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A regulatory mechanism of zinc homeostasis involving the mediator subunit MDT-15 and the transcription factor HIZR-1 Shomer, Naomi

Abstract

Zinc is a metal that is essential for cell function as it plays important catalytic and structural roles in many proteins; however, excess zinc causes cell stress. Cadmium has similar chemical properties as zinc but is toxic and not required in biological systems. To maintain homeostasis, the levels of zinc detoxification genes are modulated through transcriptional regulation, which allows organisms to adapt to environmental changes. The key players in transcriptional regulation are Transcription Factors (TF), regulatory DNA elements, and coregulators such as the Mediator complex. Mediator subunit MDT-15 is required for the regulation of stress response genes in Caenorhabditis elegans, including zinc responsive genes. However, MDT-15’s physiological role and its regulatory partners in zinc homeostasis and cadmium stress response remain unknown. In this study, I investigated which TFs collaborate with MDT-15 to regulate zinc homeostasis and cadmium stress response genes, and I also examined its physiological role in zinc homeostasis. I used a fusion of the promoter of the zinc and cadmium responsive gene cdr-1 to Green Fluorescent Protein (GFP) and real-time PCR analysis as sensitive readouts to study metal response mechanisms. I found that cdr-1 induction by zinc and cadmium depends on Mediator subunits mdt-15 and cdk-8, and the TFs high zinc activated nuclear receptor-1 (hizr-1) and elt-2. Using genetic interaction studies, I found that HIZR-1 and MDT-15 function is codependent, and showed, using the yeast-two-hybrid system, that the two proteins interact physically. Interestingly, this physical association was enhanced by micromolar zinc and cadmium. To assess zinc storage, I studied the gut granules of C. elegans, which store and replenish zinc to maintain homeostasis, and found storage defects in mdt-15 and hizr-1 mutants. Lastly, I explored the regulatory conservation of this regulatory mechanism. The Insulin Secretory Granules in pancreatic β-cells require appropriate amounts of zinc to crystallize insulin. Using mice lacking the mdt-15 ortholog Med15 in the β-cells, I found that Med15 is required to express Slc30a8, the ortholog of the mdt-15-regulated zinc transporter cdf-2. Collectively, my data show that mdt-15 and hizr-1 cooperate to regulate metal detoxification genes and zinc storage, through a mechanism that possibly is conserved.

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