Genomic instability in the offspring of irradiated parents: Facts and interpretations

This review is devoted to genomic instability in the offspring of parents that were irradiated or treated with chemical mutagens. The evidence is presented, showing high frequency of cancer diseases and instability of the genome of somatic and germline cells in the offspring of radiation-exposed animals. Possible epigenetic mechanisms of these effects are considered, as well as their significance as components of genetic factors of radiation risk for humans.     

Genomic instability in the offspring of irradiated parents: facts and interpretations

This review is devoted to genomic instability in the offspring of parents that were irradiated or treated with chemical mutagens. The evidence is presented, showing high frequency of cancer diseases and instability of the genome of somatic and germline cells in the offspring of radiation-exposed animals. Possible epigenetic mechanisms of these effects are considered, as well as their significance as components of genetic factors of radiation risk for humans.     

Do the different parental 'heteromes' cause genomic shock in newly formed allopolyploids?

Allopolyploidy, the joining of two parental genomes in a polyploid organism with diploid meiosis, is an important mechanism of reticulate evolution. While many successful long-established allopolyploids are known, those formed recently undergo an instability phase whose basis is now being characterized. We describe observations made with the Arabidopsis system that include phenotypic instability, gene silencing and activation, and methylation changes. We present a model based on the epigenetic destabilization of genomic repeats, which in the parents are heterochromatinized and suppressed. We hypothesize that loss of epigenetic suppression of these sequences, here defined as the heterome, results in genomic instability including silencing of single-copy genes.     

DNMTs are required for delayed genome instability caused by radiation

The ability of ionizing radiation to initiate genomic instability has been harnessed in the clinic where the localized delivery of controlled doses of radiation is used to induce cell death in tumor cells. Though very effective as a therapy, tumor relapse can occur in vivo and its appearance has been attributed to the radio-resistance of cells with stem cell-like features. The molecular mechanisms underlying these phenomena are unclear but there is evidence suggesting an inverse correlation between radiation-induced genomic instability and global hypomethylation. To further investigate the relationship between DNA hypomethylation, radiosensitivity and genomic stability in stem-like cells we have studied mouse embryonic stem cells containing differing levels of DNA methylation due to the presence or absence of DNA methyltransferases. Unexpectedly, we found that global levels of methylation do not determine radiosensitivity. In particular, radiation-induced delayed genomic instability was observed at the Hprt gene locus only in wild-type cells. Furthermore, absence of Dnmt1 resulted in a 10-fold increase in de novo Hprt mutation rate, which was unaltered by radiation. Our data indicate that functional DNMTs are required for radiation-induced genomic instability, and that individual DNMTs play distinct roles in genome stability. We propose that DNMTS may contribute to the acquirement of radio-resistance in stem-like cells.     

Accumulation of DNA damage in complex normal tissues after protracted low-dose radiation

The biological consequences of low levels of radiation exposure and their effects on human health are unclear. Ionizing radiation induces a variety of lesions of which DNA double-strand breaks (DSBs) are the most biologically significant, because unrepaired or misrepaired DSBs can lead to genomic instability and cell death. Using repair-proficient mice as an in vivo system we monitored the accumulation of DNA damage in normal tissues exposed to daily low-dose radiation of 100 mGy or 10 mGy. Radiation-induced foci in differentiated and tissue-specific stem cells were quantified by immunofluorescence microscopy after 2, 4, 6, 8, and 10 weeks of daily low-dose radiation and DNA lesions were characterized using transmission electron microscopy (TEM) combined with immunogold-labeling. In brain, long-living cortical neurons had a significant accumulation of foci with increasing cumulative doses. In intestine and skin, characterized by constant cell renewal of their epithelial lining, differentiated enterocytes and keratinocytes had either unchanged or only slightly increased foci levels during protracted low-dose radiation. Significantly, analysis of epidermal stem cells in skin revealed a constant increase of 53BP1 foci during the first weeks of low-dose radiation even with 10 mGy, suggesting substantial accumulations of DSBs. However, TEM analysis suggests that these remaining 53BP1 foci, which are predominantly located in compact heterochromatin, do not co-localize with phosphorylated Ku70 or DNA-PKcs, core components of non-homologous end-joining. The biological relevance of these persistent 53BP1 foci, particularly their contribution to genomic instability by genetic and epigenetic alterations, has to be defined in future studies.     

Genetic and Epigenetic Changes in Chromosomally Stable and Unstable Progeny of Irradiated Cells

Radiation induced genomic instability is a well-studied phenomenon, the underlying mechanisms of which are poorly understood. Persistent oxidative stress, mitochondrial dysfunction, elevated cytokine levels and epigenetic changes are among the mechanisms invoked in the perpetuation of the phenotype. To determine whether epigenetic aberrations affect genomic instability we measured DNA methylation, mRNA and microRNA (miR) levels in well characterized chromosomally stable and unstable clonally expanded single cell survivors of irradiation. While no changes in DNA methylation were observed for the gene promoters evaluated, increased LINE-1 methylation was observed for two unstable clones (LS12 and CS9) and decreased Alu element methylation was observed for the other two unstable clones (115 and Fe5.0–8). These relationships also manifested for mRNA and miR expression. mRNA identified for the LS12 and CS9 clones were most similar to each other (261 mRNA), while the 115 and Fe5.0–8 clones were more similar to each other, and surprisingly also similar to the two stable clones, 114 and 118 (286 mRNA among these four clones). Pathway analysis showed enrichment for pathways involved in mitochondrial function and cellular redox, themes routinely invoked in genomic instability. The commonalities between the two subgroups of clones were also observed for miR. The number of miR for which anti-correlated mRNA were identified suggests that these miR exert functional effects in each clone. The results demonstrate significant genetic and epigenetic changes in unstable cells, but similar changes are almost as equally common in chromosomally stable cells. Possible conclusions might be that the chromosomally stable clones have some other form of instability, or that some of the observed changes represent a sort of radiation signature and that other changes are related to genomic instability. Irrespective, these findings again suggest that a spectrum of changes both drive genomic instability and permit unstable cells to persist and proliferate.     

Delayed activation of DNA damage checkpoint and radiation-induced genomic instability

Ionizing radiation induces genomic instability, transmitted over many generations through the progeny of surviving cells. It is manifested as the expression of delayed effects such as delayed cell death, delayed chromosomal instability and delayed mutagenesis. Induced genomic instability exerts its delayed effects for prolonged periods of time, suggesting the presence of a mechanism by which the initial DNA damage in the surviving cells is memorized. Our recent studies have shown that transmitted memory causes delayed DNA breakage, which in turn activates DNA damage checkpoint, and is involved in delayed manifestation of genomic instability. Although the mechanism(s) involved in DNA damage memory remain to be determined, we suggest that ionizing radiation-induced mega-base deletion destabilizes chromatin structure, which can be transmitted many generations through the progeny, and is involved in initiation and perpetuation of genomic instability. The possible involvement of delayed activation of a DNA damage checkpoint in the delayed induction of genomic instability in bystander cells is also discussed.     

Epigenetic modifications in radiation-induced non-targeted effects and their clinical significance

BackgroundIonizing radiation (IR) plays an important role in the diagnosis and treatment of cancer. Besides the targeted effects, the non-targeted effects, which cause damage to non-irradiated cells and genomic instability in normal tissues, also play a role in the side effects of radiotherapy and have been shown to involve both alterations in DNA sequence and regulation of epigenetic modifications.Scope of reviewWe summarize the recent findings regarding epigenetic modifications that are involved in radiation-induced non-targeted effects as well as their clinical significance in radiotherapy and radioprotection.Major conclusionsEpigenetic modifications play an important role in both the realization and modulation of radiobiological effects. However, the molecular mechanisms underlying non-targeted effects still need to be clarified.General significanceA better understanding of the epigenetic mechanisms related to radiation-induced non-targeted effects will guide both individualized clinical radiotherapy and individualized precise radioprotection.     

Opposite Roles for p38MAPK-Driven Responses and Reactive Oxygen Species in the Persistence and Resolution of Radiation-Induced Genomic Instability

We report the functional and temporal relationship between cellular phenotypes such as oxidative stress, p38MAPK-dependent responses and genomic instability persisting in the progeny of cells exposed to sparsely ionizing low-Linear Energy Transfer (LET) radiation such as X-rays or high-charge and high-energy (HZE) particle high-LET radiation such as ⁵⁶Fe ions. We found that exposure to low and high-LET radiation increased reactive oxygen species (ROS) levels as a threshold-like response induced independently of radiation quality and dose. This response was sustained for two weeks, which is the period of time when genomic instability is evidenced by increased micronucleus formation frequency and DNA damage associated foci. Indicators for another persisting response sharing phenotypes with stress-induced senescence, including beta galactosidase induction, increased nuclear size, p38MAPK activation and IL-8 production, were induced in the absence of cell proliferation arrest during the first, but not the second week following exposure to high-LET radiation. This response was driven by a p38MAPK-dependent mechanism and was affected by radiation quality and dose. This stress response and elevation of ROS affected genomic instability by distinct pathways. Through interference with p38MAPK activity, we show that radiation-induced stress phenotypes promote genomic instability. In contrast, exposure to physiologically relevant doses of hydrogen peroxide or increasing endogenous ROS levels with a catalase inhibitor reduced the level of genomic instability. Our results implicate persistently elevated ROS following exposure to radiation as a factor contributing to genome stabilization.     

A loss of function screen identifies nine new radiation susceptibility genes

Genomic instability is considered a hallmark of carcinogenesis, and dysfunction of DNA repair and cell cycle regulation in response to DNA damage caused by ionizing radiation are thought to be important factors in the early stages of genomic instability. We performed cell-based functional screening using an RNA interference library targeting 200 genes in human cells. We identified three known and nine new radiation susceptibility genes, eight of which are linked directly or potentially with cell cycle progression. Cell cycle analysis on four of the genes not previously linked to cell cycle progression demonstrated that one, ZDHHC8, was associated with the G₂/M checkpoint in response to DNA damage. Further study of the 12 radiation susceptibility genes identified in this screen may help to elucidate the molecular mechanisms of cell cycle progression, DNA repair, cell death, cell growth and genomic instability, and to develop new radiation sensitizing agents for radiotherapy.     

Development of cancer-initiating cells and immortalized cells with genomic instability

Cancers that develop after middle age usually exhibit genomic instability and multiple mutations. This is in direct contrast to pediatric tumors that usually develop as a result of specific chromosomal translocations andepigenetic aberrations. The development of genomic instability is associated with mutations that contribute to cellular immortalization and transformation. Cancer occurs when cancer-initiating cells(CICs), also called cancer stem cells, develop as a result of these mutations. In this paper, we explore how CICs develop as a result of genomic instability, including looking at which cancer suppression mechanisms are abrogated. A recent in vitro study revealed the existence of a CIC induction pathway in differentiating stem cells. Under aberrant differentiation conditions, cells become senescent and develop genomic instabilities that lead to the development of CICs. The resulting CICs contain a mutation in the alternative reading frame of CDKN2A(ARF)/p53 module, i.e., in either ARF or p53. We summarize recently established knowledge of CIC development and cellular immortality, explore the role of the ARF/p53 module in protecting cells from transformation, and describe a risk factor for genomic destabilization that increases during the process of normal cell growth and differentiation and is associated with the downregulation of histone H2 AX to levels representative of growth arrest in normal cells.     

Genome-wide CpG island methylation and intergenic demethylation propensities vary among different tumor sites

The epigenetic landscape of cancer includes both focal hypermethylation and broader hypomethylation in a genome-wide manner. By means of a comprehensive genomic analysis on 6637 tissues of 21 tumor types, we here show that the degrees of overall methylation in CpG island (CGI) and demethylation in intergenic regions, defined as ‘backbone’, largely vary among different tumors. Depending on tumor type, both CGI methylation and backbone demethylation are often associated with clinical, epidemiological and biological features such as age, sex, smoking history, anatomic location, histological type and grade, stage, molecular subtype and biological pathways. We found connections between CGI methylation and hypermutability, microsatellite instability, IDH1 mutation, 19p gain and polycomb features, and backbone demethylation with chromosomal instability, NSD1 and TP53 mutations, 5q and 19p loss and long repressive domains. These broad epigenetic patterns add a new dimension to our understanding of tumor biology and its clinical implications.     

Sonic Hedgehog signaling impairs ionizing radiation-induced checkpoint activation and induces genomic instability

The Sonic Hedgehog (Shh) pathway plays important roles in embryogenesis, stem cell maintenance, tissue repair, and tumorigenesis. Haploinsufficiency of Patched-1, a gene that encodes a repressor of the Shh pathway, dysregulates the Shh pathway and increases genomic instability and the development of spontaneous and ionizing radiation (IR)–induced tumors by an unknown mechanism. Here we show that Ptc1^+/− mice have a defect in the IR-induced activation of the ATR–Chk1 checkpoint signaling pathway. Likewise, transient expression of Gli1, a downstream target of Shh signaling, disrupts Chk1 activation in human cells by preventing the interaction of Chk1 with Claspin, a Chk1 adaptor protein that is required for Chk1 activation. These results suggest that inappropriate Shh pathway activation promotes tumorigenesis by disabling a key signaling pathway that helps maintain genomic stability and inhibits tumorigenesis.     

早老素导致基因组不稳定的机制

基因组不稳定（genomic instability）是机体衰老的标志之一,也是儿童早老症（Hutchinson Gilford progeria syndrome, HGPS)患者细胞的典型特征。HGPS的发生与早老素（progerin)堆积密切相关,但早老素如何引起基因组不稳定尚缺乏系统性的阐述。基因组的结构稳定与DNA的正确复制、DNA损伤修复、端粒的维持和稳定以及表观遗传学修饰密切相关。本文主要讨论早老素在改变正常核纤层结构的基础上,通过影响相关通路关键蛋白质的水平或者定位,引起细胞内氧化应激增强、DNA复制应激和DNA损伤修复障碍,细胞DNA损伤增多和端粒的加速缩短,并在改变组蛋白甲基化和乙酰化方面导致基因组不稳定的机制。     

Genome hypermethylation in Pinus silvestris of Chernobyl--a mechanism for radiation adaptation?

Adaptation is a complex process by which populations of organisms respond to long-term environmental stresses by permanent genetic change. Here we present data from the natural "open-field" radiation adaptation experiment after the Chernobyl accident and provide the first evidence of the involvement of epigenetic changes in adaptation of a eukaryote-Scots pine (Pinus silvestris), to chronic radiation exposure. We have evaluated global genome methylation of control and radiation-exposed pine trees using a method based on cleavage by a methylation-sensitive HpaII restriction endonuclease that leaves a 5' guanine overhang and subsequent single nucleotide extension with labeled [3H] dCTP. We have found that genomic DNA of exposed pine trees was considerably hypermethylated. Moreover, hypermethylation appeared to be dependent upon the radiation dose absorbed by the trees. Such hypermethylation may be viewed as a defense strategy of plants that prevents genome instability and reshuffling of the hereditary material, allowing survival in an extreme environment. Further studies are clearly needed to analyze in detail the involvement of DNA methylation and other epigenetic mechanisms in the complex process of radiation stress and adaptive response.     

chroGPS,a global chromatin positioning system for the functional analysis and visualization of the epigenome

Development of tools to jointly visualize the genome and the epigenome remains a challenge. chroGPS is a computational approach that addresses this question. chroGPS uses multidimensional scaling techniques to represent similarity between epigenetic factors, or between genetic elements on the basis of their epigenetic state, in 2D/3D reference maps. We emphasize biological interpretability, statistical robustness, integration of genetic and epigenetic data from heterogeneous sources, and computational feasibility. Although chroGPS is a general methodology to create reference maps and study the epigenetic state of any class of genetic element or genomic region, we focus on two specific kinds of maps: chroGPS^factors, which visualizes functional similarities between epigenetic factors, and chroGPS^genes, which describes the epigenetic state of genes and integrates gene expression and other functional data. We use data from the modENCODE project on the genomic distribution of a large collection of epigenetic factors in Drosophila, a model system extensively used to study genome organization and function. Our results show that the maps allow straightforward visualization of relationships between factors and elements, capturing relevant information about their functional properties that helps to interpret epigenetic information in a functional context and derive testable hypotheses.