Publications by authors named "Shawn Ahmed"

33 Publications

Germ granule dysfunction is a hallmark and mirror of Piwi mutant sterility.

Nat Commun 2021 03 3;12(1):1420. Epub 2021 Mar 3.

Department of Genetics, University of North Carolina, Chapel Hill, NC, USA.

In several species, Piwi/piRNA genome silencing defects cause immediate sterility that correlates with transposon expression and transposon-induced genomic instability. In C. elegans, mutations in the Piwi-related gene (prg-1) and other piRNA deficient mutants cause a transgenerational decline in fertility over a period of several generations. Here we show that the sterility of late generation piRNA mutants correlates poorly with increases in DNA damage signaling. Instead, sterile individuals consistently exhibit altered perinuclear germ granules. We show that disruption of germ granules does not activate transposon expression but induces multiple phenotypes found in sterile prg-1 pathway mutants. Furthermore, loss of the germ granule component pgl-1 enhances prg-1 mutant infertility. Environmental restoration of germ granule function for sterile pgl-1 mutants restores their fertility. We propose that Piwi mutant sterility is a reproductive arrest phenotype that is characterized by perturbed germ granule structure and is phenocopied by germ granule dysfunction, independent of genomic instability.
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http://dx.doi.org/10.1038/s41467-021-21635-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930041PMC
March 2021

Gametes deficient for Pot1 telomere binding proteins alter levels of telomeric foci for multiple generations.

Commun Biol 2021 Feb 4;4(1):158. Epub 2021 Feb 4.

Department of Genetics, University of North Carolina, Chapel Hill, NC, 27599, USA.

Deficiency for telomerase results in transgenerational shortening of telomeres. However, telomeres have no known role in transgenerational epigenetic inheritance. C. elegans Protection Of Telomeres 1 (Pot1) proteins form foci at the telomeres of germ cells that disappear at fertilization and gradually accumulate during development. We find that gametes from mutants deficient for Pot1 proteins alter levels of telomeric foci for multiple generations. Gametes from pot-2 mutants give rise to progeny with abundant POT-1::mCherry and mNeonGreen::POT-2 foci throughout development, which persists for six generations. In contrast, gametes from pot-1 mutants or pot-1; pot-2 double mutants induce diminished Pot1 foci for several generations. Deficiency for MET-2, SET-25, or SET-32 methyltransferases, which promote heterochromatin formation, results in gametes that induce diminished Pot1 foci for several generations. We propose that C. elegans POT-1 may interact with H3K9 methyltransferases during pot-2 mutant gametogenesis to induce a persistent form of transgenerational epigenetic inheritance that causes constitutively high levels of heterochromatic Pot1 foci.
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http://dx.doi.org/10.1038/s42003-020-01624-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862594PMC
February 2021

Maternal Inheritance: Longevity Programs Nourish Progeny via Yolk.

Curr Biol 2019 08;29(15):R748-R751

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Epigenetic effects can be mediated by changes in chromatin state that are transmitted from parent to child via gametes, but support is gathering for maternal yolk, which is deposited into ooctyes, as an extranuclear epigenetic factor that can contribute to phenotypic plasticity across generations in Caenorhabditis elegans.
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http://dx.doi.org/10.1016/j.cub.2019.06.050DOI Listing
August 2019

Telomeric small RNAs in the genus .

RNA 2019 09 25;25(9):1061-1077. Epub 2019 Jun 25.

Department of Genetics, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.

Telomeric DNA is composed of simple tandem repeat sequences and has a G-rich strand that runs 5' to 3' toward the chromosome terminus. Small RNAs with homology to telomeres have been observed in several organisms and could originate from telomeres or from interstitial telomere sequences (ITSs), which are composites of degenerate and perfect telomere repeat sequences found on chromosome arms. We identified small RNAs composed of the telomere sequence (TTAGGC) with up to three mismatches, which might interact with telomeres. We rigorously defined ITSs for genomes of and for two closely related nematodes, and Most telomeric small RNAs with mismatches originated from ITSs, which were depleted from mRNAs but were enriched in introns whose genes often displayed hallmarks of genomic silencing. small RNAs composed of perfect telomere repeats were very rare but their levels increased by several orders of magnitude in and Major small RNA species in begin with a 5' guanine nucleotide, which was strongly depleted from perfect telomeric small RNAs of all three species. Perfect G-rich or C-rich telomeric small RNAs commonly began with 5' UAGGCU and 5' UUAGGC or 5' CUAAGC, respectively. In contrast, telomeric small RNAs with mismatches had a mixture of all four 5' nucleotides. We suggest that perfect telomeric small RNAs have a mechanism of biogenesis that is distinct from known classes of small RNAs and that a dramatic change in their regulation occurred during recent evolution.
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http://dx.doi.org/10.1261/rna.071324.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800518PMC
September 2019

The meiotic phosphatase GSP-2/PP1 promotes germline immortality and small RNA-mediated genome silencing.

PLoS Genet 2019 03 28;15(3):e1008004. Epub 2019 Mar 28.

Department of Genetics, University of North Carolina, Chapel Hill, North Carolina, United States of America.

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase.
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http://dx.doi.org/10.1371/journal.pgen.1008004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456222PMC
March 2019

Transgenerational Sterility of Piwi Mutants Represents a Dynamic Form of Adult Reproductive Diapause.

Cell Rep 2018 04;23(1):156-171

Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA; Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599, USA. Electronic address:

Environmental stress can induce adult reproductive diapause, a state of developmental arrest that temporarily suspends reproduction. Deficiency for C. elegans Piwi protein PRG-1 results in strains that reproduce for many generations but then become sterile. We found that sterile-generation prg-1/Piwi mutants typically displayed pronounced germ cell atrophy as L4 larvae matured into 1-day-old adults. Atrophied germlines spontaneously reproliferated across the first days of adulthood, and this was accompanied by fertility for day 2-4 adults. Sterile day 5 prg-1 mutant adults remained sterile indefinitely, but providing an alternative food source could restore their fertility. Our data imply that late-generation prg-1 mutants experience a dynamic form of adult reproductive diapause, promoted by stress response, cell death, and RNAi pathways, where delayed fertility and reproductive quiescence represent parallel adaptive developmental outcomes. This may occur in response to a form of "heritable stress" that is transmitted by gametes and epigenetic in nature.
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http://dx.doi.org/10.1016/j.celrep.2018.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918633PMC
April 2018

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

Proc Natl Acad Sci U S A 2015 May 4;112(20):E2667-76. Epub 2015 May 4.

Department of Genetics, Lineberger Comprehensive Cancer Center, and Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280

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.
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http://dx.doi.org/10.1073/pnas.1501979112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443339PMC
May 2015

Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations.

Proc Natl Acad Sci U S A 2014 Oct 25;111(41):E4323-31. Epub 2014 Sep 25.

Departments of Genetics and Biology, Curriculum in Genetics and Molecular Biology, and

Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing.
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http://dx.doi.org/10.1073/pnas.1406131111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4205660PMC
October 2014

Reduced insulin/IGF-1 signaling restores germ cell immortality to Caenorhabditis elegans Piwi mutants.

Cell Rep 2014 May 24;7(3):762-73. Epub 2014 Apr 24.

Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA; Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599, USA. Electronic address:

Defects in the Piwi/piRNA pathway lead to transposon desilencing and immediate sterility in many organisms. We found that the C. elegans Piwi mutant prg-1 became sterile after growth for many generations. This phenotype did not occur for RNAi mutants with strong transposon-silencing defects and was separable from the role of PRG-1 in transgene silencing. Brief periods of starvation extended the transgenerational lifespan of prg-1 mutants by stimulating the DAF-16/FOXO longevity transcription factor. Constitutive activation of DAF-16 via reduced daf-2 insulin/IGF-1 signaling immortalized prg-1 strains via RNAi proteins and histone H3 lysine 4 demethylases. In late-generation prg-1 mutants, desilencing of repetitive segments of the genome occurred, and silencing of repetitive loci was restored in prg-1; daf-2 mutants. This study reveals an unexpected interface between aging and transgenerational maintenance of germ cells, where somatic longevity is coupled to a genome-silencing pathway that promotes germ cell immortality in parallel to the Piwi/piRNA system.
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http://dx.doi.org/10.1016/j.celrep.2014.03.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049074PMC
May 2014

H3K4 demethylase activities repress proliferative and postmitotic aging.

Aging Cell 2014 Apr 19;13(2):245-53. Epub 2013 Nov 19.

Department of Genetics, University of North Carolina, Chapel Hill, NC, 27599-3280, USA; SPIRE Postdoctoral Fellowship Program, University of North Carolina, Chapel Hill, NC, 27599-3280, USA.

Homeostasis of postmitotic and proliferating cells is maintained by pathways that repress stress. We found that the Caenorhabditis elegans histone 3 lysine 4 (H3K4) demethylases RBR-2 and SPR-5 promoted postmitotic longevity of stress-resistant daf-2 adults, altered pools of methylated H3K4, and promoted silencing of some daf-2 target genes. In addition, RBR-2 and SPR-5 were required for germ cell immortality at a high temperature. Transgenerational proliferative aging was enhanced for spr-5; rbr-2 double mutants, suggesting that these histone demethylases may function sequentially to promote germ cell immortality by targeting distinct H3K4 methyl marks. RBR-2 did not play a comparable role in the maintenance of quiescent germ cells in dauer larvae, implying that it represses stress that occurs as a consequence of germ cell proliferation, rather than stress that accumulates in nondividing cells. We propose that H3K4 demethylase activities promote the maintenance of chromatin states during stressful growth conditions, thereby repressing postmitotic aging of somatic cells as well as proliferative aging of germ cells.
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http://dx.doi.org/10.1111/acel.12166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020274PMC
April 2014

Caenorhabditis elegans POT-1 and POT-2 repress telomere maintenance pathways.

G3 (Bethesda) 2013 Feb 1;3(2):305-13. Epub 2013 Feb 1.

Department of Genetics, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.

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.
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http://dx.doi.org/10.1534/g3.112.004440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564990PMC
February 2013

piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans.

Cell 2012 Jul 25;150(1):88-99. Epub 2012 Jun 25.

Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

Transgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in C. elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.
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http://dx.doi.org/10.1016/j.cell.2012.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464430PMC
July 2012

Caenorhabditis elegans POT-2 telomere protein represses a mode of alternative lengthening of telomeres with normal telomere lengths.

Proc Natl Acad Sci U S A 2012 May 30;109(20):7805-10. Epub 2012 Apr 30.

Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

Canonical telomere repeats at chromosome termini can be maintained by a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). Human cancers that survive via ALT can exhibit long and heterogeneous telomeres, although many telomerase-negative tumors possess telomeres of normal length. Here, we report that Caenorhabditis elegans telomerase mutants that survived via ALT possessed either long or normal telomere lengths. Most ALT strains displayed end-to-end chromosome fusions, suggesting that critical telomere shortening occurred before or concomitant with ALT. ALT required the 9-1-1 DNA damage response complex and its clamp loader, HPR-17. Deficiency for the POT-2 telomere binding protein promoted ALT in telomerase mutants, overcame the requirement for the 9-1-1 complex in ALT, and promoted ALT with normal telomere lengths. We propose that telomerase-deficient human tumors with normal telomere lengths could represent a mode of ALT that is facilitated by telomere capping protein dysfunction.
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http://dx.doi.org/10.1073/pnas.1119191109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356646PMC
May 2012

In vivo analysis of conserved C. elegans tomosyn domains.

PLoS One 2011 14;6(10):e26185. Epub 2011 Oct 14.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026185PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195084PMC
February 2012

Telomere dysfunction in human bone marrow failure syndromes.

Nucleus 2011 Jan-Feb;2(1):24-9

Department of Genetics, University of North Carolina, Chapel Hill, USA.

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.
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http://dx.doi.org/10.4161/nucl.2.1.13993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104806PMC
January 2012

DNA synthesis generates terminal duplications that seal end-to-end chromosome fusions.

Science 2011 Apr;332(6028):468-71

Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA.

End-to-end chromosome fusions that occur in the context of telomerase deficiency can trigger genomic duplications. For more than 70 years, these duplications have been attributed solely to breakage-fusion-bridge cycles. To test this hypothesis, we examined end-to-end fusions isolated from Caenorhabditis elegans telomere replication mutants. Genome-level rearrangements revealed fused chromosome ends having interrupted terminal duplications accompanied by template-switching events. These features are very similar to disease-associated duplications of interstitial segments of the human genome. A model termed Fork Stalling and Template Switching has been proposed previously to explain such duplications, where promiscuous replication of large, noncontiguous segments of the genome occurs. Thus, a DNA synthesis-based process may create duplications that seal end-to-end fusions, in the absence of breakage-fusion-bridge cycles.
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http://dx.doi.org/10.1126/science.1199022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4154375PMC
April 2011

The Caenorhabditis elegans homolog of Gen1/Yen1 resolvases links DNA damage signaling to DNA double-strand break repair.

PLoS Genet 2010 Jul 15;6(7):e1001025. Epub 2010 Jul 15.

Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, United Kingdom.

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53-mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.
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http://dx.doi.org/10.1371/journal.pgen.1001025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908289PMC
July 2010

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

EMBO J 2009 Nov 24;28(22):3549-63. Epub 2009 Sep 24.

Department of Genetics, University of North Carolina, Chapel Hill, NC, USA.

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.
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http://dx.doi.org/10.1038/emboj.2009.278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2782091PMC
November 2009

Bioactivity of analogs of the N-terminal region of gastrin-17.

Peptides 2009 Dec 15;30(12):2263-7. Epub 2009 Sep 15.

Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.

Gastrin-17-Gly (G17-Gly) has been shown to bind to non-CCK nanomolar and micromolar affinity sites on DLD-1 and HT-29 human colonic carcinoma cells and to stimulate cellular proliferation. However, in previous studies, we showed that C-terminal truncation of the gastrin-17 (G17) to the G17 analog G17(1-12) and then to G17(1-6)-NH(2) did not remove the ability to bind to DLD-1 cells or to activate proliferation. This implies that residues and/or structural motifs required for bioactivity at these receptors rest in the N-terminal region of G17. In this work, radioligand binding studies conducted with further C-terminally truncated analogs revealed that sequences as short as G17(1-4) still bind to a single receptor with micromolar affinity. Additionally, cell proliferation assays showed that G17(1-12) stimulates proliferation of DLD-1 cells, as of HT-29 cells, but the sequences shorter than G17(1-6)-NH(2), including non-amidated G17(1-6), were incapable of stimulating proliferation. These observations indicate that the tetrapeptide pGlu-Gly-Pro-Trp is the minimum N-terminal sequence for binding to the probable growth-promoting site on DLD-1 cells. Since analogs shorter than G17(1-6) are able to bind the receptor, these peptides may be of use for developing selective antagonists.
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http://dx.doi.org/10.1016/j.peptides.2009.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2787808PMC
December 2009

C. elegans SIR-2.1 translocation is linked to a proapoptotic pathway parallel to cep-1/p53 during DNA damage-induced apoptosis.

Genes Dev 2008 Oct;22(20):2831-42

Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, United Kingdom.

Caenorhabditis elegans SIR-2.1, a member of the sirtuin family related to Saccharomyces cerevisiae Sir2p, has previously been implicated in aging. The mammalian homolog SIRT1 plays important roles in multiple cellular processes including transcriptional repression and stress response. We show that sir-2.1 is essential for the execution of apoptosis in response to DNA damage, and that sir-2.1 genetically acts in parallel to the worm p53-like gene cep-1. This novel cep-1-independent proapoptotic pathway does not require the daf-16 FOXO transcription factor. Cytological analysis of SIR-2.1 suggests a novel mechanism of apoptosis induction. During apoptosis SIR-2.1 changes its subcellular localization from the nucleus to the cytoplasm and transiently colocalizes with the C. elegans Apaf-1 homolog CED-4 at the nuclear periphery. SIR-2.1 translocation is an early event in germ cell apoptosis and is independent of apoptosis execution and cep-1, raising the possibility that SIR-2.1 translocation is linked to the induction of DNA damage-induced apoptosis.
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http://dx.doi.org/10.1101/gad.482608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2569882PMC
October 2008

End joining at Caenorhabditis elegans telomeres.

Genetics 2008 Oct 9;180(2):741-54. Epub 2008 Sep 9.

Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599.

Critically shortened telomeres can be subjected to DNA repair events that generate end-to-end chromosome fusions. The resulting dicentric chromosomes can enter breakage-fusion-bridge cycles, thereby impeding elucidation of the structures of the initial fusion events and a mechanistic understanding of their genesis. Current models for the molecular basis of fusion of critically shortened, uncapped telomeres rely on PCR assays that typically capture fusion breakpoints created by direct ligation of chromosome ends. Here we use independent approaches that rely on distinctive features of Caenorhabditis elegans to study the frequency of direct end-to-end chromosome fusion in telomerase mutants: (1) holocentric chromosomes that allow for genetic isolation of stable end-to-end fusions and (2) unique subtelomeric sequences that allow for thorough PCR analysis of samples of genomic DNA harboring multiple end-to-end fusions. Surprisingly, only a minority of end-to-end fusion events resulted from direct end joining with no additional genome rearrangements. We also demonstrate that deficiency for the C. elegans Ku DNA repair heterodimer does not affect telomere length or cause synthetic effects in the absence of telomerase.
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http://dx.doi.org/10.1534/genetics.108.089920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567377PMC
October 2008

The Caenorhabditis elegans Rad17 homolog HPR-17 is required for telomere replication.

Genetics 2007 May 4;176(1):703-9. Epub 2007 Mar 4.

Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.

Subunits of the Rad9/Rad1/Hus1 (9-1-1) proliferating cell nuclear antigen (PNCA)-like sliding clamp are required for DNA damage responses and telomerase-mediated telomere replication in the nematode Caenorhabditis elegans. PCNA sliding clamps are loaded onto DNA by a replication factor C (RFC) clamp loader. The C. elegans Rad17 RFC clamp loader homolog, hpr-17, functions in the same pathway as the 9-1-1 complex with regard to both the DNA damage response and telomerase-mediated telomere elongation. Thus, hpr-17 defines an RFC-like complex that facilitates telomerase activity in vivo in C. elegans.
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http://dx.doi.org/10.1534/genetics.106.070201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1893056PMC
May 2007

Gastrin 1-6 promotes growth of colon cancer cells through non-CCK receptors.

Peptides 2007 Mar 28;28(3):632-5. Epub 2006 Nov 28.

Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, USA.

Our previous studies have shown that stimulation of proliferation of DLD-1 and HT29 human colonic cancer cells by nanomolar gastrin (G17) and carboxymethyl gastrin (G17Gly) and reversal of growth by micromolar G17 and G17Gly involves binding sites which can neither be CCK1 nor CCK2 receptors; the N terminal fragment, G17(1-12), is sufficient to increase the number of HT-29 cells by binding the higher affinity binding site but is without a suppressing effect through the lower affinity site. In this study with DLD-1 cells, competitive binding using 125I-G17(1-12) showed that G17(1-12) binds both high and low affinity sites, as do G17 and G17Gly. G17(1-6)-NH2, even without the central-to-C-terminal portion of G17, was still able to bind a single site and to promote a dose-dependent increase in cell number at nanomolar concentrations. The results indicate the presence of a non-CCK receptor on human colonic cancer cells which could mediate the tumor-promoting activity of the N-terminal-to-central portion of G17Gly which, unlike G17, is produced by such cells.
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http://dx.doi.org/10.1016/j.peptides.2006.10.008DOI Listing
March 2007

Uncoupling of pathways that promote postmitotic life span and apoptosis from replicative immortality of Caenorhabditis elegans germ cells.

Authors:
Shawn Ahmed

Aging Cell 2006 Dec 1;5(6):559-63. Epub 2006 Nov 1.

Department of Genetics and Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280, USA.

A dichotomy exists between germ and somatic cells in most organisms, such that somatic cell lineages proliferate for a single generation, whereas the germ cell lineage has the capacity to proliferate from one generation to the next, indefinitely. Several theories have been proposed to explain the unlimited replicative life span of germ cells, including the elimination of damaged germ cells by apoptosis or expression of high levels of gene products that prevent aging in somatic cells. These theories were tested in the nematode Caenorhabditis elegans by examining the consequences of eliminating either apoptosis or the daf-16, daf-18 or sir-2.1 genes that promote longevity of postmitotic somatic cells. However, germ cells of strains deficient for these activities displayed an unlimited proliferative capacity. Thus, C. elegans germ cells retain their youthful character via alternative pathways that prevent or eliminate damage that accumulates as a consequence of cell proliferation.
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http://dx.doi.org/10.1111/j.1474-9726.2006.00244.xDOI Listing
December 2006

Genetics of proliferative aging.

Exp Gerontol 2006 Oct 17;41(10):992-1000. Epub 2006 Oct 17.

Department of Genetics, University of North Carolina, Chapel Hill, NC 27599-3280, USA.

Human lifespan is limited by aging of both mitotic and post-mitotic cells. These two forms of aging may occur by distinct or overlapping mechanisms. Telomere erosion has been shown to limit the proliferative lifespan of human somatic cells. Other vertebrates, such as mice, possess robust telomerase activity in most cell types and their somatic cells display finite replicative lifespans as a consequence of other forms of macromolecular damage. Genetic analysis in humans, mice and yeast has provided clues regarding pathways that may affect a cell's replicative lifespan. In addition, analysis of the means by which germ cells maintain their effervescent character may provide a deeper understanding of how replicative aging occurs in somatic cells.
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http://dx.doi.org/10.1016/j.exger.2006.06.057DOI Listing
October 2006

Mutator phenotype of Caenorhabditis elegans DNA damage checkpoint mutants.

Genetics 2006 Oct 1;174(2):601-16. Epub 2006 Sep 1.

Department of Biology, University of North Carolina, NC 27599-3280, USA.

DNA damage response proteins identify sites of DNA damage and signal to downstream effectors that orchestrate either apoptosis or arrest of the cell cycle and DNA repair. The C. elegans DNA damage response mutants mrt-2, hus-1, and clk-2(mn159) displayed 8- to 15-fold increases in the frequency of spontaneous mutation in their germlines. Many of these mutations were small- to medium-sized deletions, some of which had unusual sequences at their breakpoints such as purine-rich tracts or direct or inverted repeats. Although DNA-damage-induced apoptosis is abrogated in the mrt-2, hus-1, and clk-2 mutant backgrounds, lack of the apoptotic branch of the DNA damage response pathway in cep-1/p53, ced-3, and ced-4 mutants did not result in a Mutator phenotype. Thus, DNA damage checkpoint proteins suppress the frequency of mutation by ensuring that spontaneous DNA damage is accurately repaired in C. elegans germ cells. Although DNA damage response defects that predispose humans to cancer are known to result in large-scale chromosome aberrations, our results suggest that small- to medium-sized deletions may also play roles in the development of cancer.
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http://dx.doi.org/10.1534/genetics.106.058701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1602097PMC
October 2006

Developmental modulation of nonhomologous end joining in Caenorhabditis elegans.

Genetics 2006 Jul 15;173(3):1301-17. Epub 2006 May 15.

Department of Genetics, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.

Homologous recombination and nonhomologous end joining (NHEJ) are important DNA double-strand break repair pathways in many organisms. C. elegans strains harboring mutations in the cku-70, cku-80, or lig-4 NHEJ genes displayed multiple developmental abnormalities in response to radiation-induced DNA damage in noncycling somatic cells. These phenotypes did not result from S-phase, DNA damage, or mitotic checkpoints, apoptosis, or stress response pathways that regulate dauer formation. However, an additional defect in him-10, a kinetochore component, synergized with NHEJ mutations for the radiation-induced developmental phenotypes, suggesting that they may be triggered by mis-segregation of chromosome fragments. Although NHEJ was an important DNA repair pathway for noncycling somatic cells in C. elegans, homologous recombination was used to repair radiation-induced DNA damage in cycling somatic cells and in germ cells at all times. Noncycling germ cells that depended on homologous recombination underwent cell cycle arrest in G2, whereas noncycling somatic cells that depended on NHEJ arrested in G1, suggesting that cell cycle phase may modulate DNA repair during development. We conclude that error-prone NHEJ plays little or no role in DNA repair in C. elegans germ cells, possibly ensuring homology-based double-strand break repair and transmission of a stable genome from one generation to the next.
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http://dx.doi.org/10.1534/genetics.106.058628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1526663PMC
July 2006

trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase.

PLoS Genet 2006 Feb 10;2(2):e18. Epub 2006 Feb 10.

Department of Genetics, University of North Carolina, Chapel Hill, North Carolina, USA.

Mutants of trt-1, the Caenorhabditis elegans telomerase reverse transcriptase, reproduce normally for several generations but eventually become sterile as a consequence of telomere erosion and end-to-end chromosome fusions. Telomere erosion and uncapping do not cause an increase in apoptosis in the germlines of trt-1 mutants. Instead, late-generation trt-1 mutants display chromosome segregation defects that are likely to be the direct cause of sterility. trt-1 functions in the same telomere replication pathway as mrt-2, a component of the Rad9/Rad1/Hus1 (9-1-1) proliferating cell nuclear antigen-like sliding clamp. Thus, the 9-1-1 complex may be required for telomerase to act at chromosome ends in C. elegans. Although telomere erosion limits replicative life span in human somatic cells, neither trt-1 nor telomere shortening affects postmitotic aging in C. elegans. These findings illustrate effects of telomere dysfunction in C. elegans mutants lacking the catalytic subunit of telomerase, trt-1.
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http://dx.doi.org/10.1371/journal.pgen.0020018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1361356PMC
February 2006

Importance of N- and C-terminal regions of gastrin-Gly for preferential binding to high and low affinity gastrin-Gly receptors.

Peptides 2005 Jul;26(7):1207-12

Department of Biomedical Science, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE, USA.

G17-Gly has been shown to stimulate the growth of DLD-1 human colon cancer cells in a biphasic manner via high and low affinity receptors. In the current study, the existence of heterogeneous receptor populations for G17-Gly on the HT-29 human colon cancer cell line was investigated. The effect of either N- or C-terminal peptide truncation on receptor binding and cell growth stimulation was also explored. [Leu15]G17-Gly bound to both high (nM) and low (microM) affinity sites on HT-29 cells. The peptide stimulated cell growth in a dose-dependent and biphasic manner with maximal stimulation at 10(-9) M peptide concentration, suggesting that, as in the case of DLD-1 cells, it is the high affinity receptor which is responsible for the growth-promoting effects. In contrast, G17(1-12) stimulated the growth of HT-29 cells in a sigmoidal fashion with an EC50 of 4.6x10(-9) M. Sequential N-terminal truncation of [Leu15]G17-Gly results in decreased binding to the high affinity G17-Gly receptor on DLD-1 cells. [Leu15]G17(11-17)Gly bound to the low affinity G17-Gly receptor with an affinity similar to that of the full sequence peptide but was unable to displace the radioligand from high affinity sites. G17(1-6)-NH2 was unable to displace [3H]G17-Gly from either site. These results suggest that the important residues for binding to the low affinity receptor are in the C-terminal region of the peptide while those required for interaction with the high affinity receptor lie further towards the N-terminus.
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http://dx.doi.org/10.1016/j.peptides.2005.02.001DOI Listing
July 2005