Long-Range Transcriptional Regulation of Placental and Ovary Specific Genes

我们利用大插入克隆技术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重要的基因：一个是PLAC1，在胎盘中唯一表达，其表达仅限于面向母体组织的特定胎盘区域；在老鼠的跨种杂交的胎盘问题中，在胎儿健康和人类怀孕的成功结果中，它都有牵连。另一种基因FOXL2仅在发育中的眼睑和卵巢卵泡细胞中表达，FOXL2的缺乏会导致一些女性卵巢早衰（POF）（见AG000647-05）。目标是确定这些基因的选择性组织特异性表达的基础。最近我们关注的是PLAC1的调控和功能。最近的EST数据库调查表明，人类PLAC1可能在先前定义的3个外显子之外还有额外的5外显子。我们现在已经证明该基因有3个额外的非编码外显子，导致总共6个外显子跨越200 Kb，至少有5个异构体是通过选择性剪接形成的。基因组中第1外显子的转录起始点与下一个基因的转录末端之间的距离仅为5300 bp，这表明大多数启动子元件位于该区域。我们克隆并融合了这个5.3 Kb的片段，包括第一个外显子，到一个荧光素酶报告基因上，发现与无启动子的载体相比，它在胎盘源细胞中提供了高达60倍的荧光素酶活性。因此，启动子活性驻留在这个片段中。删除构建去除紧邻第1外显子的700 bp独特序列，从而消除报告活性。我们现在正在对特定转录所需的启动子序列进行精细的结构映射。为了研究该基因在胎盘中的功能，还使用重组方法从小鼠BAC中恢复了包含Plac1编码和侧翼序列的片段。将克隆的片段进行修饰，去除Plac1序列，并用一个可选择的新霉素耐药标记代替。该构建体正在被转染到C57/BL6 ES细胞中，以产生敲除小鼠模型。

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: 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). The goal is to determine the basis for the extraordinarily selective tissue-specific expression of these genes. We have recently focused our attention on the regulation and function 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, resulting in a total of 6 exons spanning 200 Kb, with at least five isoforms made by alternative splicing. The distance between the putative start of transcription of the 1st exon and the end of the next gene in the genome is only 5300 bp, suggesting that most promoter elements are located within this region. We cloned and fused this 5.3 Kb fragment, including the 1st exon, to a Luciferase reporter gene, and find that it gives up to 60 fold higher luciferase activity in placental derived cells, when compared to a promoter-less vector. Promoter activity thus resides in this fragment. Deletion constructs removing 700 bp unique sequence immediately neighbouring the 1st exon abolishes the reporter activity. We are now doing fine structure mapping of the promoter sequences necessary for specific transcription. 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.