Long-Range Transcriptional Regulation of Placental and Ovary Specific Genes

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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.