Stefan Taubert

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Endoplasmic reticulum (ER) stress due to protein misfolding or membrane lipidimbalance is observed in many diseases. ER homeostasis can be restored by activation of theunfolded protein response (UPR-ER), which, in higher eukaryotes, consists of three parallelbranches: The Inositol-Requiring-Enzyme 1 (IRE-1) branch, the protein kinase RNA-like ERkinase (PEK-1) branch, and the Activating Transcription Factor 6 (ATF-6) branch. These sensorsactivate downstream effectors to restore cellular homeostasis. However, we lack a global view ofgenetic perturbations that activate the UPR-ER in metazoans. To identify\metabolic pathwaysthat affect ER homeostasis, I used RNA interference (RNAi) to inactivate 1247 metabolic genesin Caenorhabditis elegans using an IRE-1 branch specific transcriptional reporter, hsp-4p::gfp.After screening and validation, I obtained 34 high-confidence hits that also activate the PEK-1branch. Next, using a strain lacking the key IRE-1 pathway effector XBP-1 (xbp-1; hsp-4p::gfp),I showed that these gene inactivations induce canonical IRE-1 signaling. Moreover, dietarycholine supplementation, which suppresses UPR-ER in worms defective for phosphatidylcholine(PC) synthesis pathway, partially suppresses UPR-ER activation in 3 of the 34 hits, suggestingthat most hits do not activate the UPR-ER via defective PC synthesis. Finally, I performedfollow-up studies on two of the 34 hits, the primases pri-1 and pri-2, whose inactivation causesDNA damage due to replication fork stalling. These two RNAi clones were selected becauseboth activate hsp-4p::gfp in C. elegans embryos in a partially ire-1-, xbp-1-independent manner.I observed that pri-1 and pri-2 RNAi specifically induce the UPR-ER, but not themechanistically distinct cytosolic or mitochondrial UPRs. This suggests that pri-1 and pri-2RNAi do not cause global protein misfolding. Interestingly, genomic instability caused by loss of DNA repair pathways did not activate the UPR-ER, suggesting that replication stress specificallyactivates the UPR-ER in the embryo. In sum, by identifying new genes that affect UPR-ERhomeostasis in C. elegans, my project provides new insights into mechanisms of UPR-ERregulation. Furthermore, as many genes identified here have human homologues, my data mayprovide a starting point for the discovery of novel drug targets for human diseases featuring ERdysfunction.

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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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Nuclear hormone receptors (NHRs) are transcription factors that regulate a wide variety of developmental and physiological processes. NHRs are targets of numerous drugs. However, due to limited knowledge on NHR specificity, many such drugs activate multiple biological pathways downstream of NHRs, leading to undesired side effects. To study NHR specificity in vivo, I used the model organism Caenorhabditis elegans. One C. elegans NHR is NHR-49, which regulates various aspects of lipid metabolism. Specifically, it activates genes involved in fatty acid desaturation and fatty acid β-oxidation by binding to a subunit of the Mediator multiprotein complex, MDT-15. Vice versa, NHR-49 represses genes involved in sphingolipid breakdown by heterodimerizing with another C. elegans NHR, NHR-66. Recently, three point mutations in nhr-49 were identified that promote fatty acid desaturation, but whether these alleles act specifically in this pathway or also affect other nhr-49 regulated processes is not clear. To test whether the mutated residues are linked to specific biological functions, I studied how they affect gene expression and protein-protein interactions by real time quantitative PCR and Yeast 2 Hybrid assays. I found that the three point mutations have different effects on nhr-49 dependent metabolic processes. While all three alleles broadly promoted nhr-49 dependent activation, only one allele affected nhr-49 dependent repression. This shows that the mutations and the corresponding amino acid residues have some association with specific nhr-49 dependent biological processes.

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The Mediator is a conserved transcriptional co-factor complex required for eukaryotic gene expression. In C. elegans, the Mediator subunit mdt-15 is essential for the expression of genes involved in fatty acid metabolism and ingestion-associated stress response. mdt-15 loss-of-function causes defects in reproduction and mobility and shortens lifespan. In the present study, we find that mdt-15 depletion or mutation specifically decreases membrane phospholipid unsaturation. Accordingly, mdt-15 worms exhibit disturbed ER homeostasis indicated by a constitutively activated ER unfolded protein response (UPRER). This stress response is only partially the consequence of reduced membrane lipid unsaturation, implicating other mdt-15–regulated processes in the protection against ER stress. Interestingly, mdt-15 inactivation or depletion of lipid metabolism enzymes SCD or sams-1 activates the UPRER without promoting misfolded protein aggregates in the ER. Moreover, these worms all exhibit wild-type sensitivity to chemically induced protein misfolding, and they do not display synthetic lethality with ire-1, whose inactivation causes protein misfolding. Therefore, the constitutive UPRER in mdt-15, SCD, or sams-1 worms is not the consequence of disturbed proteostasis, but likely the direct result from altered properties of the ER membrane. Altogether, our data suggest that the UPRER can be directly induced by membrane disequilibrium and thus acts as a circuit that comprehensively monitors ER homeostasis.

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