiation may effect oPL gene transactivation. Two GATA elements in the oPL gene at -67 bp (FP2) and -102 bp (FP3) are functional and interact with GATA-2. A novel cis-acting element (-109/-102) was identified in FP3 by mutational analysis in human (BeWo) choriocarcinoma cell transient transfections. A heat stable nuclear protein with an apparent Mr of 41,000 was found to interact with a CCACGA element in BeWo and BNC nuclear extracts. Screening a placental cDNA expression library by Southwestern techniques identified Purα, a single-strand DNA binding protein. Purα from BeWo and BNC nuclear extracts was found to interact with the novel element by electrophoretic mobility shift assays (EMSA) and supershift assays. Additionally, co-transfection of a Purα expression vector with the oPL minimal promoter stimulated transactivation in BeWo cells. A fourth footprint (-173/-137) protected a canonical E-box at -163 bp, and EMSA demonstrated a specific protein-DNA interaction at this site. However, the functionality of this element was not evident by deletion analysis in choriocarcinoma cell lines, but appears to play a context dependent role in choriocarcinoma cells. Two DNase I protected sites (FP5 and FP6) reside within a region of the oPL promoter that is required for foil transactivation of the promoter in choriocarcinoma cell lines. The cis-acting element in FP5 was identified as -260 CANAGGCT -253, and mutating the element reduced transactivation in BeWo and Rcho-1 cells. Three nuclear proteins specifically interact with FP5 in Southwestern analysis of BeWo nuclear extracts and two proteins were identified in Rcho-1 nuclear extracts, which may indicate a family of transcription factors or a hierarchy of regulation at this element. Footprint-6 was examined with EMSA and the cis-acting element, -337 AGGAGGGCATGGCAAC -322, was identified to interact with a BNC nuclear protein with an apparent Mr of ~140,000 by Southwestern analysis. A triplicate concatamer of FP6 enhanced trophoblast-specific transactivation in an orientation dependent, minimal promoter independent fashion, implicating this cis-acting element as a trophoblast-specific enhancer element for the oPL gene. These data indicate that oPL gene expression in BNC of ovine placenta is mediated by two known placental elements, AP-2 and GATA, but three novel cis-acting elements are important in its regulation. One of these elements functions by interacting with Purα, a single-strand DNA binding protein, which has not been previously shown to enhance transcription of placental genes.