Low dose ionizing radiation strongly stimulates insertional mutagenesis in a γH2AX dependent manner

Zelensky, Alex N.; Schoonakker, Mascha; Brandsma, Inger; Tijsterman, Marcel; Gent, Dik C. van; Essers, Jeroen; Kanaar, Roland

doi:10.1371/journal.pgen.1008550

Cited by 9 publications

(5 citation statements)

References 96 publications

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“…Stem and early progenitor cells are considered cells of origin for radiation-induced carcinogenesis, owing to their replicative potential and long lifespan in the organism [ 11 ]. Increasing evidence suggests that stem cells display distinct responses to low versus high-dose IR, including activation thresholds for proliferation and differentiation [ 12 ], the dose-dependent integration of extrachromosomal DNA after LD [ 13 ], low-dose-specific hyper-radiosensitivity [ 14 ], persistent oxidative stress and decreased self-renewal [ 15 ] and the selective stimulation of proliferation based on their p53 mutation status [ 16 ]. The survival/persistence of normal tissue stem cells with residual DNA damage may increase the risk of second primary tumors.…”

Section: Introductionmentioning

confidence: 99%

Divergent Molecular and Cellular Responses to Low and High-Dose Ionizing Radiation

Sampadi

Vermeulen

Mišovic

et al. 2022

Cells

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Cancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low (LD; 0.1 Gy) or a high (HD; 1 Gy) dose of X-rays, we show that, although the DNA damage response (DDR) displayed dose proportionality, many other molecular and cellular responses did not. Phosphoproteomics uncovered a novel mode of phospho-signaling via S12-PPP1R7, and large-scale dephosphorylation events that regulate mitotic exit control in undamaged cells and the G2/M checkpoint upon IR in a dose-dependent manner. The phosphoproteomics of irradiated DNA double-strand breaks (DSBs) repair-deficient cells unveiled extended phospho-signaling duration in either a dose-dependent (DDR signaling) or independent (mTOR-ERK-MAPK signaling) manner without affecting signal magnitude. Nascent transcriptomics revealed the transcriptional activation of genes involved in NRF2-regulated antioxidant defense, redox-sensitive ERK-MAPK signaling, glycolysis and mitochondrial function after LD, suggesting a prominent role for reactive oxygen species (ROS) in molecular and cellular responses to LD exposure, whereas DDR genes were prominently activated after HD. However, how and to what extent the observed dose-dependent differences in molecular and cellular responses may impact cancer development remain unclear, as the induction of chromosomal damage was found to be dose-proportional (10–200 mGy).

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Section: Introductionmentioning

confidence: 99%

Divergent Molecular and Cellular Responses to Low and High-Dose Ionizing Radiation

Sampadi

Vermeulen

Mišovic

et al. 2022

Cells

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“…IDLVs have previously been reported to have the ability to label DSBs induced by artificial endonucleases [ 23 , 42 , 43 ], ionizing radiation [ 44 ], or chemical agents [ 45 ]. IDLV-DSB labelling facilitates unbiased identification of potential off-targets attributed to artificial endonucleases, especially in those cells which are difficult to transfect.…”

Section: Resultsmentioning

confidence: 99%

Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks

et al. 2021

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Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off-target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 3′LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.

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“…An alternatively mechanistic model of (stw)intron propagation in Hypoxylon would involve an RNA-mediated version of random integration [ 68 , 69 ] during the repair of blunt-ended DSBs ( Figure 6 b, to the right of the figure). DSBs increase the frequency of random DNA integration (e.g., [ 70 ]). We propose that double-stranded (ds) RNA molecules could be formed by annealing two single-stranded molecules upon debranching of the lariats of the excised, highly symmetrical sister intron RNA.…”

Section: Discussionmentioning

confidence: 99%

Internally Symmetrical Stwintrons and Related Canonical Introns in Hypoxylaceae Species

Fekete

Pénzes

Scazzocchio

et al. 2021

JoF

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Spliceosomal introns are pervasive in eukaryotes. Intron gains and losses have occurred throughout evolution, but the origin of new introns is unclear. Stwintrons are complex intervening sequences where one of the sequence elements (5′-donor, lariat branch point element or 3′-acceptor) necessary for excision of a U2 intron (external intron) is itself interrupted by a second (internal) U2 intron. In Hypoxylaceae, a family of endophytic fungi, we uncovered scores of donor-disrupted stwintrons with striking sequence similarity among themselves and also with canonical introns. Intron–exon structure comparisons suggest that these stwintrons have proliferated within diverging taxa but also give rise to proliferating canonical introns in some genomes. The proliferated (stw)introns have integrated seamlessly at novel gene positions. The recently proliferated (stw)introns appear to originate from a conserved ancestral stwintron characterised by terminal inverted repeats (45–55 nucleotides), a highly symmetrical structure that may allow the formation of a double-stranded intron RNA molecule. No short tandem duplications flank the putatively inserted intervening sequences, which excludes a DNA transposition-based mechanism of proliferation. It is tempting to suggest that this highly symmetrical structure may have a role in intron proliferation by (an)other mechanism(s).

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Low dose ionizing radiation strongly stimulates insertional mutagenesis in a γH2AX dependent manner

Cited by 9 publications

References 96 publications

Divergent Molecular and Cellular Responses to Low and High-Dose Ionizing Radiation

Divergent Molecular and Cellular Responses to Low and High-Dose Ionizing Radiation

Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks

Internally Symmetrical Stwintrons and Related Canonical Introns in Hypoxylaceae Species

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