Analysis of single telomeres using fluorescence microscopy

Eukaryotic chromosome ends are protected by special DNA structures known as telomeres. In mammals the DNA of telomeres consist of TTAGGG repeats which, in cooperation with specialized proteins, “cap” the ends of chromosomes to protect the chromosomes from end-to-end fusion and erosion. Thus, telomeres are important to maintain chromosome stability and play a vital role in preserving the information in our genome. A key factor of telomeric function is the length of the telomeres. Short and dysfunctional telomeres with less than a few dozen repeats are associated with genomic instability and tumorigenesis. Furthermore, loss of telomere function is implicated in numerous diseases like bone marrow failure, hematological malignancies and other cancers. There is a substantial body of evidence indicating that the average length of telomeres can provide prognostic information in human diseases. However, limitations in the currently available technologies for detecting and measuring the length of telomeres has hampered progress in translating telomere length assays into clinical practice. Additionally, many questions about the relation between telomere length and telomere function remain to be answered. Consequently, novel approaches to study single telomeres are of significant interest. In this study, I investigated two cutting-edge technologies to assess properties of telomeres. I used quantitative fluorescence in situ hybridization (Q-FISH) to identify the length of telomeres based on fluorescence values. Using Q-FISH, I was able to generate DNA measurements using plasmids with different size telomeric inserts that served as a reference for length quantifications taken on different platforms. Also, I initiated explorations of a novel high-throughput method to study physical properties of single telomeres and, potentially, measure the length of telomere repeats. Convex Lens-induced Confinement technique (CLiC) is a technique developed to image individual biological molecules and study their dynamics. Using the CLiC platform, I sought to define the length of plasmid DNA based on diffusion coefficient values. My work has set the stage for others to explore the CLiC platform to study properties of telomeres including biological properties as well as their length. The latter can possibly be used as a prognostic tool in bone marrow failure, hematological malignancies and other disorders.Medicine, Faculty ofMedical Genetics, Department ofGraduat

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