Chorionic gonadotropin (CG) is a placental hormone that stimulates steroidogenesis in the corpus luteum, providing an essential step in the maintenance of early pregnancy. Because it is expressed at high levels within days after fertilization and implantation, CG is used routinely to detect pregnancy at early stages and to monitor the progress of pregnancy during the first trimester. CG is also produced in a variety of different malignancies, and it is used as a tumor marker in these conditions. CG is a heterodimeric hormone that is encoded by separate alpha and beta subunit genes. The current proposal will focus on transcriptional control of the CGbeta subunit genes. There are six separate CGbeta genes that arose by gene duplication from an ancestral luteinizing hormone (LHbeta) gene. In the course of evolution, several of the CGbeta genes acquired unique regulatory sequences that dictate expression from a new upstream promoter site and thereby confer gene expression in the placenta rather than in the pituitary. The CGbeta gene also provides a model for understanding new pathways of transcriptional regulation by cAMP. We propose an integrated series of studies to identify regulatory DNA elements in the CGbeta promoter and to use these sequences as tools to identify transcription factors that regulate tissue-specific and cAMP-mediated expression of the CGbeta gene. The five specific aims are to: 1) Determine the functional properties of the six different CGbeta genes in vivo and in transfected cells. Identification of the functional and nonfunctional members of the CGbeta gene family provides a foundation for subsequent studies to elucidate the promoter sequences that are essential for CGbeta gene expression in the placenta; 2) Determine whether enhancer elements reside outside of 5'-flanking regions of the CGbeta genes. The CGbeta/LHbeta gene cluster will be analyzed using DNase I hypersensitivity and screened for intragenic and downstream enhancer elements that can activate expression of a minimal CGbeta promoter. 3) Define the base substitutions in the active CGbeta genes that confer placenta specific expression. Based upon comparisons of functional and nonfunctional promoter sequences, site-directed mutagenesis will be used to determine which of the relatively small number of nucleotide differences account for activation of the CGbeta promoter in placental cells; 4) Define, using site-directed mutagenesis, the sequence determinants of a novel cAMP response element (CRE) in the CGbeta promoter; 5) Identify and characterize the transcription factor(s) that binds to a repeated functional motif in the CGbeta CRE. Labeled CGbeta sequences will be used in an expression cloning strategy to identify a transcription factor that binds specifically to the CGbeta CRE. After cloning the transcription factor, patterns of expression will be analyzed in different tissues and during development and its functional properties will be examined in transfected cells. The results of these studies will further our understanding of mechanisms that activate CGbeta gene expression early in development and in response to the cAMP pathway. Characterization of CGbeta regulatory DNA sequences and transcription factors will also be relevant for studies of other genes that share regulatory pathways with the CGbeta gene.
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