For PLAC1, to determine the basis for its extraordinarily selective tissue-specific expression, we have shown 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 follow up 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 co-immunoprecipitation followed by mass spectrometry, confirmed that the interaction is real by reverse co-immnunoprecipitation. The results suggest that Plac1 interacts with a cellular membrane organelle. We have proven that these interactions require an intact ZP-N domain in PLAC1. The interactions with the membrane could result in changes in signaling. The microarray results and protein-protein interaction studies are in preparation for publication. In a related work on the regulation of FOXL2 gene, we have shown that FOXL2 mediates Col1a2 gene regulation (see attached reference). For rDNA structure analysis using TAR cloning methods we have isolated an initial set of 18 clones in a YAC-BAC vector from a chromosome 21 containing human-mouse hybrid cell-line and sequenced them using long-read method in a Pac-Bio instrument as well as Illumina short-read techniques. Sequence analysis shows that the clones contain ribosomal units in one or two copies and there are single nucleotide variants as well as insertions and deletions in these copies. Preliminary analyses indicate some variation, and efforts at validation are now in progress.