PATHOLOGICAL & NORMAL FUNCTION OF A NOVEL HOMEOBOX GENE

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This application focuses upon a novel gene, Pem, we recently isolated. The cDNA was cloned from an AKR T lymphoma cell line. The cDNA sequence contains a homeobox motif and the predicted protein has some unique features. Pem is the first homeobox gene to be localized to the X chromosome. Pem is expressed in embryogenesis but not detectably in normal adult tissues or lymphoid cells; it is abundantly expressed in numerous transformed and immortalized cell lines. Pem is expressed in mature oocytes and in a highly regulated manner in the extraembryonic cells early in murine development. In situ analysis shows that some cells which express Pem invade the maternal uterine tissue during implantation. the possibility that Pem has a role in tumorigenicity or metastatic invasion will be investigated. The cell line from which the Pem clone was isolated causes prominent bilateral ovarian tumors in 95% of IV injected female AKR mice. the functional analysis of the Pem gene presents an opportunity to investigate a possible role of disregulated Pem expression in processes of transformation and/or invasion. The physiological and pathological function of the Pem gene will be examined using two basic approaches: "loss of function" and "gain of function" experiments. the loss of function studies will be accomplished by gene targeting (homologous recombination). Progeny of animals lacking Pem expression will be produced. Cells lacking Pem gene function will be tested in a variety of in vitro assays. Gain of Pem function will be accomplished by transfecting Pem cDNA into related SL12 T lymphoma cells and into normal AKR and human fibroblasts which do not express the gene and assessed for their tumorigenic potential. Pem gene expression will be further examined during embryogenesis, in pluripotent stem cells and in selected adult stem cell tissues by in situ hybridization and/or immunohistochemistry. The maturation status of Pem ablated stem cells will be experimentally altered to assess whether Pem function is required for in vitro differentiation. We will assess whether Pem expression is associated with the capacity of cells to form ovarian tumors. The fact that the Pem protein contains a homeodomain and is located in the nuclear compartment indicates that Pem functions as a transcriptional regulator. Our affinity purified anti-Pem antisera will be a useful adjunct to determine whether Pem protein 1) is post-transcriptionally modified; 2) functions in a complex or cooperatively with other proteins; 3) binds to DNA and/or complexes with DNA binding proteins to inhibit DNA binding.