Long-Range Transcriptional Regulation of Placental and O

Aging

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Abstract

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, is expressed not only 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. 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. 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. [unreadable] We have now focused our attention on the regulation and function of PLAC1. Using an inducible lambda bacteriophage-based recombination system in bacteria, we have introduced a Flag tag into the C-terminal end of PLAC1 and retrofitted the BAC with a mammalian selectable drug marker, blasticidin. Upon transfection of this construct into choriocarcinoma derived BeWo cells which expresses native PLAC1 endogenously, we can detect transcripts derived from the modified BAC by RT-PCR. As a negative control, the same construct shows little if any transcription from the BAC in HeLa cells which do not express endogenous PLAC1. These results suggest that all the promoter elements necessary for specific transcription reside in the modified BAC. Next a set of targeted deletions removing 10Kb, 20Kb, and 62Kb blocks of DNA starting at the 5' to Exon 1 were generated using the recombination based approaches. These constructs are now being transfected and stable tranfectants established to check for 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 was transfected into ES cells, and neomycin-resistant ES cell clones are being screened for the loss of Plac1 as a step towards the generation of a knockout mouse model.