Deciphering the GnRH Signaling Network: Role of beta-catenin and Homer1 Splicing in Gonadotropin Gene Regulation

Gonadotropin-releasing hormone (GnRH) is a periodically released driver of mammalian reproductive function. It is released from hypothalamus in a pulsatile manner and differentially regulates the biosynthesis and secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), in the gonadotropes. Over several decades, many signaling components and mechanisms involved in the regulation of gonadotropin gene expression have been identified. In this thesis work, we have:;1) Identified the role of beta-catenin in FSHbeta gene expression. beta-catenin nuclear translocation was found to be GnRH-induced and JNK-dependent. Small interfering RNA (siRNA) knockdown of either beta-catenin or JNK led to a reduction of FSHbeta mRNA. Site-directed mutagenesis of putative TCF/LEF binding sites and chromatin immunoprecipitation (ChIP) of beta-catenin showed no evidence of direct or adaptor-mediated complexing with the FSHbeta promoter by beta-catenin. Through RNA sequencing and qPCR validation, a novel regulator of FSHbeta, BRMS1L, was found to mediate beta-catenin regulation in FSHbeta gene expression. This new JNK-beta-catenin-BRMS1L pathway is independent of the known JNK/c-JUN pathway that leads to FSHbeta; expression. Since beta-catenin plays a central role in tumorigenesis, our study provides a potential new link between GnRH signaling and gonadal hormone-sensitive cancers.;2) Determined the effects of GnRH on global mRNA splicing and the involvement of regulation of HomerI splicing in gonadotropin gene control. High-depth RNA sequencing analysis revealed that GnRH induces global changes in RNA splicing including Homer1, a gene previously found to be involved in postsynaptic density and calcium signaling in neurons. Homer1 expresses a short splice form, Homer1a, and more abundant long transcripts Homer1b/c. GnRH induced a modest increase in Homer1b/c expression and a dramatic increase in the Homer1a splice form. Depletion of the splicing regulator SRp20, whose phosphorylation was induced by GnRH, attenuated the GnRH-induced increase in the Homer1a/Homer1bc ratio and modulated FSHbeta and LHbeta expression. Isoform-specific reduction of Homer1b/c increased both FSHbeta and LHbeta mRNAs, whereas reduction of Homer1a had the opposite effect on FSHbeta expression. Our study is the first to report that the regulation of alternative splicing by GnRH contributes to gonadotropin gene control.;These results provide novel perspectives into regulatory mechanisms of gonadotropin gene expression and identify potential targets for GnRH-related therapeutics.