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. For PLAC1, to determine the basis for its extraordinarily selective tissue-specific expression, we showed that the gene is expressed from two promoters, P1 and P2, spaced 105 Kilobases apart and is alternatively spliced. By cloning both promoters from mouse and human, 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 and their binding has a positive effect stimulating transcription >10 fold, in the presence of their respective agonists. In a second publication, in Oncogenesis (2013), Plac1 expression in cancer cells was evaluated by a classical approach 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 a major way in which the gene is activated in cancer cells, which thereby provides a route to repress the gene activity. To address the in vivo function of the PLAC1 gene, in collaboration with Dr. M. Fant a Plac1 knock out (KO) mouse strain was derived. The placental structure in these mice is grossly perturbed, with an expanded spongiotrophoblast layer and distorted labyrinthine layer structural integrity, and sharply reduced viability of homozygous litters. Thus appropriate expression of Plac1 is essential for normal mouse development. Further, it should be noted that 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 preferentially inactivated in placenta and thus, is non-functional. Consequently, in crosses between normal males and mutant heterozygous females, the litters are devoid of KO male mice, but they can be detected in utero, suggesting that they are not carried to term or are lost immediately following birth. Currently, we have a) carried out microarray analysis of gene expression changes in placentas retrieved from different embryonic stages;b) engineered Plac1 KO embryonic stem cells (ESCs) to differentiate into trophoblast cells and followed the changes in gene expression;and c) expressed Plac1 from a controllable promoter in wild-type ESCs and compared effects on transcription profiles to those in KO ESCs. The results show that loss or gain of Plac1 in ESCs has profound effects on gene expression, even though it is not a transcription factor. Additional experiments are underway to understand the primary cause of these effects. . At the biochemical level, we have evidence to show that Plac1 is a component of the cell membrane, with some of it secreted from cells. We have identified potential interacting partners by immunoprecipitation followed by mass spectrometry, giving clues as to how the protein might produce its effects on cells.
