Transcription Regulation of Placental /Ovary Specific Ge

我们利用大片段克隆获得了酵母和细菌人工染色体克隆(YACs和BACs)，其中包含两个在人类生殖中起重要作用的基因：PLAC1，在胎盘的三层之一中唯一表达；以及FOXL2，仅在发育中的眼皮和卵泡细胞中表达。[FOXL2基因缺陷会导致某些女性卵巢早衰。]我们的目标是确定这些基因异常选择性的组织特异性表达的基础。与肝脏特异性基因等其他情况不同，10kb以下的启动子DNA片段不能复制组织特异性。PLAC1或FOXL2。这表明较长期的监管是可行的。与这一概念一致，扰乱FOXL2转录的易位位于离基因序列非常远的地方(约168kb)。 在这些研究的下一阶段，我们将重点放在人类和小鼠的Plac1基因上，利用细菌中基于重组的方法，我们正在探索将GFP(绿色荧光蛋白)作为报告基因整合到包含人PLAC1基因的BAC中，以及在哺乳动物细胞中可选择的药物标记。在初步研究中，含有至少60kb上游序列的人PLAC1基因的BAC，在3‘UTR区用GFP盒改造，在Jeg3绒毛膜癌细胞系中表达，这也符合长期调控。现在的目标是系统地切除BAC衍生物中的启动子区域，并使用转基因研究来确定DNA的上游调节区和相应的染色质结构的特征。在蛋白质水平的另一种方法中，用针对人类和小鼠蛋白质的选定表位的兔抗体进行的实验表明，PLAC1蛋白质在体内以二聚体的形式存在，经历了大量的蛋白质修饰，并定位于细胞质中。实验正在进行中，以跟踪具有FLAG表位的人类PLAC1蛋白，并通过与抗FLAG抗体的免疫沉淀寻找相互作用的伙伴。该标志片段将BAC编码的PLAC1与内源性表达的蛋白区分开来。在这些研究的同时，小鼠Plac1基因也正在与GFP融合，以构建转基因，并在发育过程中确定其表达模式，与正在产生小鼠基因敲除的M.Fant博士合作。基于基因芯片数据，我们最近还发现了另外两个基因，PLAC8和Plac9，它们在胎盘中表达丰富。它们为调控信号的最终比较分析提供了进一步的靶点；在PLAC1模型表征之后，也为FOXL2基因预测了类似的方法。

We have used large-insert cloning to obtain yeast and bacterial artificial chromosome clones (YACs and BACs) containing two genes of importance in human reproduction: PLAC1, expressed uniquely in one of the three layers of the placenta, and FOXL2, expressed only in developing eyelids and in follicular cells of the ovarian follicles. [Deficiency in FOXL2 leads to Premature Ovarian Failure (POF) in some women.] The goal is to determine the basis for the extraordinarily selective tissue-specific expression of these genes. In contrast to other instances such as liver-specific genes, the tissue specificity is not reproduced by segments of putative promoter DNA up to 10 Kb 5? of PLAC1 or FOXL2. This indicates that longer-range regulation is operative. Consistent with that notion, a translocation that disrupts transcription of FOXL2 lies very far (about 168 Kb away) from the gene sequence. In the next phase of these studies we are focusing on both human and mouse Plac1 genes, Using recombination-based methods in bacteria, we are exploring the incorporation of GFP (green fluorescence protein) as a reporter gene into a BAC containing the human PLAC1 gene, along with a drug marker selectable in mammalian cells. In preliminary studies, a BAC containing the human PLAC1 gene with at least 60 Kb of upstream sequences, retrofitted with a GFP cassette in the 3'UTR region, gives expression in Jeg3 choriocarcinoma cell line, again consistent with long-range regulation. The aim now will be to resect the promoter region systematically in derivatives of the BAC, and use transfection studies to define the upstream regulatory regions in DNA and features of the corresponding chromatin structure. In a further approach at the protein level, experiments with rabbit antibodies raised against selected epitopes from both human and mouse proteins suggest that the PLAC1 protein exists in vivo as a dimer, undergoes substantial protein modifications, and is localized in the cytoplasm. Experiments are in progress to follow the human PLAC1 protein with the FLAG epitope, and to look for interacting partners by immunoprecipitation with anti-FLAG antibodies. The FLAG segment distinguishes the BAC-encoded PLAC1 from endogenously expressed protein. In conjunction with these studies, the mouse Plac1 gene is also being fused with GFP to construct a transgene and determine its expression pattern during development, in collaboration with Dr. M. Fant, who is generating a mouse knockout. Based on microarray data, we have also recently identified two more genes, Plac8 and Plac9, whose expression is enriched in placenta. They provide further targets for eventual comparative analysis of regulatory signals; and comparable approaches are also projected for the FOXL2 gene after the PLAC1 model is characterized.