Chorionic gonadotropin (CG) is a placental hormone that stimulates gonadal steroidogenesis and is important for maintenance of the corpus luteum of pregnancy. After implantation of the fertilized ovum, CG levels increase through the first trimester, after which they gradually decline. CG is also produced ectopically by a variety of neoplasms. The long-term goal of this proposal is to understand the cellular mechanisms that regulate CG biosynthesis at the level of gene expression. CG is a heterodimer consisting of nonidentical alpha and beta subunits that are encoded by separate genes. The alpha-subunit is encoded by a single copy gene, whereas the beta-subunit genes comprise a multigenic family that includes six CG beta genes and a single copy of the structurally-related LH beta gene. This project will address: 1) Determination of the DNA regulatory sequences of the alpha, CG beta, and LH beta genes using deletion mutagenesis and analyses of gene expression in transfected cells. These studies will focus on the cis-acting DNA sequences that confer cell-specific expression and hormone-responsiveness as well as the regulatory elements that result in coordinant expression of alpha and beta subunit genes. In addition, the regulatory DNA sequences of the recently diverged LH beta/CG beta gene pair will be exchanged onto the heterologous beta-subunit promoter to examine the mechanisms that regulate placental-specific expression of the CG beta gene and pituitary-specific expression of the LH beta gene. Similarly, the gene regulatory elements involved in ectopic gene expression will be sought by defining the DNA sequences that confer the capacity to express the CG alpha and beta genes in ectopic hormone-producing cell lines. 2) The trans-acting cellular factors that interact specifically with the alpha and beta gene regulatory elements will be examined using gel-retardation assays and DNA footprinting studies. These assays will also provide a means to monitor the purification of these DNA binding proteins during a series of isolation steps. Competition assays in transfected cells will be used to examine the cellular factors that interact with DNA regulatory elements. These studies will include experiments to assess interactions of alpha and beta regulatory sequences with shared regulatory factors that may mediate coordinant gene expression. 3) In parallel with the studies of transfected fusion genes, expression of the endogenous gonadotropin alpha and beta subunit genes will be examined in pituitary and placental cell lines as well as in pituitary tumors. These studies will focus on coordinant expression of the alpha and beta subunits, and characterization of defects in biosynthesis and secretion that occur in pituitary tumors.
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