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

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.Medicine, Faculty ofMedical Genetics, Department ofGraduat

The role of MAGT1 in genetic syndromes

Disturbances in magnesium homeostasis, often linked to altered expression and/or function of magnesium channels, have been implicated in a plethora of diseases. This review focuses on magnesium transporter 1 (MAGT1), as recently described changes in this gene have further extended our understanding of the role of magnesium in human health and disease. The identification of genetic changes and their functional consequences in patients with immunodeficiency revealed that magnesium and MAGT1 are key molecular players for T cell-mediated immune responses. This led to the description of XMEN (X-linked immunodeficiency with magnesium defect, Epstein Barr Virus infection, and neoplasia) syndrome, for which Mg2+ supplementation has been shown to be beneficial. Similarly, the identification of a copy-number variation (CNV) leading to dysfunctional MAGT1 in a family with atypical ATRX syndrome and skin abnormalities, suggested that the MAGT1 defect could be responsible for the cutaneous problems. On the other hand, recent genetic investigations question the previously proposed role for MAGT1 in intellectual disability. Understanding the molecular basis of the involvement of magnesium and its channels in human pathogenesis will improve opportunities for Mg2+ therapies in the clinic

Mediator subunit MDT-15/MED15 and Nuclear Receptor HIZR-1/HNF4 cooperate to regulate toxic metal stress responses in Caenorhabditis elegans.

Zinc is essential for cellular functions as it is a catalytic and structural component of many proteins. In contrast, cadmium is not required in biological systems and is toxic. Zinc and cadmium levels are closely monitored and regulated as their excess causes cell stress. To maintain homeostasis, organisms induce metal detoxification gene programs through stress responsive transcriptional regulatory complexes. In Caenorhabditis elegans, the MDT-15 subunit of the evolutionarily conserved Mediator transcriptional coregulator is required to induce genes upon exposure to excess zinc and cadmium. However, the regulatory partners of MDT-15 in this response, its role in cellular and physiological stress adaptation, and the putative role for mammalian MED15 in the metal stress responses remain unknown. Here, we show that MDT-15 interacts physically and functionally with the Nuclear Hormone Receptor HIZR-1 to promote molecular, cellular, and organismal adaptation to cadmium and excess zinc. Using gain- and loss-of-function mutants and qRT-PCR and reporter analysis, we find that mdt-15 and hizr-1 cooperate to induce zinc and cadmium responsive genes. Moreover, the two proteins interact physically in yeast-two-hybrid assays and this interaction is enhanced by the addition of zinc or cadmium, the former a known ligand of HIZR-1. Functionally, mdt-15 and hizr-1 mutants show defective storage of excess zinc in the gut and are hypersensitive to zinc-induced reductions in egg-laying. Furthermore, mdt-15 but not hizr-1 mutants are hypersensitive to cadmium-induced reductions in egg-laying, suggesting potential divergence of regulatory pathways. Lastly, mammalian MDT-15 orthologs bind genomic regulatory regions of metallothionein and zinc transporter genes in a cadmium and zinc-stimulated fashion, and human MED15 is required to induce a metallothionein gene in lung adenocarcinoma cells exposed to cadmium. Collectively, our data show that mdt-15 and hizr-1 cooperate to regulate cadmium detoxification and zinc storage and that this mechanism is at least partially conserved in mammals