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. We have cloned both mouse and human promoters and fused them to a Luciferase reporter gene. They provide up to 60-fold higher luciferase activity in placental-derived cells, when compared to a promoter-less vector. Promoter activity thus resides in these fragments. Sequential deletion constructs have further narrowed the promoter regions to several hundred base pairs in human and mouse. We are now doing fine structure mapping of transcription factors in this region. 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.? For FOXL2, we have isolated sequences as much as 200 kb upstream of the transcription start site that contribute to its regulation, and 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-fractionation of 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 generated for R1-9 ES cells and total kidney to identify peptide signatures for about 6000 proteins. Analysis of the metabolic clustering of inferred proteins show the complex tissue proteomics can be analyzed in terms of metabolic pathways and signaling pathways as well as specifying the components of functional organelles and structures in complex tissues.
| Chen, Y; Moradin, A; Schlessinger, D et al. (2011) RXR? and LXR activate two promoters in placenta- and tumor-specific expression of PLAC1. Placenta 32:877-84 |
| Herrera, Luisa; Ottolenghi, Chris; Garcia-Ortiz, J Elias et al. (2005) Mouse ovary developmental RNA and protein markers from gene expression profiling. Dev Biol 279:271-90 |
| Crisponi, Laura; Uda, Manuela; Deiana, Manila et al. (2004) FOXL2 inactivation by a translocation 171 kb away: analysis of 500 kb of chromosome 3 for candidate long-range regulatory sequences. Genomics 83:757-64 |
| Galaviz-Hernandez, Carlos; Stagg, Carole; de Ridder, Gustaaf et al. (2003) Plac8 and Plac9, novel placental-enriched genes identified through microarray analysis. Gene 309:81-9 |
| Cocchia, M; Kouprina, N; Kim, S J et al. (2000) Recovery and potential utility of YACs as circular YACs/BACs. Nucleic Acids Res 28:E81 |
