Identification of cis-elements and trans-acting factors that are involved in rat placental lactogen II (rPLII) gene expression in placenta

A large family of PRL related genes have been identified in rodents and ruminants, which are expressed only during pregnancy in the placenta and maternal decidua. The rodent family members demonstrate highly specific cell and temporal expression patterns, but little is known about the factors that regulate this expression. I have used the rat placental lactogen II (rPLII) gene as a model to further our understanding of the transcriptional regulation of this family and placental genes in general.; Previous work identified an enhancer element containing two DNAse I protected regions within a proximal 3 Kb 5' flanking fragment that appeared to contain sequences important for placental specific expression. I was able to show by electrophoretic mobility assays (EMSA) that the FP1 region bound members of the Elf subfamily of Ets transcription factors. When database searches were unsuccessful in identifying binding factors to the FP2 region I applied a proteomics approach using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) to identify nuclear factors from trophoblast giant cells that bound the enhancer element in vitro. Using mass matching and tandem MS sequencing my collaborators and I identified AP2gamma, a known placental-specific transcription factor in rodents, as a factor that could bind the rPLII enhancer sequence. EMSA experiments confirmed this binding and showed that it was specific for the FP2 region of the element. Reporter expression studies in transfected cells indicated that enhancing activity was increased by exogenous AP2gamma and lost when the FP2 sequence was mutated. Chromatin immunoprecipitation experiments confirmed that AP2gamma was associated with DNA at the enhancer site in the chromatin of rPLII expressing Rcho cells. My data strongly suggest that AP2gamma binding to the rPLII enhancer is likely to be an important component of the developmental expression of this gene during pregnancy.; With other members of the laboratory I undertook studies in transgenic mice, using a large P1 genomic clone to try to identify further regulatory sequences outside the 3 Kb 5' fragment that might be involved in the more complete, high level rPLII expression. We showed that the clone contained the rPLII gene, with approximately 4 Kb of 5' flanking sequence and complete 3' flanking DNA, a previously unknown rPLII-related pseudogene, the rPLP-I gene and the rPLP-B gene; I characterized the pseudogene, the first to be described in this gene family. The latter two genes contained complete 5' flanking regions and complete, or in the case of rPLP-B, partial 3' flanking sequences. Three transgenic mice were analyzed for rPLII and rPLP-B mRNA expression. Placentas from two fetuses expressed low levels of rPLII as compared to mouse PLII, but levels of rPLP-B that were comparable to the endogenous mouse gene. Our results suggest that regulatory sequences within a few kilobases of the transcription start site, as opposed to more distal sequences, play an important role in the high level expression of at least some members of this gene family, although they do not rule out a role for a locus control region as has been identified in some other large gene families.; To identify further regulatory regions in the rPLII 5' flanking DNA I carried out DNAse I hypersensitive studies using chromatin from rat placenta, rPLII-expressing Rcho rat trophoblast cells and non-expressing C6 rat glioma cells; results with rat placental genomic DNA indicated hypersensitivity in the region of the enhancer element. My results suggest that the Rcho cells which have served as a model system for the rat placental giant cell in many studies may not be as informative in these hypersensitivity studies as placental tissue itself.; My studies are the first to identify a role for a known placental-specific transcription factor in the regulation of a member of the PRL gene family; however, since AP2gamma is expressed in al...