Long-Range Transcriptional Regulation of Placental and Ovary Specific Genes

为了确定PLAC 1的非常选择性的组织特异性表达的基础，我们表明该基因由两个启动子P1和P2表达，间隔105千碱基。我们克隆了小鼠和人的启动子，并确定了最小的启动子区域。最小启动子区在特定位点结合核受体视黄酸X受体α（RXR-α）、LXR-β和类固醇生成因子1（SF 1）/雌激素相关受体β（ERR-β）。在那些因子和相应激动剂的存在下，转录被刺激>10倍。这项工作现在发表在胎盘。 还通过建立癌细胞系的经典方法评估了癌细胞中的Plac 1表达; SV 40介导了原代细胞WI 38和IMR 90细胞的转化。 我们发现，一系列步骤是由SV 40调控区的片段催化的，这些片段打开染色质，促进Plac 1转录;然后转录被核受体进一步激活。 我们还评估了胎盘的蛋白质组学谱，并将其与其转录谱进行了比较。使用二维微高压液相色谱结合串联质谱（MS/MS）分析，我们鉴定了21，781个肽特征，其中13，409个是独特的，并被分配给6，415个蛋白质。利用我们的计算资源，我们策划了NCBI小鼠蛋白质NR数据库，以整理和统一Genbank数据库中表示的多个蛋白质ID。回收的蛋白质代表了所有已知的细胞内区室和全范围的等电电荷;因此，分级分离方法显示无明显偏倚。特别令人感兴趣的是，1299个蛋白质，已经预测完全基于序列分析，现在已被证实为真正的产品翻译从转录基因。在比较正在进行的工作中，我们分析了小鼠R1-9 ES细胞的蛋白质组学数据。 关于PLAC 1基因的功能，与M.博士合作。假设我们已经得到了其中Plac 1被消融的小鼠品系。它们的胎盘结构严重紊乱，海绵滋养层扩张，胎盘层结构完整性扭曲，生存能力急剧下降。 此外，当KO基因是父系遗传时，雌性中母体Plac 1基因的良好拷贝可以补偿Plac 1功能并且胚胎是正常的，但是当敲除基因型是从母亲遗传时，胎盘缺陷是严重的，因为父系X染色体在胎盘中通常是失活的并且是无功能的。 因此，Plac 1对正常胎盘健康至关重要。 我们还注意到，与正常的兄弟姐妹相比，出生时有缺陷基因的幼崽随着年龄的增长往往会变得肥胖。 正在对此进行进一步评估。 目前，我们已经a）通过微阵列分析从不同胚胎阶段取出的胎盘中的基因表达变化;和B）已经工程化Plac 1 KO胚胎干细胞以分化成滋养层细胞。 我们现在正在进行微阵列分析，以跟踪它们的分化，并分析和验证KO ES细胞和WT ES细胞之间的基因表达差异。 对于Foxl 2，我们已经分离出了转录起始位点上游多达200 kb的序列，这些序列有助于Foxl 2的调节，并且已经表明几个序列元件联合收割机结合以解释该基因的组织特异性调节。 为了鉴定Foxl 2结合靶标和含有结合位点的基因，我们用来自小鼠胚胎干细胞的抗Flag抗体进行染色质免疫沉淀，其中Flag标记的Foxl 2过表达。还使用抗Foxl 2抗体从小鼠卵巢组织（和垂体细胞系，其中该基因是二次表达的）进行平行免疫沉淀，并比较结合位点基序。结果显示结合位点与一组基因表达的正相关性的表达谱分析结果的对应关系，并且目前为手稿形式。

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 a series of steps are catalyzed by segments of the SV40 regulatory regions that open chromatin, facilitating Plac1 transcription; and transcription is then further activated by the nuclear receptors. 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. Concerning the function of the PLAC1 gene, in collaboration with Dr. M. Fant we have derived a mouse strain in which Plac1 is ablated. Their placental structure 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 a mother the placental defects are severe because the paternal X chromosome is normally inactivated in placenta and is non-functional. Plac1 is thus 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. 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.