Long-Range Transcriptional Regulation of Placental and Ovary Specific Genes

National Institute On Aging

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Abstract

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. Further, we have evidence that the gene is transcribed from two alternate promoters P1and P2, spaced 97 Kilobases apart. We have cloned both promoters from mouse and human and fused them to a Luciferase reporter gene and have defined the minimal promoter regions. Computer analysis of the minimal promoter suggested the presence of binding sites for nuclear receptors Retinoic Acid X Receptor alpha (RXR-alpha), Liver X Receptor-beta (LXR-beta), and Estrogen related receptor beta (ERR-beta) at both promoter sites. In the presence of agonists for these receptors, individually or in combination, the luciferase activity from the reporter constructs is stimulated significantly, confirming a definitive role for these nuclear receptors in regulating the promoter activity. Mutations in the core nucleotide binding sites for the receptor also significantly reduced the activity. We note that in vivo the P2 promoter is preferred compared to P1 promoter in placenta. However, in several cancer cell-lines we tested, P1or P2 or both P1 and P2 promoters are used. This perhaps reflects the levels of auxiliary factors in cells. For FOXL2, we have isolated sequences as much as 200 kb upstream of the transcription start site that contribute to its regulation, 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-fractionated 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 for mouse R1-9 ES cells and identified 9,370 expressed proteins, and for total kidney, 6000 proteins. Analysis of the metabolic clustering of inferred proteins show that complex tissue proteomics can be analyzed in terms of metabolic and signaling pathways as well to specifying the components of functional organelles and structures.