The HSP70 chaperone as sensor of the NEDD8 cycle upon DNA damage.

Molecular chaperones are essential components of the protein quality control system and maintenance of homeostasis. Heat Shock Protein 70 (HSP70), a highly evolutionarily conserved family of chaperones is a key regulator of protein folding, oligomerisation and prevents the aggregation of misfolded proteins. HSP70 chaperone function depends on the so-called 'HSP70-cycle', where HSP70 interacts with and is released from substrates via ATP hydrolysis and the assistance of HSP70 co-factors/co-chaperones, which also provide substrate specificity. The identification of regulatory modules for HSP70 allows the elucidation of HSP70 specificity and target selectivity. Here, we discuss how the HSP70 cycle is functionally linked with the cycle of the Ubiquitin-like molecule NEDD8. Using as an example the DNA damage response, we present a model where HSP70 acts as a sensor of the NEDD8 cycle. The NEDD8 cycle acts as a regulatory module of HSP70 activity, where conversion of poly-NEDD8 chains into mono-NEDD8 upon DNA damage activates HSP70, facilitating the formation of the apoptosome and apoptosis execution.

C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency.

Mutation is associated with developmental and hereditary disorders, aging, and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We used Caenorhabditis elegans whole-genome sequencing to model mutational signatures, analyzing 183 worm populations across 17 DNA repair-deficient backgrounds propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was approximately one per genome per generation, not overtly altered across several DNA repair deficiencies over 20 generations. Telomere erosion led to complex chromosomal rearrangements initiated by breakage-fusion-bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin B1 induced substitutions of guanines in a GpC context, as observed in aflatoxin-induced liver cancers. Mutational burden increased with impaired nucleotide excision repair. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions, and rearrangements. Strikingly, both agents induced clustered rearrangements resembling "chromoanasynthesis," a replication-based mutational signature seen in constitutional genomic disorders, suggesting that interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting that this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease.

Stable-isotope labeling with amino acids in nematodes.

We describe an approach for accurate quantitation of global protein dynamics in Caenorhabditis elegans. We adapted stable-isotope labeling with amino acids in cell culture (SILAC) for nematodes by feeding worms a heavy lysine- and heavy arginine-labeled Escherichia coli strain and report a genetic solution to elminate the problem of arginine-to-proline conversion. Combining our approach with quantitative proteomics methods, we characterized the heat-shock response in worms.

Germ cell apoptosis and DNA damage responses.

In the past 12 years, since the first description of C. elegans germ cell apoptosis, this area of research rapidly expanded. It became evident that multiple genetic pathways lead to the apoptotic demise of germ cells. We are only beginning to understand how these pathways that all require the CED-9/Bcl-2, Apaf-1/CED-4 and CED-3 caspase core apoptosis components are regulated. Physiological apoptosis, which likely accounts for the elimination of more than 50% of all germ cells, even in unperturbed conditions, is likely to be required to maintain tissue homeostasis. The best-studied pathways lead to DNA damage-induced germ cell apoptosis in response to a variety of genotoxic stimuli. This apoptosis appears to be regulated similar to DNA damage-induced apoptosis in the mouse germ line and converges on p53 family transcription factors. DNA damage response pathways not only lead to apoptosis induction, but also directly affect DNA repair, and a transient cell cycle arrest of mitotic germ cells. Finally, distinct pathways activate germ cell apoptosis in response to defects in meiotic recombination and meiotic chromosome pairing.

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

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.

Two distinct mechanisms lead to either oocyte or spermatocyte decrease in C. elegans after whole developmental exposure to γ-rays.

Wildlife is subject to various sources of pollution, including ionizing radiation. Adverse effects can impact the survival, growth, or reproduction of organisms, later affecting population dynamics. In invertebrates, reproduction, which directly impacts population dynamics, has been found to be the most radiosensitive endpoint. Understanding the underlying molecular pathways inducing this reproduction decrease can help to comprehend species-specific differences in radiosensitivity. From our previous studies, we found that decrease in reproduction is life stage dependent in the roundworm Caenorhabditis elegans, possibly resulting from an accumulation of damages during germ cell development and gamete differentiation. To go further, we used the same experimental design to assess more precisely the molecular determinants of reproductive toxicity, primarily decreases in gamete number. As before, worms were chronically exposed to 50 mGy·h-1 external gamma ionizing radiation throughout different developmental periods (namely embryogenesis, gametogenesis, and full development). To enable cross species extrapolation, conserved molecular pathways across invertebrates and vertebrates were analysed: apoptosis and MAP kinase Ras/ERK (MPK-1), both involved in reproduction and stress responses. Our results showed that these pathways are life-stage dependent, resulting from an accumulation of damages upon chronic exposure to IR throughout the life development. The Ras/ERK pathway was activated in our conditions in the pachytene region of the gonad where it regulates cell fate including apoptosis, but not in the ovulation zone, where it controls oocyte maturation and ovulation. Additionally, assessment of germ cell proliferation via Ras/ERK pathway showed no effect. Finally, a functional analysis of apoptosis revealed that while the decrease of the ovulation rate is caused by DNA-damaged induced apoptosis, this process does not occur in spermatocytes. Thus, sperm decrease seems to be mediated via another mechanism, probably a decrease in germ cell proliferation speed that needs further investigation to better characterize sex-specific responses to IR exposure. These results are of main importance to describe radio-induced reprotoxic effects and contribute as weight of evidence for the AOP #396 "Deposition of ionizing energy leads to population decline via impaired meiosis".

Targeting the NEDP1 enzyme to ameliorate ALS phenotypes through stress granule disassembly.

The elimination of aberrant inclusions is regarded as a therapeutic approach in neurodegeneration. In amyotrophic lateral sclerosis (ALS), mutations in proteins found within cytoplasmic condensates called stress granules (SGs) are linked to the formation of pathological SGs, aberrant protein inclusions, and neuronal toxicity. We found that inhibition of NEDP1, the enzyme that processes/deconjugates the ubiquitin-like molecule NEDD8, promotes the disassembly of physiological and pathological SGs. Reduction in poly(ADP-ribose) polymerase1 activity through hyper-NEDDylation is a key mechanism for the observed phenotype. These effects are related to improved cell survival in human cells, and in C. elegans, nedp1 deletion ameliorates ALS phenotypes related to animal motility. Our studies reveal NEDP1 as potential therapeutic target for ALS, correlated to the disassembly of pathological SGs.

Proteome-wide identification of NEDD8 modification sites reveals distinct proteomes for canonical and atypical NEDDylation.

The ubiquitin-like molecule NEDD8 controls several biological processes and is a promising target for therapeutic intervention. NEDDylation occurs through specific NEDD8 enzymes (canonical) or enzymes of the ubiquitin system (atypical). Identification of NEDD8 sites on substrates is critical for delineating the processes controlled by NEDDylation. By combining the use of the NEDD8 R74K mutant with anti-di-glycine (anti-diGly) antibodies, we identified 1,101 unique NEDDylation sites in 620 proteins. Bioinformatics analysis reveals that canonical and atypical NEDDylation have distinct proteomes; the spliceosome/mRNA surveillance/DNA replication and ribosome/proteasome, respectively. The data also reveal the formation of poly-NEDD8, hybrid NEDD8-ubiquitin, and NEDD8-SUMO-2 chains as potential molecular signals. In particular, NEDD8-SUMO-2 chains are induced upon proteotoxic stress (atypical) through NEDDylation of K11 in SUMO-2, and conjugates accumulate in previously described nucleolus-related inclusions. The study uncovers a diverse proteome for NEDDylation and is consistent with the concept of extensive cross-talk between ubiquitin and Ubls under proteotoxic stress conditions.

A Role for Caenorhabditis elegans COMPASS in Germline Chromatin Organization.

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed by the SET1/COMPASS complex and is associated with context-dependent effects on gene expression and local changes in chromatin organization. The role of SET1/COMPASS in shaping chromosome architecture has not been investigated. Here we used Caenorhabditis elegans to address this question through a live imaging approach and genetic analysis. Using quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histones eGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of set-2, encoding the C. elegans SET1 homologue, or CFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germline chromosome organization defects and sterility of condensin-II depleted animals. set-2 loss also aggravates germline defects resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes. Expression profiling of set-2 mutant germlines revealed only minor transcriptional changes, suggesting that the observed effects are at least partly independent of transcription. Altogether, our results are consistent with a role for SET1/COMPASS in shaping meiotic chromosomes in C. elegans, together with the non-histone proteins condensin-II and topoisomerase. Given the high degree of conservation, our findings expand the range of functions attributed to COMPASS and suggest a broader role in genome organization in different species.

The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity.

Ubiquitin and ubiquitin-like chains are finely balanced by conjugating and de-conjugating enzymes. Alterations in this balance trigger the response to stress conditions and are often observed in pathologies. How such changes are detected is not well understood. We identify the HSP70 chaperone as a sensor of changes in the balance between mono- and poly-NEDDylation. Upon DNA damage, the induction of the de-NEDDylating enzyme NEDP1 restricts the formation of NEDD8 chains, mainly through lysines K11/K48. This promotes APAF1 oligomerization and apoptosis induction, a step that requires the HSP70 ATPase activity. HSP70 binds to NEDD8, and, in vitro, the conversion of NEDD8 chains into mono-NEDD8 stimulates HSP70 ATPase activity. This effect is independent of NEDD8 conjugation onto substrates. The study indicates that the NEDD8 cycle is a regulatory module of HSP70 function. These findings may be important in tumorigenesis, as we find decreased NEDP1 levels in hepatocellular carcinoma with concomitant accumulation of NEDD8 conjugates.

Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes.

How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.

Methods for studying the DNA damage response in the Caenorhabdatis elegans germ line.

In response to genotoxic insults, cells activate DNA damage response pathways that either stimulate transient cell cycle arrest and DNA repair or induce apoptosis. The Caenorhabditis elegans germ line is now well established as a model system to study these processes in a genetically tractable, multicellular organism. Upon treatment with genotoxic agents, premeiotic C. elegans germ cells transiently halt cell cycle progression, whereas meiotic prophase germ cells in the late-pachytene stage undergo apoptosis. Further, accumulation of unrepaired meiotic recombination intermediates can also lead to apoptosis of affected pachytene cells. DNA damage-induced cell death requires key components of the evolutionarily conserved apoptotic machinery. Moreover, both cell cycle arrest and pachytene apoptosis responses depend on conserved DNA damage checkpoint proteins. Genetics- and genomics-based approaches that have demonstrated roles for conserved checkpoint proteins have also begun to uncover novel components of these response pathways. In this chapter, we briefly review the C. elegans DNA damage response field, discuss in detail methods currently used to assay DNA damage responses in C. elegans, and describe the development of new experimental tools that will facilitate a more comprehensive understanding of the DNA damage response.

Activation of Mrc1, a mediator of the replication checkpoint, by telomere erosion.

BACKGROUND INFORMATION: In budding yeast, the loss of either telomere sequences (in telomerase-negative cells) or telomere capping (in mutants of two telomere end-protection proteins, Cdc13 and Yku) lead, by distinct pathways, to telomeric senescence. After DNA damage, activation of Rad53, which together with Chk1 represents a protein kinase central to all checkpoint pathways, normally requires Rad9, a checkpoint adaptor. RESULTS: We report that in telomerase-negative (tlc1Delta) cells, activation of Rad53, although diminished, could still take place in the absence of Rad9. In contrast, Rad9 was essential for Rad53 activation in cells that entered senescence in the presence of functional telomerase, namely in senescent cells bearing mutations in telomere end-protection proteins (cdc13-1 yku70Delta). In telomerase-negative cells deleted for RAD9, Mrc1, another checkpoint adaptor previously implicated in the DNA replication checkpoint, mediated Rad53 activation. Rad9 and Rad53, as well as other DNA damage checkpoint proteins (Mec1, Mec3, Chk1 and Dun1), were required for complete DNA-damage-induced cell-cycle arrest after loss of telomerase function. However, unexpectedly, given the formation of an active Rad53-Mrc1 complex in tlc1Delta rad9Delta cells, Mrc1 did not mediate the cell-cycle arrest elicited by telomerase loss. Finally, we report that Rad9, Mrc1, Dun1 and Chk1 are activated by phosphorylation after telomerase inactivation. CONCLUSIONS: These results indicate that loss of telomere capping and loss of telomere sequences, both of which provoke telomeric senescence, are perceived as two distinct types of damages. In contrast with the Rad53-Rad9-mediated cell-cycle arrest that functions in a similar way in both types of telomeric senescence, activation of Rad53-Mrc1 might represent a specific response to telomerase inactivation and/or telomere shortening, the functional significance of which has yet to be uncovered.