Stress-induced Genome Instability Research Articles

A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.

Locus-specific transcription silencing at the FHIT gene suppresses replication stress-induced copy number variant formation and associated replication delay.

Impaired replication progression leads to de novo copy number variant (CNV) formation at common fragile sites (CFSs). We previously showed that these hotspots for genome instability reside in late-replicating domains associated with large transcribed genes and provided indirect evidence that transcription is a factor in their instability. Here, we compared aphidicolin (APH)-induced CNV and CFS frequency between wild-type and isogenic cells in which FHIT gene transcription was ablated by promoter deletion. Two promoter-deletion cell lines showed reduced or absent CNV formation and CFS expression at FHIT despite continued instability at the NLGN1 control locus. APH treatment led to critical replication delays that remained unresolved in G2/M in the body of many, but not all, large transcribed genes, an effect that was reversed at FHIT by the promoter deletion. Altering RNase H1 expression did not change CNV induction frequency and DRIP-seq showed a paucity of R-loop formation in the central regions of large genes, suggesting that R-loops are not the primary mediator of the transcription effect. These results demonstrate that large gene transcription is a determining factor in replication stress-induced genomic instability and support models that CNV hotspots mainly result from the transcription-dependent passage of unreplicated DNA into mitosis.

Systematic Analysis of the Stress-Induced Genomic Instability of Type Three Secretion System in Aeromonas salmonicida subsp. salmonicida.

The type three secretion system (TTSS) locus of Aeromonas salmonicida subsp. salmonicida, located on the plasmid pAsa5, is known to be lost when the bacterium is grown at temperatures of 25 °C. The loss of the locus is due to the recombination of the insertion sequences flanking the TTSS region. However, the mechanism involved in this recombination is still elusive. Here, we analyzed 22 A. salmonicida subsp. salmonicida strains that had already lost their TTSS locus, and we systematically explored another 47 strains for their susceptibility to lose the same locus when grown at 25 °C. It appeared that strains from Europe were more prone to lose their TTSS locus compared to Canadian strains. More specifically, it was not possible to induce TTSS loss in Canadian strains that have AsaGEI2a, a genomic island, and prophage 3, or in Canadian strains without a genomic island. A comparative genomic approach revealed an almost perfect correlation between the presence of a cluster of genes, not yet characterized, and the susceptibility of various groups of strains to lose their locus. This cluster of genes encodes putative proteins with DNA binding capacity and phage proteins. This discovery creates new opportunities in the study of pAsa5 thermosensitivity.

Nutrient-Driven O-GlcNAcylation Controls DNA Damage Repair Signaling and Stem/Progenitor Cell Homeostasis

Stem/progenitor cells exhibit high proliferation rates, elevated nutrient uptake, altered metabolic flux, and stress-induced genome instability. O-GlcNAcylation is an essential post-translational modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which act in a nutrient- and stress-responsive manner. The precise role of O-GlcNAc in adult stem cells and the relationship between O-GlcNAc and the DNA damage response (DDR) is poorly understood. Here, we show that hyper-O-GlcNacylation leads to elevated insulin signaling, hyperproliferation, and DDR activation that mimic the glucose- and oxidative-stress-induced response. We discover a feedback mechanism involving key downstream effectors of DDR, ATM, ATR, and CHK1/2 that regulates OGT stability to promote O-GlcNAcylation and elevate DDR. This O-GlcNAc-dependent regulatory pathway is critical for maintaining gut homeostasis in Drosophila and the DDR in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). Our findings reveal a conserved mechanistic link among O-GlcNAc cycling, stem cell self-renewal, and DDR with profound implications for stem-cell-derived diseases including cancer.

Chromatin-Mediated Regulation of Genome Plasticity in Human Fungal Pathogens.

Human fungal pathogens, such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, are a public health problem, causing millions of infections and killing almost half a million people annually. The ability of these pathogens to colonise almost every organ in the human body and cause life-threating infections relies on their capacity to adapt and thrive in diverse hostile host-niche environments. Stress-induced genome instability is a key adaptive strategy used by human fungal pathogens as it increases genetic diversity, thereby allowing selection of genotype(s) better adapted to a new environment. Heterochromatin represses gene expression and deleterious recombination and could play a key role in modulating genome stability in response to environmental changes. However, very little is known about heterochromatin structure and function in human fungal pathogens. In this review, I use our knowledge of heterochromatin structure and function in fungal model systems as a road map to review the role of heterochromatin in regulating genome plasticity in the most common human fungal pathogens: Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans.

Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

The mitochondrion-associated protein LRPPRC (leucine-rich pentatricopeptide repeat-containing) interacts with one of the microtubule-associated protein family members MAP1S (microtubule-associated protein 1 small form), originally named C19ORF5 (chromosome 19 open reading frame 5), to form a complex. MAP1S interacts with LC3 (light chain 3), the mammalian homologue of yeast autophagy marker ATG8 and one of the most important autophagy markers in mammalian cells, and helps the attachment of autophagosomes with microtubules for trafficking and recruitment of substrate mitochondria into autophagosomes for degradation. MAP1S activates autophagosomal biogenesis and degradation to remove misfolded/aggregated proteins and dysfunctional organelles such as mitochondria and suppress oxidative stress-induced genomic instability and tumorigenesis. Previously, various studies have attributed LRPPRC nucleic acid-associated functions. Instead, in the present study, we show that LRPPRC associates with mitochondria, interacts with Beclin 1 and Bcl-2 and forms a ternary complex to maintain the stability of Bcl-2. Suppression of LRPPRC leads to reduction in mitochondrial potential and reduction in Bcl-2. Lower levels of Bcl-2 lead to release of more Beclin 1 to form the Beclin 1–PI3KCIII (class III phosphoinositide 3-kinase) complex to activate autophagy and accelerate the turnover of dysfunctional mitochondria through the PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) pathway. The activation of autophagy induced by LRPPRC suppression occurs upstream of the ATG5–ATG12 conjugate-mediated conversion of LC3-I into LC3-II and has been confirmed in multiple mammalian cell lines with multiple autophagy markers including the size of GFP–LC3 punctate foci, the intensity of LC3-II and p62 protein and the size of the vacuolar structure. The activated autophagy enhances the removal of mitochondria through lysosomes. LRPPRC therefore acts to suppress the initiation of basal levels of autophagy to clean up dysfunctional mitochondria and other cellular debris during the normal cell cycle.

Investigation of the phototoxicity and cytotoxicity of naproxen, a non-steroidal anti-inflammatory drug, in human fibroblasts

Non-steroidal anti-inflammatory drugs (NSAID) are widely used in the treatment of pain and inflammation associated with several diseases. Naproxen, 2-(6-methoxy-2-naphthyl) propionic acid (NAP), belongs to this pharmacological class and appears to be associated with a high incidence of both photoallergic and phototoxic reactions. In this study, using human fibroblasts, we examined the biological effects of NAP photosensitization induced by UVA, the predominant UV component of sunlight reaching the Earth's surface. We showed that NAP or UVA alone have no cytotoxic effects at the concentrations and doses used in this study. The same result was observed when cells were pre-incubated with NAP but irradiated without NAP. In marked contrast, exposure of cells in the presence of NAP led to a drastic reduction of cell viability. These results suggest that the phototoxicity is mainly due to irradiation of extracellular NAP that damages cell membranes. Moreover, we showed that NAP itself led to a low but reproducible production of reactive oxygen species (ROS), to protein modifications by lipid peroxidation-derived aldehydes, to p38 phosphorylation and to the slowing-down of DNA replication, while UVA treatment alone showed no effects. NAP photosensitization with UVA led to protein S-glutathionylation, oxidation of the proliferating cell nuclear antigen (PCNA), oxidation of cellular tryptophan, phosphorylation of Chk1 and inhibition of DNA replication. However, using small interfering RNA to down regulate Chk1 expression in cells, we showed that Chk1 is not required to slow the S-phase down. Nevertheless, inhibition of Chk1, but not of p38, sensitized the cells to the phototoxic effects of NAP. Collectively, our data suggest that the interaction of NAP with the cells triggers oxidative damage and a replication stress, which are exacerbated by UVA radiation. As oxidative and replication stress-induced genome instability are important factors in aging and tumor predisposition, it is of interest to evaluate the consequence of a non-steroidal anti-inflammatory drug, like naproxen, on genomic instability.

SIRT4 Has Tumor-Suppressive Activity and Regulates the Cellular Metabolic Response to DNA Damage by Inhibiting Mitochondrial Glutamine Metabolism

DNA damage elicits a cellular signaling response that initiates cell cycle arrest and DNA repair. Here, we find that DNA damage triggers a critical block in glutamine metabolism, which is required for proper DNA damage responses. This block requires the mitochondrial SIRT4, which is induced by numerous genotoxic agents and represses the metabolism of glutamine into tricarboxylic acid cycle. SIRT4 loss leads to both increased glutamine-dependent proliferation and stress-induced genomic instability, resulting in tumorigenic phenotypes. Moreover, SIRT4 knockout mice spontaneously develop lung tumors. Our data uncover SIRT4 as an important component of the DNA damage response pathway that orchestrates a metabolic block in glutamine metabolism, cell cycle arrest, and tumor suppression.

Genome constraint through sexual reproduction: application of 4D-Genomics in reproductive biology

Assisted reproductive technologies have been used to achieve pregnancies since the first successful test tube baby was born in 1978. Infertile couples are at an increased risk for multiple miscarriages and the application of current protocols are associated with high first-trimester miscarriage rates. Among the contributing factors of these higher rates is a high incidence of fetal aneuploidy. Numerous studies support that protocols including ovulation-induction, sperm cryostorage, density-gradient centrifugation, and embryo culture can induce genome instability, but the general mechanism is less clear. Application of the genome theory and 4D-Genomics recently led to the establishment of a new paradigm for sexual reproduction; sex primarily constrains genome integrity that defines the biological system rather than just providing genetic diversity at the gene level. We therefore propose that application of assisted reproductive technologies can bypass this sexual reproduction filter as well as potentially induce additional system instability. We have previously demonstrated that a single-cell resolution genomic approach, such as spectral karyotyping to trace stochastic genome level alterations, is effective for pre- and post-natal analysis. We propose that monitoring overall genome alteration at the karyotype level alongside the application of assisted reproductive technologies will improve the efficacy of the techniques while limiting stress-induced genome instability. The development of more single-cell based cytogenomic technologies are needed in order to better understand the system dynamics associated with infertility and the potential impact that assisted reproductive technologies have on genome instability. Importantly, this approach will be useful in studying the potential for diseases to arise as a result of bypassing the filter of sexual reproduction.

SIRT3 Is a Mitochondria-Localized Tumor Suppressor Required for Maintenance of Mitochondrial Integrity and Metabolism during Stress

The sirtuin gene family (SIRT) is hypothesized to regulate the aging process and play a role in cellular repair. This work demonstrates that SIRT3(-/-) mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3(-/-) MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3(-/-) MEFs and reverses the tumor-permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3(-/-) mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondria-localized tumor suppressor.

DNA replication stress, genome instability and aging

Genome instability is a fundamentally important component of aging in all eukaryotes. How age-related genome instability occurs remains unclear. The free radical theory of aging posits oxidative damage to DNA and other cellular constituents as a primary determinant of aging. More recent versions of this theory predict that mitochondria are a major source of reactive oxygen species (ROS) that cause oxidative damage. Although substantial support for the free radical theory exists, the results of some tests of this theory have been contradictory or inconclusive. Enhanced growth signaling also has been implicated in aging. Many efforts to understand the effects of growth signaling on aging have focused on inhibition of oxidative stress responses that impact oxidative damage. However, recent experiments in the model organism Saccharomyces cerevisiae (budding yeast) and in higher eukaryotes suggest that growth signaling also impacts aging and/or age-related diseases—including cancer and neurodegeneration—by inducing DNA replication stress, which causes DNA damage. Replication stress, which has not been broadly considered as a factor in aging, may be enhanced by ROS that signal growth. In this article, we review evidence that points to DNA replication stress and replication stress-induced genome instability as important factors in aging.

Molecular architecture of a eukaryotic DNA transposase

Mobile elements and their inactive remnants account for large proportions of most eukaryotic genomes, where they have had central roles in genome evolution. Over 50 years ago, McClintock reported a form of stress-induced genome instability in maize in which discrete DNA segments move between chromosomal locations. Our current mechanistic understanding of enzymes catalyzing transposition is largely limited to prokaryotic transposases. The Hermes transposon from the housefly is part of the eukaryotic hAT superfamily that includes hobo from Drosophila, McClintock's maize Activator and Tam3 from snapdragon. We report here the three-dimensional structure of a functionally active form of the transposase from Hermes at 2.1-A resolution. The Hermes protein has some structural features of prokaryotic transposases, including a domain with a retroviral integrase fold. However, this domain is disrupted by the insertion of an additional domain. Finally, transposition is observed only when Hermes assembles into a hexamer.