Analysis of Telomere Maintenance Mechanisms in the Roundworm C. elegans

The ends of linear chromosomes are composed of repetitive hexameric sequences called telomeres. Because canonical DNA polymerases cannot fully replicate the ends of linear chromosomes, they shorten with each cycle of replication. Once telomeres become critically short, cells stop replicating and enter a state of replicative arrest--senescence. In this way, telomere length limits the replicative potential of all cells. The enzyme telomerase can maintain telomere length by adding de novo telomeric repeats via reverse transcription from an associated RNA template. Although telomerase expression is exclusive to human somatic and germ stem cells, its expression in somatic stem cells is not sufficient to prevent telomere erosion. Therefore, telomeres shorten with age, effectively serving as a mitotic clock. As in mammals, components of the C. elegans 9-1-1 (HPR-9/MRT-2/HUS-1) DNA damage response complex and its clamp loader, HPR-17, are necessary for telomerase-mediated telomere repeat addition in vivo. Here we present the mapping and characterization of three new telomerase-defective alleles of mrt-2, hpr-17, and hpr-9, and a mutation in a novel gene, all of which possess defects in repairing ionizing radiation- and interstrand crosslink-induced DNA damage. In addition to telomerase, several canonical telomere-binding proteins and other associated proteins maintain telomere length homeostasis and protect chromosome ends from exacerbated erosion and erroneous DNA damage responses in humans. C. elegans harbors four homologs of the human Protection of Telomeres 1 (POT1) telomere capping protein, POT-1, POT-2, POT-3 and MRT-1. Here I identify POT-1 and POT-2 as negative regulators of telomerase-mediated telomere repeat addition in vivo. I demonstrate that POT-1, but not POT-2, protects telomeres from exacerbated erosion. I employ several biochemical strategies to assess whether POT-1 or POT-2 interact with telomerase in vivo. Although an epitope-tagged pot-1 transgene rescued the telomere elongation phenotype of the pot-1 mutant, it exacerbated the onset of senescence in mutants defective for telomerase-mediated telomere repeat addition. We suggest that alterations in telomere capping proteins may drive telomere dysfunction in telomerase-negative cells, which may help to define causal mutations for patients suffering from diseases of premature aging with undefined etiologies.Doctor of Philosoph

In Vivo Analysis of Conserved C. elegans Tomosyn Domains

Neurosecretion is critically dependent on the assembly of a macromolecular complex between the SNARE proteins syntaxin, SNAP-25 and synaptobrevin. Evidence indicates that the binding of tomosyn to syntaxin and SNAP-25 interferes with this assembly, thereby negatively regulating both synaptic transmission and peptide release. Tomosyn has two conserved domains: an N-terminal encompassing multiple WD40 repeats predicted to form two β-propeller structures and a C-terminal SNARE-binding motif. To assess the function of each domain, we performed an in vivo analysis of the N- and C- terminal domains of C. elegans tomosyn (TOM-1) in a tom-1 mutant background. We verified that both truncated TOM-1 constructs were transcribed at levels comparable to rescuing full-length TOM-1, were of the predicted size, and localized to synapses. Unlike full-length TOM-1, expression of the N- or C-terminal domains alone was unable to restore inhibitory control of synaptic transmission in tom-1 mutants. Similarly, co-expression of both domains failed to restore TOM-1 function. In addition, neither the N- nor C-terminal domain inhibited release when expressed in a wild-type background. Based on these results, we conclude that the ability of tomosyn to regulate neurotransmitter release in vivo depends on the physical integrity of the protein, indicating that both N- and C-terminal domains are necessary but not sufficient for effective inhibition of release in vivo

Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans

Transgenes can be permanently silenced in a single generation via a previously described small RNA-induced epigenetic silencing (RNAe) mechanism, which is promoted by the presence of a perfect Piwi-interacting RNA (piRNA) target site. In this study, we identify a previously unidentified mechanism capable of silencing single-copy transgenes that lack perfect piRNA target sites and that is triggered by a lack of chromosomal pairing during meiosis for multiple generations. Multigenerational RNAe can lead to reversible or permanent transgene silencing and may provide insight into variability in the expression of single-copy transgenes or single-copy genomic insertions, which are commonly used in experimental biology. Our analysis of “multigenerational RNAe” also offers new insights into potentially common epigenetic silencing events relevant to genome expression in the germline and embryo

Caenorhabditis elegans POT-1 and POT-2 Repress Telomere Maintenance Pathways

Telomeres are composed of simple tandem DNA repeats that protect the ends of linear chromosomes from replicative erosion or inappropriate DNA damage response mechanisms. The mammalian Protection Of Telomeres (POT1) protein interacts with single-stranded telomeric DNA and can exert positive and negative effects on telomere length. Of four distinct POT1 homologs in the roundworm Caenorhabditis elegans, deficiency for POT-1 or POT-2 resulted in progressive telomere elongation that occurred because both proteins negatively regulate telomerase. We created a POT-1::mCherry fusion protein that forms discrete foci at C. elegans telomeres, independent of POT-2, allowing for live analysis of telomere dynamics. Transgenic pot-1::mCherry repressed telomerase in pot-1 mutants. Animals deficient for pot-1, but not pot-2, displayed mildly enhanced telomere erosion rates in the absence of the telomerase reverse transcriptase, trt-1. However, trt-1; pot-1 double mutants exhibited delayed senescence in comparison to trt-1 animals, and senescence was further delayed in trt-1; pot-2; pot-1 triple mutants, some of which survived robustly in the absence of telomerase. Our results indicate that POT-1 and POT-2 play independent roles in suppressing a telomerase-independent telomere maintenance pathway but may function together to repress telomerase

Telomere dysfunction in human bone marrow failure syndromes

Approximately 90% of all human cancers, in which some deregulation of cell cycle arrest or programmed cell death has occurred, express telomerase, a ribonucleoprotein whose activity is normally turned off in healthy somatic tissues. Additionally, small populations of self-renewing stem cells, such as hematopoietic stem cells, skin and hair follicle basal layer cells and intestinal basal crypt cells, have been shown to retain telomerase activity. Conversely, hereditary defects that result in shortened telomeres in humans have been shown to manifest most often as bone marrow failure or pulmonary fibrosis, along with a myriad of other symptoms, likely due to the loss of the stem and/or progenitor cells of affected tissues. The aim of this review is to highlight our knowledge of the mechanisms of telomere maintenance that contribute to the pathology of human disease caused by dysfunctional telomere homeostasis. Specifically, a new role for the SNM1B/Apollo nuclease in the pathologies of Hoyeraal-Hreidarsson syndrome will be discussed

The MRT-1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans

The telomerase reverse transcriptase adds de novo DNA repeats to chromosome termini. Here we define Caenorhabditis elegans MRT-1 as a novel factor required for telomerase-mediated telomere replication and the DNA-damage response. MRT-1 is composed of an N-terminal domain homologous to the second OB-fold of POT1 telomere-binding proteins and a C-terminal SNM1 family nuclease domain, which confer single-strand DNA-binding and processive 3′-to-5′ exonuclease activity, respectively. Furthermore, telomerase activity in vivo depends on a functional MRT-1 OB-fold. We show that MRT-1 acts in the same telomere replication pathway as telomerase and the 9-1-1 DNA-damage response complex. MRT-1 is dispensable for DNA double-strand break repair, but functions with the 9-1-1 complex to promote DNA interstrand cross-link (ICL) repair. Our data reveal MRT-1 as a dual-domain protein required for telomerase function and ICL repair, which raises the possibility that telomeres and ICL lesions may share a common feature that plays a critical role in de novo telomere repeat addition

Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans

Single-copy transgenes in Caenorhabditis elegans can be subjected to a potent, irreversible silencing process termed small RNA-induced epigenetic silencing (RNAe). RNAe is promoted by the Piwi Argonaute protein PRG-1 and associated Piwi-interacting RNAs (piRNAs), as well as by proteins that promote and respond to secondary small interfering RNA (siRNA) production. Here we define a related siRNA-mediated silencing process, termed “multigenerational RNAe,” which can occur for transgenes that are maintained in a hemizygous state for several generations. We found that transgenes that contain either GFP or mCherry epitope tags can be silenced via multigenerational RNAe, whereas a transgene that possesses GFP and a perfect piRNA target site can be rapidly and permanently silenced via RNAe. Although previous studies have shown that PRG-1 is typically dispensable for maintenance of RNAe, we found that both initiation and maintenance of multigenerational RNAe requires PRG-1 and the secondary siRNA biogenesis protein RDE-2. Although silencing via RNAe is irreversible, we found that transgene expression can be restored when hemizygous transgenes that were silenced via multigenerational RNAe become homozygous. Furthermore, multigenerational RNAe was accelerated when meiotic pairing of the chromosome possessing the transgene was abolished. We propose that persistent lack of pairing during meiosis elicits a reversible multigenerational silencing response, which can lead to permanent transgene silencing. Multigenerational RNAe may be broadly relevant to single-copy transgenes used in experimental biology and to shaping the epigenomic landscape of diverse species, where genomic polymorphisms between homologous chromosomes commonly result in unpaired DNA during meiosis

Over-expression of TOM1-A SNARE or ΔSNARE constructs do not inhibit synaptic release in the wild-type background.

<p><b>A.</b> Representative evoked traces for full-length TOM-1A (SY1237), SNARE (SY1234) and ΔSNARE (SY1235) expressing transgenes in the wild-type background. (<b>B</b>) Average evoked amplitude and (<b>C</b>) Average charge integral were only significantly reduced by full-length TOM-1A relative to wild type (***, p = 0.0005, and p = 0.0007 for B and C, respectively). All data are expressed as mean ± SEM, the sample size (n) is indicated as a number in each bar, significance values obtained with the Mann Whitney T-test.</p

Both TOM1-A SNARE and ΔSNARE are stably expressed and localized at synapses.

<p><b>A.</b> The FLAG tagged SNARE and ΔSNARE constructs are of the predicted size on Westerns. <b>B.</b> Representative confocal images of SNARE::FLAG and ΔSNARE::FLAG expression in the ventral nerve cord (VNC) anterior to the vulva, the region used for electrophysiological recording. Staining in the lateral nerve cord (LNC) was also observed. Scale bar is 50 µm.</p

Flag-tagged TOM-1A SNARE and ΔSNARE transgenics phenocopy untagged lines.

<p><b>A.</b> Representative evoked response traces for SNARE::FLAG (SY1232) and ΔSNARE::FLAG (SY1233) expressing lines. <b>B.</b> Plots of average evoked amplitude and (<b>C</b>) evoked charge integral. All data are expressed as mean ± SEM, the sample size (n) is indicated as a number in each bar. Mann Whitney T-tests showed values were not significantly different.</p