Long-Range Transcriptional Regulation of Placental and Ovary Specific Genes

我们正在研究两个在人类生殖中重要的基因：一个是PLAC 1，它在胎盘中独特表达，其表达仅限于面向母体组织的特定胎盘区域;它与小鼠种间杂交中的胎盘问题以及人类胎儿健康和成功妊娠结果有关。另一个基因FOXL 2仅在发育中的眼睑和卵泡的滤泡细胞中表达，FOXL 2的缺乏导致某些女性的卵巢早衰（POF）（参见AG 000647 -05）。我们的目标是确定这些基因的异常选择性组织特异性表达的基础。 关于PLAC 1的调控和作用，最近的EST数据库调查表明，人类PLAC 1可能有额外的外显子5到3个外显子以前定义。 我们现在已经表明，该基因有3个额外的非编码外显子，在小鼠和人类，导致总共6个外显子跨越200 Kb，至少有五个剪接异构体。我们已经克隆了小鼠和人的启动子，并将它们融合到荧光素酶报告基因。与无启动子的载体相比，它们在胎盘来源的细胞中提供高达60倍的荧光素酶活性。 因此，启动子活性存在于这些片段中。 序列缺失构建体已经进一步将人类和小鼠中的启动子区域缩小到几百个碱基对。 我们现在正在对该区域的转录因子进行精细结构定位。 为了研究该基因在胎盘中的功能，还使用重组工程方法从小鼠BAC中回收含有Plac 1编码和侧翼序列的片段。对克隆的片段进行修饰，以消除Plac 1序列，并用可选择的新霉素抗性标记替换它。该构建体正在被转染到C57/BL 6 ES细胞中以产生敲除小鼠模型。 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的调控，并且正在进行测试以确定哪些序列和因子导致FOXL 2表达的组织特异性。 为了补充这些研究，我们着手评估胎盘的蛋白质组学谱，并将其与其转录谱进行比较。通过SDS凝胶电泳对胎盘总蛋白进行预分级分离，并将回收的大小分级分离的蛋白进行胰蛋白酶消化和二维微高压液相色谱-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。对于2，809个蛋白质，从NCBI EST数据库中的一组8，387个EST中找到来自胎盘来源的匹配EST。 质谱结果为其余3，606种蛋白质的表达提供了直接证据。特别令人感兴趣的是，几个蛋白质，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。在正在进行的比较工作中，我们分析了R1-9 ES细胞和总肾脏产生的蛋白质组学数据，以确定约6000种蛋白质的肽特征。 对推测蛋白质的代谢聚类分析表明，复杂组织蛋白质组学可以从代谢途径和信号传导途径以及确定复杂组织中功能细胞器和结构的组成方面进行分析。

We are studying two genes of importance in human reproduction: One, PLAC1, is expressed uniquely in placenta, where its expression is restricted to specific placental regions facing the maternal tissue; and it has been implicated both in cases of placental problems in inter-specific crosses of mice, and in fetal well being and successful outcome of pregnancy in humans. The other gene, FOXL2, is expressed only in developing eyelids and in follicular cells of the ovarian follicles, and deficiency in FOXL2 leads to Premature Ovarian Failure (POF) in some women (see AG000647-05). Our goal is to determine the basis for the extraordinarily selective tissue-specific expression of these genes. Concerning the regulation and role of PLAC1, recent EST database survey suggested that the human PLAC1 might have additional exons 5 to the 3 exons previously defined. We have now shown that the gene has 3 additional non-coding exons, both in mouse and human, resulting in a total of 6 exons spanning 200 Kb, with at least five splice isoforms. We have cloned both mouse and human promoters and fused them to a Luciferase reporter gene. They provide up to 60-fold higher luciferase activity in placental-derived cells, when compared to a promoter-less vector. Promoter activity thus resides in these fragments. Sequential deletion constructs have further narrowed the promoter regions to several hundred base pairs in human and mouse. We are now doing fine structure mapping of transcription factors in this region. To study the function of the gene in placenta, recombineering methods were also used to recover a fragment containing Plac1 coding and flanking sequences from a mouse BAC. The cloned fragment was modified to ablate the Plac1 sequence and replace it with a selectable neomycin resistance marker. This construct is in the process of being transfected into C57/BL6 ES cells to generate a knockout mouse model. For FOXL2, we have isolated sequences as much as 200 kb upstream of the transcription start site that contribute to its regulation, and and are testing to identify which sequences and factors account for the tissue-specificity of its expression. To complement these studies, we undertook to assess the proteomics profile of placenta and compare it to its transcription profile. Using pre-fractionation of total proteins from placenta by SDS gel-electrophoresis and subjecting the recovered size-fractionated proteins to trypsin digestion and 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. For 2,809 proteins, matching ESTs from placental source were found from a set of 8,387 ESTs in the NCBI EST database. Mass spectrometric results provide direct evidence for expression of the remaining 3,606 proteins. Of particular interest, several 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 generated for R1-9 ES cells and total kidney to identify peptide signatures for about 6000 proteins. Analysis of the metabolic clustering of inferred proteins show the complex tissue proteomics can be analyzed in terms of metabolic pathways and signaling pathways as well as specifying the components of functional organelles and structures in complex tissues.