Genetic and bimolecular fluorescence complementation analysis of AP-1 in DNA damage responses and development in C. elegans

Holli Marie Duren, Purdue University

Abstract

Activator protein 1 (AP-1) proteins consist of the Fos and Jun families, which belong to the basic region leucine zipper (bZIP) family. AP-1 proteins function as dimers and play an important role in cell proliferation, differentiation, stress response and apoptosis. In humans, there are 53 bZIP proteins allowing for the potential formation of 2,809 dimers. Thus, it remains a huge challenge to study the role of individual AP-1 dimers in cells and in vivo. I have taken advantage of a model organism with reduced genetic redundancy, Caenorhabditis elegans, and the bimolecular fluorescence complementation (BiFC) assay to investigate the roles of AP-1 in DNA damage response and development in C. elegans. Genome integrity is critically important for organisms to survive and flourish. Cells have protective measures, such as cell cycle arrest, DNA repair and apoptosis to combat the effects of DNA damage. Unrepaired DNA damage can be passed to the daughter cells and increase the possibility of mutations in the DNA, thus potentially leading to increased cancer risk. To determine the role of AP-1 genes in the UV response, we have previously identified the C. elegans fos-1 and jun-1 homologous genes. Using RNA interference (RNAi) methods and deletion mutants, I have shown that knockdown/deletion of these individual AP-1 genes sensitized the worms to UV-induced worm death. In addition, I have shown that the deletion of jun-1 in the worm increases cell death in response to UVC. Interestingly, jun-1 seems to play a minor role in cell cycle arrest and is not involved in DNA repair. I have also demonstrated that functional knockout of the JUN-1:JUN-1 dimer sensitizes the worm to UV. Consistent with these data, UV induces the formation of the JUN-1:JUN-1 homodimer, visualized by the BiFC assay, suggesting that JUN-1 plays a major role in apoptosis by serving a pro-survival function. Further genetic analyses have revealed that jun-1 may act downstream of the MAPK genes, mpk-1 and kgb-1 and upstream of ced-9, a major component of the core apoptotic machinery. Interestingly, I have also found that jun-1 genetically, and possibly redundantly, interacts with the MAPK gene, pmk-1, or xpf-1, a gene involved in nucleotide excision repair, to promote UV-induced apoptosis. Given that AP-1 functions as a dimer, I reasoned that visualization of AP-1 dimers in vivo could suggest where an AP-1 dimer may function. To this end, I have visualized the temporal and spatial interaction patterns of individual AP-1 dimers throughout the development of the worm. BiFC analyses have revealed that only FOS-1 homodimers are formed in the anchor cell. In contrast, FOS-1:FOS-1 homodimers and FOS-1:JUN:1 heterodimers are formed in utse cells and spermathecal cells. These results not only confirm the role of FOS-1 in anchor cell invasion, lumen development and regulation of ovulation but also provide new evidence that different AP-1 dimers are involved in these developmental and physiological processes. Interestingly, our temporal and spatial analyses of AP-1 dimerization throughout development have uncovered that AP-1 may also play a role in other tissues/organs such as nervous system, vulval development, and embryonic development. Taken together, results from my thesis study have provided evidence that genetic and BiFC approaches are two complementary approaches that can uniquely allow us to dissect the role of AP-1 dimers in developmental processes and physiological responses in C. elegans.

Degree

Ph.D.

Advisors

Hu, Purdue University.

Subject Area

Molecular biology|Genetics|Developmental biology

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