Long-Range Transcriptional Regulation of Placental and Ovary Specific Genes

对于FOXL2，我们分离了转录起始点上游长达200kb的序列，这些序列有助于FOXL2的调控，并表明几个序列元件结合在一起来解释该基因的组织特异性调控。为了确定FOXL2结合的靶点和含有结合位点的基因，我们从FLAG标记的FOXL2过表达的小鼠胚胎干细胞中用抗FLAG抗体进行染色质免疫沉淀。用抗FOXL2抗体对小鼠卵巢组织(以及第二次表达该基因的垂体细胞系)进行平行免疫沉淀，并比较其结合位点基序。结果表明，结合位点和表达谱结果一致，表明一组基因的表达存在正相关性，目前还处于手稿形式。 对于PLAC1，为了确定其异常选择性的组织特异性表达的基础，我们证明了该基因是由两个启动子P1和P2表达的，两个启动子间隔105kb，并交替剪接。通过克隆小鼠和人的启动子，确定了最小启动子区域。最小启动子区域与核受体维甲酸X受体α(RXR-α)、维甲酸X受体-β(LXR-β)和类固醇生成因子1(SF1)/雌激素相关受体β(ERR-β)在特定的位置结合，在它们各自的激动剂存在下，它们的结合具有刺激转录10倍的正效应。 在第二篇发表在Oncogensis(2013)上的文章中，通过建立癌细胞系的经典方法-SV40介导的原代细胞WI38和IMR90细胞的转化-评估了Plac1在癌细胞中的表达。我们发现，在SV40介导的转化后，原代细胞诱导PLAC1和一系列步骤是由SV40早期区域编码的大T抗原催化的，这些T抗原改变了通常与启动子区域结合的TP53抑制物特性，使其失去抑制能力，使染色质从封闭状态转变为开放状态，促进Plac1转录。然后在核受体存在的情况下进一步刺激转录，如果存在额外的辅助激活因子NCOA2(核受体辅助激活因子2)，它就招募RB，导致该基因的额外上调。因此，我们已经确定了该基因在癌细胞中被激活的主要方式，从而提供了一条抑制该基因活性的途径。 为了研究PLAC1基因在体内的功能，与M.Fant博士合作，获得了Plac1基因敲除(KO)小鼠品系。这些小鼠的胎盘结构被严重扰乱，海绵滋养层扩张，迷宫层结构完整性扭曲，纯合子胎仔的存活率急剧下降。因此，Plac1的适当表达对小鼠的正常发育是必不可少的。此外，应该注意的是，当KO基因是父系遗传时，雌性母体Plac1基因的良好副本可以补偿Plac1功能，胚胎是正常的，但当敲除基因是从母亲那里遗传来的时，胎盘缺陷是严重的，因为父系X染色体优先在胎盘中失活，因此不起作用。因此，在正常雄性和突变杂合子雌性之间的杂交中，后代中没有KO雄性小鼠，但它们可以在子宫中被检测到，这表明它们没有怀孕到足月，或者在出生后立即丢失。目前，我们有a)对不同胚胎阶段胎盘基因表达变化的基因表达变化进行微阵列分析；b)工程Plac1 KO胚胎干细胞(ESCs)分化为滋养层细胞并跟踪基因表达的变化；c)从野生型ESCs中表达可控启动子的Plac1，并比较其对转录谱的影响。结果表明，尽管Plac1不是转录因子，但在ESCs中Plac1的缺失或增加对基因表达有深远的影响。其他实验正在进行中，以了解这些影响的主要原因。。在生化水平上，我们有证据表明Plac1是细胞膜的一个组成部分，其中一些是由细胞分泌的。我们已经通过免疫沉淀和质谱分析确定了潜在的相互作用伙伴，为蛋白质如何对细胞产生影响提供了线索。

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.