To determine the basis for the extraordinarily selective tissue-specific expression of PLAC1, we showed that the gene is expressed from two promoters, P1 and P2, spaced 105 Kilobases apart. We cloned both promoters from mouse and human and, defined the minimal promoter regions. The minimal promoter region binds nuclear receptors Retinoic Acid X Receptor alpha (RXR-alpha), LXR-beta, and Steroidogenic factor 1 (SF1)/Estrogen related receptor beta (ERR-beta) at specific sites. In the presence of those factors and respective agonists, transcription is stimulated >10 fold. This work is now published in Placenta. Plac1 expression in cancer cells was also evaluated by a classical approach of establishing cancer cell lines;SV40 mediated transformation of primary cells WI38 and IMR90 cells. We found that following SV40 mediated transformation the primary cells induced PLAC1 and a series of steps are catalyzed by Large T antigen encoded by SV40 early regions that modify Tp53 repressor properties normally bound to the promoter region such that it loses its repressive ability, bring about changes in chromatin from closed to open status facilitating Plac1 transcription. The transcription is then further stimulated in the presence of nuclear receptors and if an additional coactivator NCOA2 (nuclear receptor co-activator2) is present, it recruits RB leading to additional up regulation of the gene. Thus, we have defined one of the ways in which the gene is activated in cancer cells and this provides a route to repress the gene. This work is now in press in Oncogenesis (2013) under the title T antigen transformation reveals Tp53-/RB-dependent route to PLAC1 transcription activation in primary fibroblasts. We have also assessed the proteomics profile of placenta and compared it to its transcription profile. Using two-dimensional micro-high pressure liquid chromatography coupled to tandem mass spectrometric (MS/MS) analysis, we identified 21,781 peptide signatures, 13,409 of which were unique and were assigned to 6,415 proteins. Using our computing resources, we curated the NCBI mouse protein NR database to collate and unify multiple protein IDs represented in the Genbank database. The recovered proteins represent all known intracellular compartments and a full range of isoelectric charge;thus the fractionation method showed no apparent bias. Of particular interest, 1299 proteins that had been predicted solely on the basis of sequence analysis have now been substantiated as true products of translation from transcribed genes. In comparative ongoing work we have analyzed the proteomics data for mouse R1-9 ES cells. To address the in vivo function of the PLAC1 gene, in collaboration with Dr. M. Fant we have derived a Plac1 knock out (KO) mouse strain. Their placental structure in these mice is grossly perturbed, with an expanded spongiotrophoblast layer and distorted labyrinthine layer structural integrity, and sharply reduced viability. Further, when the KO gene is inherited paternally, the good copy of the maternal Plac1 gene in females can compensate Plac1 function and the embryos are normal, but when the knockout genotype is inherited from the mother the placental defects are severe because the paternal X chromosome is normally inactivated in placenta and is non-functional. Further, in the crosses between the normal males and mutant heterozygous mice, the litters are devoid of homozygous KO mice although can be detected in utero, suggesting that they are not carried to term or lost immediately following birth. Therefore some level of Plac1 is essential for normal placental well being. We also note that the pups born with defective gene tend to become obese as they age compared to their normal siblings. This is being further evaluated. Currently, we have a) carried out analysis of gene expression changes in placenta retrieved from different embryonic stages by microarray;and b) have engineered Plac1 KO embryonic stem cells to differentiate into trophoblast cells and following the changes in gene expression. We are now doing microarray analysis following their differentiation and analyzing and validating gene expression differences between KO ES cells and WT ES cells. For Foxl2, we have isolated sequences as much as 200 kb upstream of the transcription start site that contribute to its regulation, and have shown that several sequence elements combine to account for the tissue-specific regulation of the gene. To identify Foxl2 binding targets and the genes that contain the binding sites, we carried out chromatin immuno-precipitation with anti-flag antibody from mouse embryonic stem cells in which Foxl2 tagged with Flag is over expressed. Parallel immuno-precipitation from mouse ovarian tissue (and a hypophysis cell-line where the gene is secondarily expressed) was also done using anti-Foxl2 antibody, and the binding site motifs were compared. The results showed a correspondence of binding sites and expression profiling results for positive dependence of the expression of a cohort of genes, and are currently in manuscript form.
|Chen, Y; Schlessinger, D; Nagaraja, R (2013) T antigen transformation reveals Tp53/RB-dependent route to PLAC1 transcription activation in primary fibroblasts. Oncogenesis 2:e67|