Structure and transcriptional regulation of the ovine placental lactogen gene.
Ovine placental lactogen (oPL), a member of the growth hormone/prolactin gene family, is produced by chorionic binucleate cells at the maternal-fetal interface, and is thought to modulate metabolic processes and enhance fetal growth. We have determined that the oPL gene contains five exons and four introns, and the transcriptional start site was mapped 91 bp 5' of the initiation codon (AUG). An additional 4.5 kb of 5'-flanking sequence was sequenced and used for transient transfection analysis in human (BeWo) and rat (Rcho-1) choriocarcinoma cell lines to examine trophoblast cell-specific activity. Trophoblast cell-specific transactivation of the reporter gene was conferred by the proximal 1. 1 kb of oPL gene 5'-flanking sequence. Transfection of deletion constructs derived from the 1.1 kb of 5'-flanking sequence resulted in varying profiles of transactivation between the two choriocarcinoma cell lines, but maximal activation in both cell lines resided within the proximal 383 bp of oPL gene 5'-flanking sequence. DNase I protection analysis using ovine chorionic binucleate cell nuclear protein, identified 19 footprints within the 1.1-kb sequence, six of which are located within the 383-bp region. Electrophoretic mobility-shift assays and mutational analysis identified two functional GATA (-67, -102) sequences as transactivators of the oPL gene. However, a previously undefined element (GAGGAG) residing at -338 and -283 is required for full transactivation, and mutation of either significantly reduces reporter activity. In addition, an AP-2 site (-58) and an E-box (-163) were identified and may coordinate oPL transactivation. Transcriptional regulation of human and rodent PL genes has been previously characterized, and our results indicate that tissue-specific regulation of oPL expression may result from cis-acting elements in common with human and rat genes expressed within the placenta. However, our data indicate that regulation of oPL also results from novel cis-acting elements.
Liang
R
R
Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Fort Collins, CO, USA.
Limesand
S W
SW
Anthony
R V
RV
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D005260:Female; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D010928:Placental Lactogen / Q000235:genetics*; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D051381:Rats; D012756:Sheep / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D014327:Trophoblasts / Q000378:metabolism
D017931:DNA Primers; D004247:DNA; D010928:Placental Lactogen