Transcriptional repression in ovarian granulosa cells

Regulation of gene expression is crucial for cellular differentiation in all organisms. Transcriptional regulation is the initial step controlling gene expression and requires the cooperation of numerous factors. The studies presented in this thesis demonstrate that transcriptional repression involves the coordinated regulation of factors functioning through overlapping and divergent mechanisms. To study repression, I have examined the luteinizing hormone (LH)-dependent regulatory mechanisms that control ovarian gene expression throughout the periovulatory period during the rodent reproductive cycle, focusing specifically on the mechanisms that repress inhibin alpha gene expression. Four isoforms of inducible CAMP early repressor (ICER) are rapidly expressed in the ovary in response to LH signaling. Experiments in this thesis demonstrate that all four isoforms can repress inhibin alpha gene expression. Mutational studies to the DNA binding and dimerization domain (DBD) of ICER demonstrate that binding of ICER to the inhibin promoter is necessary for repression. Electrophoretic mobility shift assays (EMSAs) as well as chromatin immunoprecipitations (ChIPs) demonstrate that ICER competes with the activator CREB for binding to the inhibin promoter. Due to the transient nature of ICER expression in the ovary, it is likely that additional factors mediate the repression of the inhibin alpha gene that is maintained throughout the postovulatory period. I examined the mechanisms by which CCAAT/enhancer binding protein beta, C/EBPbeta, represses the inhibin alpha gene and show in EMSAs that C/EBPbeta can compete with CREB for binding to the inhibin CRE similar to ICER. In addition, C/EBPbeta can bind to an upstream C/EBPbeta response element causing repression of inhibin gene transcription. Expression of the nuclear receptor, nerve growth factor inducible-B (NGFI-B) in the ovary parallels that of ICER. I examined whether NGFI-B has a role in the downregulation of inhibin alpha gene expression following LH signaling and show in transient transfections that NGFI-B represses inhibin promoter activity. Mutational studies to the DBD of NGFI-B demonstrate that direct binding of NGFI-B to the inhibin promoter is not required for inhibin alpha gene repression. Thus, in response to one cue, signaling through the LH receptor, multiple factors are induced and work cooperatively to regulate ovarian gene expression.