Lopes, Amanda et al. (2021) International Worm Meeting "A C. elegans model to study the molecular pathogenesis of Cockayne Syndrome progeria"

Lopes, Amanda, Rieckher, Matthias, Schumacher, Bjorn

[International Worm Meeting, 2021]

Cockayne Syndrome (CS) is a rare congenital disease that causes photosensitivity, growth and mental retardation, impaired nervous system development and premature ageing. The syndrome displays a complex array of symptoms that are caused by mutations in the CSB, or CSA gene, respectively, which facilitate DNA damage recognition in transcription-coupled nucleotide excision repair (TC-NER). While CS mouse models are photosensitive, and have been important for understanding some of the clinical features observed in humans, they lack concrete evidence for neurodegeneration. Here, we present evidence of neuronal and mitochondrial aberrations in csb-1(ok2335) mutant worms, which can be rescued by transgenic expression of pcsb-1CSB-1::GFP. Neurodegeneration manifests progressively and is paralleled by neuro-muscular functional decline substantiated by reduced pharyngeal pumping, locomotion, touch sensation and chemosensation abilities, which are further enhanced upon UVB-induced DNA damage or transcription-blocking lesions caused by the cytotoxin Illudin M. The csb-1 mutant shows the accumulation of dysfunctional mitochondria, and increased hyperfusion, which is exacerbated upon exposure to UVB, resulting in reduced respiratory activity. Our data support the causal role of endogenous DNA damage for neurodegeneration and mitochondrial dysfunction in CS, and warrants the use of the model for identifying pharmacological interventions to improve CS and ageing.

Wolters, Stefanie et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Screening for novel DNA repair genes functioning in somatic and germ line development"

Schumacher, Bjorn, Ermolaeva, Maria, Wolters, Stefanie

[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.

Wang, Siyao et al. (2021) International Worm Meeting "H3K4me2 regulates the recovery of protein biosynthesis and homeostasis following DNA damage"

Meyer, David, Wang, Siyao, Schumacher, Bjorn

[International Worm Meeting, 2021]

DNA damage causes cancer, impairs development and accelerates aging. Transcription-blocking lesions and transcription-coupled repair defects lead to developmental failure and premature aging in humans. Following DNA repair, homeostatic processes need to be reestablished to ensure development and maintain tissue functionality. Here, we report that, in Caenorhabditis elegans, removal of the WRAD complex of the MLL/COMPASS H3K4 methyltransferase exacerbates developmental growth retardation and accelerates aging, while depletion of the H3K4 demethylases SPR-5 and AMX-1 promotes developmental growth and extends lifespan amid ultraviolet-induced damage. We demonstrate that DNA-damage-induced H3K4me2 is associated with the activation of genes regulating RNA transport, splicing, ribosome biogenesis and protein homeostasis and regulates the recovery of protein biosynthesis that ensures survival following genotoxic stress. Our study uncovers a role for H3K4me2 in coordinating the recovery of protein biosynthesis and homeostasis required for developmental growth and longevity after DNA damage.

Uszkoreit, Simon et al. (2021) International Worm Meeting "Somatic Regulators of the Non-Cell-Autonomous CEP-1/p53-Mediated DNA Damage Response in Primordial Germ Cells"

Schumacher, Bjorn, Ou, Hui-Ling, Uszkoreit, Simon

[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.

Vaddavalli, Pavana Lakshmi et al. (2021) International Worm Meeting "cep-1/p53 mediated DNA damage response - understanding apoptosis in Caenorhabditis elegans germ cells"

Schumacher, Bjorn, Meyer, David, Vaddavalli, Pavana Lakshmi

[International Worm Meeting, 2021]

The DNA damage response (DDR) is an elaborate system that functions to preserve genome integrity. The DDR is initiated by DNA damage checkpoint pathways which halt cell cycle progression to allow DNA repair to precede or trigger programmed cell death (apoptosis) depending on the cell type and the extent of the damage. The tumor suppressor p53 is a central regulator of the DDR and regulates apoptosis by transcriptionally inducing pro-apoptotic genes. p53 is the most commonly mutated gene in human cancers. Most cancer-associated p53 mutations are missense mutations that compromise the tumour suppressive transcriptional activity. Consequently, damaged cells fail to undergo apoptosis, leading to uncontrolled proliferation and cancer formation. In this study, we employ Caenorhabditis elegans, to gain a deeper understanding of the mechanisms through which the DNA damage checkpoint signaling pathways impact the apoptotic DNA damage response. The core apoptotic pathway is distinctly conserved in C. elegans, where C. elegans p53-like, CEP-1, transcriptionally induces the BH3-only domain pro-apoptosis factors to trigger the apoptosome upon DNA damage. This model provides distinct advantages for the genetic investigation of apoptosis -the germline of the worm shows a specific and easy to visualize apoptotic response to DNA damage, enabling quantifiable evaluation of genetic and chemical interventions. We recapitulated the most prevalent p53 hotspot mutation in the worm, and generated a genetic tool for high throughput screening of novel regulators of cep-1/p53 dependent apoptosis in whole live organism. Ionizing radiation-induced apoptosis is abrogated in worms with the hotspot mutation, and in the forward genetic screen performed, we were able to isolate mutant worms which showed restored apoptosis despite a mutated p53. Our data indicate that the restored apoptotic response occurs in a delayed manner and involve a dysregulated ERK/MAPK signaling pathway. Overall, our preliminary findings point towards a secondary delayed wave of DNA damage response that is set in motion if the primary response regulated by cep-1/p53 fails when it is mutated.

Bujarrabal, Arturo et al. (2021) International Worm Meeting "The DRM complex functions as master regulator of somatic DNA repair capacities"

Bujarrabal, Arturo, Schumacher, Bjorn, Sendtner, Georg, Meyer, David H.

[International Worm Meeting, 2021]

The repair of DNA damage is crucial to ensure organismal health. However, the response to DNA damage differs depending on whether the cells are somatic or germ cells, as well as their proliferative and differentiation status. In germ cells, DNA repair is highly efficient and accurate, leading to very low mutation rates compared to somatic cells. In addition, replicating cells can use high-fidelity repair mechanisms such as homologous recombination as well as Single Strand Annealing, Microhomology End-Joining and long-patch Base Excision Repair. To repair UV-lesions, dividing cells rely on global genome nucleotide excision repair, whereas quiescent cells mostly use transcription-coupled nucleotide excision repair. In C. elegans, repair pathways are highly conserved and can be easily studied in the context of postmitotic somatic cells and the highly proliferative germline. The DRM/DREAM complex is a highly conserved transcriptional repressor of cell cycle genes that promotes quiescence. Using C. elegans, we have found that DRM can bind and repress multiple genes involved in the DNA damage response. A deficiency in the DRM complex leads to a gene expression signature with a significant component of DNA repair genes upregulated in the soma that resembles a germline-like expression pattern. DRM complex deficient mutants show a remarkable resistance to and improved repair of various DNA-damage types, both during development and aging. We propose that the DRM complex represses all major DNA repair pathways in somatic tissues, thus limiting their resistance to genotoxic stress. The DRM mutants' resistance to various DNA damage types indicates that this complex functions as a master regulator of somatic DNA repair capacity.

Rieckher, Matthias et al. (2015) International Worm Meeting "Investigations on cell- and tissue-specific activity of NER upon UVB irradiation in development and ageing of C. elegans."

Werthenbach, Paul, Lopes, Amanda, Rieckher, Matthias, Schumacher, Bjorn, Anton, Vincent

[International Worm Meeting, 2015]

DNA damage and genome instability are hallmarks of ageing. In humans, congenital defects in DNA repair mechanisms such as nucleotide excision repair (NER) lead to increased cancer risk, accelerated ageing including neurodegeneration and developmental growth defects. NER activity is highly conserved in C. elegans and NER deficient animals are incapable of repairing DNA damage inflicted by UV irradiation, resulting in growth arrest and decreased life span. We investigate the tissue-specific and age-dependent activity of NER in maintaining genome stability in C. elegans. To this end, we established an in vivo imaging system to monitor NER activity in C. elegans: We generated transgenic animals expressing the central NER factors XPA-1::GFP or CSB-1::GFP under control of the respective endogenous promoters. In vivo expression analysis reveals a progressive decline of XPA-1::GFP during ageing in all tissues, except the neuronal system. Further, we are performing tissue-specific rescue studies of the highly UVB sensitive xpa-1 mutant animals by constitutively driving expression of XPA-1::GFP in muscles, intestine, cuticle, and the neuronal system. We apply a UVC laser system for precise single-cell DNA damage induction in cuticle tissue, and observe rapid recruitment of XPA-1::GFP to the site of irradiation. This system allows investigating cell type-specific dynamics of DNA damage response factors during ageing and in the context of a whole organism. To further understand tissue-specific relevance of DNA repair, we created transgenic animals that ectopically express the A. thaliana photolyase PHR1, which specifically repairs cyclobutane pyrimidine dimers (CPDs), which comprise the main lesion type induced by UV. Ubiquitous and constitutive expression of PHR1::GFP enhances UV resistance of wild type and NER deficient animals. Understanding tissue-specific DNA damage responses and genome maintenance in C. elegans will lead to a better understanding of the complex human disorders that are caused by DNA repair defects and allow the development of intervention strategies to counteract age-related tissue-decline and promote longevity.

JM Schumacher et al. (1999) International C. elegans Meeting "The Great Divide: Analysis of single-cell cytokinesis-defective mutants in C. elegans"

A Golden, M Wallenfang, G Seydoux, JM Schumacher

[International C. elegans Meeting, 1999]

Mitosis culminates in the equal segregation of sister chromatids to each of two newly- formed cells. Genetic instability can be caused not only by defects in the segregation process itself, but also by failures in cytokinesis that can lead to the formation of polyploid cells. Previous studies in our lab have focused on two kinases that are directly involved in each of these events (Schumacher et al. 1998a,b). The C. elegans AIR-1 protein is required for proper assembly and function of the mitotic spindle, whereas the highly related AIR-2 protein is required for the stabilization of the cleavage furrow and the completion of cytokinesis. In an attempt to isolate mutations in the AIR-2 locus as well as identify new components in the AIR-2 pathway, we have recovered thirty temperature-sensitive maternal-effect lethal mutants that arrest at the one-cell stage. DAPI staining of all thirty mutants revealed that seven were highly polyploid at the restrictive temperature and essentially had the same phenotype as air-2(RNAi) cytokinesis-defective embryos (Schumacher et al. 1998b). Genetic mapping of and complementation tests between six of the mutants have shown that all six map to distinct genetic intervals and represent six independent loci. The seventh has not yet been mapped. Three of the mutants map to Chr. I, two map to Chr. V, and the remaining locus maps to a chromosome arm. All six appear to represent new mutants in that they do not map to genetic intervals that contain previously described cytokinesis-defective mutations. Finer mapping data as well as the analysis of candidate genes within the relevant intervals will be presented. Schumacher, J., Ashcroft, N., Donovan, P. and A. Golden (1998a) Development 125:4391-4402. Schumacher, J., Golden, A. and P. Donovan (1998b) J Cell Biol. 143:1635-1646. Research sponsored by the National Cancer Institute, DHHS, under contract with ABL.

Mueller, Michael et al. (2013) International Worm Meeting "DNA damage responses in development and ageing."

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.

Rieckher, Matthias et al. (2021) International Worm Meeting "Deciphering endogenous formaldehyde-induced cytotoxicity mechanisms in C. elegans"

Schumacher, Bjorn, Kolesnikova, Ksenia, Wiesner, Eva, Rieckher, Matthias, Pontel, Lucas, Graser, Laetitia, Morellato, Agustin, Umansky, Carla, Heidbuchel, Lisa

[International Worm Meeting, 2021]

Formaldehyde (FA) is an endogenous and environmental metabolite genotoxin that causes DNA and DNA-protein crosslinks. Endogenous FA has been identified as a major genotoxic and carcinogenic agent involved in the onset of the cancer-prone genetic disease Fanconi Anemia and in the development of a type of inherited bone marrow failure syndrome (IBMFS). Here, we employ C. elegans and human carcinoma cells to reveal that FA causes cellular damage by triggering oxidative stress, which is prevented by the alcohol dehydrogenase enzyme ADH5/GSNOR. We identify the ADH5 orthologue in C. elegans (adh-5; H24K24.3) and show that adh-5 mutants are hypersensitive to FA. Oxidative stress exacerbates FA sensitivity in ADH-5 deficient worms. Moreover, both worms and cells display ROS accumulation and the activation of oxidative stress responses, which is suppressed by supplementation with the GSH precursor N-acetylcysteine (NAC). Further, we demonstrate that endogenous GSH can protect cells lacking the Fanconi Anemia DNA repair pathway, or worms that are deficient in Nucleotide Excision Repair (NER), from endogenous or environmental FA. Hence, our findings establish a conserved mechanism that protects from FA cytotoxicity, which has broad implications for developing novel intervention strategies for Fanconi Anemia patients and against certain cancers.