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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Ultraviolet radiation of the sunlight represents an environmental carcinogen, which can lead to helix-distorting lesions in the genome and therefore cause cancer. To cope with this damaging influence cells developed a multistep cut and patch mechanism to repair the impaired DNA. The Nucleotide Excision Repair (NER) removes helix distorting lesions and is divided into two branches, which differ in damage recognition: while initiation factors of Global-Genome-NER (GG-NER) scan the whole genome for damage, Transcription Coupled Repair (TCR) initiates repair when RNA polymerase II encounters a lesion during transcription. The NER pathway is highly conserved between mammals and C. elegans. We identified UV hypersensitivity phenotypes of known C. elegans NER mutants distinct for GG-NER und TCR. While mutations in TCR lead to developmental arrest at L1 larval stage, defects in GG-NER lead to germ line arrest and sterility upon UV radiation of L1 larvae. We took advantage of these phenotypes to design a random mutagenesis based screening strategy. By performing this screening method we identified five different mutants that are hypersensitive to UV light. The respective mutations were mapped to the C. elegans genome via SNP mapping technique. Following genetic non-complementation analysis using strains that carry mutations in known DNA repair genes suggests that we identified mutations in potentially novel genes important for UV irradiation response. The specific location of the mutation will be identified by whole genome sequencing. In parallel the response of the mutant strains to UV light as well as genetic interactions with known NER factors are being characterized by means of biochemistry, immunohistochemistry and genetic analysis.
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[
International Worm Meeting,
2021]
The integrity of DNA is constantly threatened by DNA damage induced by endogenous metabolic processes, environmental factors like chemicals and irradiation, or spontaneously via deamination and depurination. In order to remove those DNA lesions fast and efficiently, multiple DNA repair pathways can be activated. Nucleotide excision repair (NER) is a major DNA repair pathway which repairs a wide variety of helix-distorting lesions, especially those induced by UV irradiation. Two subpathways in NER can be discerned: global genome NER (GG-NER), which detects DNA damage anywhere in the genome, and transcription-coupled NER (TC-NER) that recognizes DNA damage that stalls RNA polymerase II in transcribed strands. Following detection, both subpathways converge onto the common core NER verification step, followed by excision of the damaged DNA strand and gap filling by novel DNA synthesis. Hereditary mutations in NER genes cause multiple different diseases, including the progeroid Cockayne syndrome and the cancer prone xeroderma pigmentosum. However, their exact pathogenesis is not fully understood. We use C. elegans to understand the in vivo impact of DNA damage and the biological relevance of NER in a multicellular, developing organism. Previously, we showed that GG-NER primarily acts in proliferative germ cells and embryos, while TC-NER acts in post-mitotic somatic cells to maintain transcription. To better understand how DNA repair is organized in this tissue-specific manner, we developed a label free quantification proteomics pipeline with which we are able to isolate and characterize endogenous DNA repair complexes, both in unchallenged conditions and after UV irradiation-induced DNA damage. Using this method, we identified several previously unknown proteins that might regulate both NER initiation as well as the verification step. Currently, we investigate whether these proteins function in GG-NER and/or TC-NER, at which NER step they might function and whether their function is evolutionary conserved.
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[
International Worm Meeting,
2017]
Defects in the DNA Damage Response (DDR) affect tissues differently suggesting that genome maintenance operates in a tissue-specific manner. A prime example is given by ERCC1/XPF deficiency, which gives rise to pleiotropic symptoms including neurodegeneration, developmental defects, cancer, bone marrow failure and accelerated aging, depending on the type of mutation. ERCC1/XPF is a structure specific endonuclease that is involved in several DNA repair pathways and has a critical role in Nucleotide excision repair (NER). This major DDR pathway is responsible for removing bulky DNA lesions, in particular those formed by UV light. NER consists of two sub-pathways: Global Genome NER (GG-NER) deals with damage anywhere in the genome, whereas Transcription-Coupled NER (TC-NER) deals with damage that blocks transcription. Following damage detection and verification, a DNA stretch containing the damage is excised by endonucleases ERCC1/XPF and XPG. The resulting gap is filled in by DNA synthesis and ligation. Although the function of ERCC1/XPF has been studied in great detail in biochemical and cell biological experiments, it is not entirely clear how deficiency of this complex can lead to such a variety of tissue-specific symptoms. Previously, we have shown that in C. elegans, GG-NER mainly protects germ cells while TC-NER mainly protects somatic cells against UV-induced DNA damage. Furthermore, ERCC-1/XPF-1 loss-of-function causes developmental defects and accelerated replicative aging , which is reminiscent of symptoms in human patients. To better understand to what extent DNA repair functions differently in tissues, we expressed fluorescently tagged ERCC-1/XPF-1 in the germline, hypodermis, intestine, neurons and muscles. Next, we set up new live cell imaging methods to monitor the tissue-specific spatio-temporal dynamics of ERCC-1/XPF-1 in response to DNA damage. We show that in oocytes the endonuclease complex quickly but transiently re-localizes to and binds UV-damaged chromosomes in a GG-NER-dependent manner. Intriguingly, we find that re-localization and chromatin binding in response to DNA damage changes upon differentiation of cells. Our results confirm that in vivo the main role of GG-NER is to safeguard the whole genome in the totipotent germline, while the main role of TC-NER is to safeguard active genes to promote cell function in differentiated tissues.
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[
East Coast Worm Meeting,
1998]
Mutations in the
clk-1 gene of the nematode Caenorhabditis elegans affect the timing of a variety of developmental and physiological processes and increase life span (Wong et al., Genetics 139, 1247 (1995)).
clk-1 encodes a protein with high similarity to yeast Coq7p as well as to predicted mammalian proteins (Ewbank et al., Science 275, 980 (1997)). Coq7p is required for respiration as it is necessary for the biosynthesis of the lipid co-factor coenzyme Q (CoQ) (also called ubiquinone). Indeed, no or very little CoQ is found in these mutants (Marbois and Clarke, J. Biol. Chem. 271, 2995 (1996)). We show that in worms CLK-1 is fully active when fused to Green Fluorescent Protein (GFP) and is found in mitochondria. We show that mitochondrial respiration is only mildly depressed in
clk-1 mutants (~85% of the wild type). Furthermore, an excess of exogenous CoQ cannot restore normal respiration, suggesting that a deficiency in CoQ is not the main cause of the
clk-1 phenotype. A transgene overexpressing CLK-1 activity in wild-type worms can increase respiration beyond the wild type level, accelerate behavioral rates (the defecation cycle) during aging and shorten life span, indicating that
clk-1 controls these processes. These observations (that decreased respiration in
clk-1 mutants results in a longer life and that increased respiration in worms overexpressing CLK-1 results in a shorter life) suggest that decreased mitochondrial respiration at older ages, which is observed in all animals, might be the result of an active process promoting longer life.
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[
International Worm Meeting,
2005]
We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).
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Mueller, Michael, Frommolt, Peter, Ermolaeva, Maria, Schumacher, Bjorn, Greiss, Sebastian, Schneider, Jennifer, Castells-Roca, Laia
[
International Worm Meeting,
2013]
Congenital defects in genome maintenance systems cause complex disease phenotypes characterized by developmental failure, cancer susceptibility and premature aging. In contrast to well-characterized cellular DNA damage checkpoint mechanisms, it remains poorly understood how DNA damage responses affect organismal development and maintain functionality of tissues when DNA damage gradually accumulates with aging. The distinct human disease outcomes of DNA repair defects become particularly apparent in syndromes caused by mutations in nucleotide excision repair (NER): While transcription-coupled (TC-) NER defects lead to developmental growth defects and premature ageing in Cockayne syndrome (CS), global-genome (GG-) NER mutations lead to highly skin cancer prone Xeroderma pigmentosum (XP). In C. elegans, defects in the highly conserved TC-NER branch lead to developmental arrest of somatic tissues, while GG-NER defects give rise to genome instability in proliferating germ cells. We have employed the nematode model to explore the distinct DNA damage responses in germ cells and somatic tissues. We identified a network of insulin-like growth factor signalling (IIS) genes that responds to DNA damage during C. elegans development and show that the FoxO transcription factor DAF-16 is activated in response to DNA damage. We demonstrate that DAF-16 alleviates DNA damage induced growth arrest through differential activation of downstream target genes that contrasts its established role in the starvation response, and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We propose that IIS mediates developmental DNA damage responses and that DAF-16 activity enables developmental progression amid persistent DNA lesions and promotes tissue maintenance through enhanced tolerance of DNA damage that accumulates with aging.
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Vermeulen, Wim, Nilsen, Hilde, Jansen, Gert, Karambelas, Andrea E, Hoeijmakers, Jan H.J., Cupac, Daniel, Lindvall, Jessica M., Fensgard, Oyvind, Thijssen, Karen, Lans, Hannes
[
International Worm Meeting,
2013]
Nucleotide Excision Repair (NER) is a versatile DNA repair pathway that removes a wide variety of helix-distorting DNA lesions, including those induced by UV irradiation. Two subpathways in NER can be discerned, one which detects DNA damage anywhere in the genome, called global genome NER (GG-NER), and one which detects damage in the transcribed strand during transcription, called transcription-coupled NER (TC-NER). Following detection, the damaged DNA strand is excised and the resulting gap is repaired by novel DNA synthesis. In humans, NER-deficiency is associated with severe clinical disorders characterized by cancer predisposition and/or pleiotropic developmental defects and accelerated aging of which the pathogenesis is only partially understood.
We use C. elegans to understand the in vivo impact of DNA damage and the biological relevance of NER in a multicellular, developing organism. Previously, we showed that UV-exposed germ cells mainly depend on functional GG-NER, while postembryonic cells predominantly depend on TC-NER. Further analysis of development and aging associated with NER deficiency shows that, contrary to expectation, DNA damage accumulation does not decrease adult lifespan of post-mitotic tissue. Surprisingly, NER deficiency even further extends life-span of long-lived
daf-2 mutants, through an adaptive activation of stress signaling. Contrary, NER deficiency leads to a striking decrease in replicative lifespan and transgenerational aging of proliferating cells. DNA damage accumulation induces severe, stochastic impairment of development and growth, which is most pronounced when additional DNA repair pathways besides NER are impaired. These results show that different DNA lesions contribute to replicative aging and suggest that also in patients there might be a direct link between symptoms and the level of DNA repair deficiency.
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[
International Worm Meeting,
2015]
Nucleotide Excision Repair (NER) is a major genome maintenance mechanism responsible for removing DNA-distorting lesions, in particular those formed by UV light. NER is initiated by two distinct damage detection mechanisms: Global Genome NER (GG-NER), that detects damage anywhere in the genome, and Transcription Coupled NER (TC-NER), that detects damage in the transcribed strand during transcription. Both mechanisms converge in a common pathway that verifies the presence of damage, excises it and fills the gap. In humans, NER deficiency affects tissues in various manners and is associated with several clinical disorders that are characterized by cancer predisposition, developmental defects and accelerated aging. Although, the molecular mechanism of NER is known in detail, mainly from in vitro and cell culture experiments, it is not entirely clear how defects in one pathway lead to these various and tissue-specific symptoms.We use C. elegans to better understand how NER operates and is regulated in vivo, in different tissues and stages of development. Previously we have shown that GG-NER mainly acts in germ cells while TC-NER mainly acts in somatic cells to protect against UV-induced DNA damage. Here, we focus on the ERCC-1/XPF-1 complex, a structure-specific endonuclease involved in damage excision during NER but which also acts in the repair of interstrand crosslinks and double strand breaks. To determine how this endonuclease responds to DNA damage and to identify which mechanisms regulate this, we expressed fluorescently tagged XPF-1 in different tissues and in diverse NER genetics backgrounds. By imaging living and fixed animals, we analyze the in vivo kinetics of the repair reaction in response to different kinds of DNA damage. Preliminary results show that XPF-1 quickly re-localizes in response to DNA damage in different tissues. By performing genetic analysis and proteomic screening we hope to uncover the regulatory mechanisms that underlie cell type specific responses of NER.
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[
International Worm Meeting,
2003]
Comparing homologous cis-regulatory DNA sequences from three or more genomes has advantages over pairwise comparison of only two. Cis-regulatory sequences are short (6-20 bp) and tolerate substantial variation. Purely random pairing of unrelated 100-bp DNA segments is expected to yield two perfect 6 bp matches. Alignment of a third or fourth sequence should greatly lower the frequency of false positive regions, allowing small but real cis-regulatory sequences to be efficiently detected. This increased resolution should also allow direct comparison between phylogenetically conserved sequences and statistically overrepresented sequences, which may yield complementary views of regulatory elements. In the Caenorhabditis genus, C. remanei appears to be most closely related to C. briggsae; two other species, CB5161 and PS1010, comprise the two closest known and culturable Caenorhabditis species outside the elegans-briggsae group (Fitch, 2000). CB5161 is closest to C. elegans, and PS1010 the next most divergent; these two species thus provide an evolutionarily graded series. We have constructed fosmid libraries from CB5161 and PS1010, and begun sequencing individual fosmids for comparative analysis of genes involved in vulval or sensory neuron development. At the same time, we have devised the Mussa software package to adapt the algorithms of Davidson and coworkers (Brown et al., 2002) to multiple sequence analysis. At this writing, we have sequence data from the
egl-30,
lin-11, and
mab-5 loci of both CB5161 and PS1010. Initial results of sequencing and comparative sequence analysis will be presented. References: Brown, C.T., Rust, A.G., Clarke, P.J., Pan, Z., Schilstra, M.J., De Buysscher, T., Griffin, G., Wold, B.J., Cameron, R.A., Davidson, E.H., and Bolouri, H. (2002). New computational approaches for analysis of cis-regulatory networks. Dev. Biol. 246, 86-102; Fitch, D.H.A. (2000). Evolution of Rhabditidae and the male tail. J. Nematol. 32, 235-244.
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[
International Worm Meeting,
2021]
The integrity of heritable genomes is a prerequisite for species maintenance. For more than a century, it was thought that the genomes in the germline of an organism are isolated from somatic influences by the so-called Weismann barrier. In C. elegans, the primordial germline consists of two somatic gonad precursors (SGPs) and two primordial germ cells (PGCs). We previously established that PGCs are relying on global genome nucleotide excision repair (GG-NER) to repair UV-induced DNA lesions. When GG-NER is compromised and the DNA damage persists the C. elegans
p53-like, CEP-1, protein is induced in PGCs and keeps them arrested. Using a forward genetic approach, we established an unexpected non-cell-autonomous regulation of the CEP-1-mediated DNA damage response (DDR) in PGCs via the somatic niche. The niche control of the DDR in PGCs is mediated by the translation initiation factor IFE-4 operating in the SGPs and communicated via FGF-like signalling. Moreover, we determined that the niche control mechanisms of the
p53 response is highly conserved from worms to mammals. In mammals, the IFE-4 orthologue eIF4E2 regulates the
p53 induction in hair follicle stem cells upon UV-induced DNA damage. Therefore, we propose that a better understanding of the non-cell-autonomous control of the
p53-mediated DDR in C. elegans will be highly relevant to better understand this central tumour suppressor mechanism in humans. To elucidate the somatic regulators of the DDR in PGCs, we used a targeted genetic approach and interrogated pathways that we hypothesised to regulate the response to genotoxic stress. We determined an important regulatory role for nutritional interpretation and neuronal signalling. Impairment in these signalling processes suppressed the PGC cell cycle arrest and germline development upon persistent DNA damage. To dissect the mechanisms of this somatic influence in more detail, current emphasis is put on tissue-specific transcriptomics on fluorescence activated cell sorting (FACS)-based PGC/SGP isolations. We show for the first time a neuronal influence on the non-cell-autonomous DDR in PGCs and broaden the understanding of somatic regulations on the germline of C. elegans thus challenging the Weismann barrier.