Understanding AP-1 dimerization in vivo: Genetic and BiFC analysis of Caenorhabditis elegans Fos-1 and Jun-1 in regulation of ovulation
Abstract
Activator protein 1 (AP-1) proteins regulate key processes in most organisms. These proteins, which include Fos, Jun, ATF2, and Maf proteins, are basic region leucine zipper proteins, and form dimers and bind DNA to regulate transcription. In mammals, there are 53 bZIP proteins, allowing the formation of 2,809 potential dimers. Although it has been over twenty years since the discovery of AP-1, the roles of individual AP-1 dimers, such as the Fos-Jun heterodimer or the Jun-Jun homodimer, have yet to be elucidated, in part due to functional complementation of these proteins in vivo and the lack of appropriate methods by which to study dimerization. In this thesis, I present evidence that use of a model organism with reduced genetic redundancy (i.e. one Fos gene and one Jun gene) can significantly aid understanding of these proteins. I have successfully identified the C. elegans homologs of c-Fos and c-Jun, encoded by the genes F29G9.4 and T24H10.7, respectively. Bioinformatics and biochemical evidence have confirmed the identification of these proteins as AP-1. I have further characterized an overlapping role for both FOS-1 and JUN-1 in reproduction. Specifically, each of these genes control spermathecal valve dilation and thus regulate ovulation. This function is carried out on the molecular level by transcriptional regulation of plc-1, a key enzyme necessary for synthesis of inositol 1,4,5-triphosphate (IP 3). Finally, I have applied the bimolecular fluorescence complementation (BiFC) assay to visualize interactions of individual AP-1 dimers to specifically determine the roles of the FOS-1 and JUN-1 homodimers and heterodimers in regulation of ovulation. Results from these BiFC analyses have shown that FOS-1B homodimers form in the body and valves of the spermatheca, and the FOS-1B-JUN-1A heterodimer forms in the valves of the spermatheca. JUN-1A homodimerizes in oocytes and fertilized eggs, but not in the spermatheca. These unique patterns of dimerization suggest unique roles for each dimer and support our genetic analysis that fos-1 and jun-1 regulate plc-1 transcription and control ovulation.
Degree
Ph.D.
Advisors
Hu, Purdue University.
Subject Area
Molecular biology
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