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, we have now confirmed that the gene is expressed from two promoters, P1 and P2, spaced 97 Kilobases apart. Transcription from P1 leads to expression of 3 additional non-coding exons and the gene structure was revised to include these newly defined exons and the gene now spans 200 Kb. Further, there are at least five splice isoforms. We cloned both promoters from mouse and human and fused them to a Luciferase reporter gene and have defined the minimal promoter regions. In silico analysis of the minimal promoter region suggested the presence of binding sites for nuclear receptors Retinoic Acid X Receptor alpha (RXR-alpha), and Steroidogenic factor 1 (SF_1)/Estrogen related receptor beta (ERR-beta) at promoter sites. We have now shown that RXR-alpha can directly interact with the binding sites along with Liver X receptor-beta (LXR-beta) as a heterodimer. In the presence of RXR-alpha, LXR-beta and SF-1/ERR-beta, and their respective agonists transcription is stimulated >10 fold. Various mutations at the binding sites leads to impaired binding of nuclear receptors and loss of stimulation. Transcription from P2 promoter dominates in placenta compared to P1 promoter. However, in several cancer cell-lines we tested, P1 or P2 is predominantly used. This perhaps reflects the levels of auxiliary factors in cells. This work is now submitted for publication. Ongoing work involves designing and executing experiments to understand how the gene is silenced in non-placental tissues and trying to elucidate the pathways that are involved in activating the silent gene in cancer 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, 1299 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.
