Zhentian Li

Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog

NONO, SFPQ and PSPC1 make up a family of proteins with diverse roles in transcription, RNA processing and DNA double-strand break (DSB) repair. To understand long-term effects of loss of NONO, we characterized murine embryonic fibroblasts (MEFs) from knockout mice. In the absence of genotoxic stress, wild-type and mutant MEFs showed similar growth rates and cell cycle distributions, and the mutants were only mildly radiosensitive. Further investigation showed that NONO deficiency led to upregulation of PSPC1, which replaced NONO in a stable complex with SFPQ. Knockdown of PSPC1 in a NONO-deficient background led to severe radiosensitivity and delayed resolution of DSB repair foci. The DNA-dependent protein kinase (DNA-PK) inhibitor, NU7741, sensitized wild-type and singly deficient MEFs, but had no additional effect on doubly deficient cells, suggesting that NONO/PSPC1 and DNA-PK function in the same pathway. We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes. Of 12 genes tested, none were downregulated, and several were upregulated. Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

Co-culturing with High-Charge and Energy Particle Irradiated Cells Increases Mutagenic Joining of Enzymatically Induced DNA Double-Strand Breaks in Nonirradiated Cells.

Cell populations that have been exposed to high-charge and energy (HZE) particle radiation, and then challenged by expression of a rare-cutting nuclease, show an increased frequency of deletions and translocations originating at the enzyme cut sites. Here, we examine whether this effect also occurs in nonirradiated cells that have been co-cultured with irradiated cells. Human cells were irradiated with 0.3–1.0 Gy of either 600 MeV/u 56Fe or 1,000 MeV/u 48Ti ions or with 0.3–3.0 Gy of 320 kV X rays. These were co-cultured with I-SceI-expressing reporter cells at intervals up to 21 days postirradiation. Co-culture with HZE-irradiated cells led to an increase in the frequency of I-SceI-stimulated translocations and deletions in the nonirradiated cells. The effect size was similar to that seen previously in directly irradiated populations (maximum effect in bystander cells of 1.7- to 4-fold depending on ion and end point). The effect was not observed when X-ray-irradiated cells were co-cultured with nonirradiated cells, but was correlated with an increase in γ-H2AX foci-positive cells in the nonirradiated population, suggesting the presence of genomic stress. Transcriptional profiling of a directly irradiated cell population showed that many genes for cytokines and other secretory proteins were persistently upregulated, but their induction was not well correlated with functional effects on repair in co-cultured cells, suggesting that this transcriptional response alone is not sufficient to evoke the effect. The finding that HZE-irradiated cells influence the DNA double-strand break repair fidelity in their nonirradiated neighbors has implications for risk in the space radiation environment.

Increased Mutagenic Joining of Enzymatically-Induced DNA Double-Strand Breaks in High-Charge and Energy Particle Irradiated Human Cells

The carcinogenic risk of high-charge and energy (HZE) particle exposure arises from its ability to both induce complex DNA damage and from its ability to evoke deleterious, non-DNA targeted effects. We investigate here whether these nontargeted effects involve dysregulation of double-strand break repair, such that a history of HZE exposure heightens the risks from future injury. We used a new human cell reporter line, in which expression of the I-SceI meganuclease stimulates both translocations on different chromosomes, and deletions on the same chromosome. Exposure to 1.0 Gy of 600 MeV/u 56Fe ions led to a doubling in the frequency of I-SceI-mediated translocations and a smaller, but nevertheless significant, increase in the frequency of I-SceI-mediated deletions. This mutagenic repair phenotype persisted for up to two weeks and eight population doublings. The phenotype was not induced by low-linear energy transfer radiation or by a lower dose of HZE-particle radiation (0.3 Gy) indicating that the effect is radiation quality and dose dependent. The mutagenic repair phenotype was associated with the presence of micronuclei and persistent DSB repair foci, consistent with a hypothesis that genomic stress is a causative factor.

SFPQ•NONO and XLF function separately and together to promote DNA double-strand break repair via canonical nonhomologous end joining.

Abstract A complex of two related mammalian proteins, SFPQ and NONO, promotes DNA double-strand break repair via the canonical nonhomologous end joining (c-NHEJ) pathway. However, its mechanism of action is not fully understood. Here we describe an improved SFPQ•NONO-dependent in vitro end joining assay. We use this system to demonstrate that the SFPQ•NONO complex substitutes in vitro for the core c-NHEJ factor, XLF. Results are consistent with a model where SFPQ•NONO promotes sequence-independent pairing of DNA substrates, albeit in a way that differs in detail from XLF. Although SFPQ•NONO and XLF function redundantly in vitro, shRNA-mediated knockdown experiments indicate that NONO and XLF are both required for efficient end joining and radioresistance in cell-based assays. In addition, knockdown of NONO sensitizes cells to the interstrand crosslinking agent, cisplatin, whereas knockdown of XLF does not, and indeed suppresses the effect of NONO deficiency. These findings suggest that each protein has one or more unique activities, in addition to the DNA pairing revealed in vitro, that contribute to DNA repair in the more complex cellular milieu. The SFPQ•NONO complex contains an RNA binding domain, and prior work has demonstrated diverse roles in RNA metabolism. It is thus plausible that the additional repair function of NONO, revealed in cell-based assays, could involve RNA interaction.

Effect of radiation quality on mutagenic joining of enzymatically-induced DNA double-strand breaks in previously irradiated human cells.

Previous work has shown that high charge and energy particle irradiation of human cells evokes a mutagenic repair phenotype, defined by increased mutagenic repair of new double-strand breaks that are introduced enzymatically, days or weeks after the initial irradiation. The effect was seen originally with 600 MeV/u 56Fe particles, which have a linear energy transfer (LET) value of 174 keV/μm, but not with X rays or γ rays (LET ≤ 2 keV/μm). To better define the radiation quality dependence of the phenomenon, we tested two ions with intermediate LET values, 1,000 MeV/u 48Ti (LET = 108 keV/μm) and 300 MeV/u 28Si (LET = 69 keV/μm). These experiments used a previously validated assay, where a rare-cutting nuclease introduces double-strand breaks in two reporter transgene cassettes, which are located on different chromosomes. Deletions of a block of sequence in one of the cassettes, or translocations between cassettes, are measured independently using a multicolor fluorescence assay. The results showed that 48Ti was a potent, but transient, inducer of mutagenic repair, based on increased frequency of nuclease-induced translocations. The 48Ti ions did not affect the frequency of nuclease-induced deletions. The 28Si ions had no measurable effect on either endpoint. There was a close correlation between the induction of the mutagenic repair phenomenon and the frequency of micronuclei in the targeted population (R2 = 0.74), whereas there was no apparent correlation with radiation-induced cell inactivation. Together, these results better define the radiation quality dependence of the mutagenic repair phenomenon and establish its correlation, or lack of correlation, with other endpoints.

Galactic Cosmic Radiation Induces Persistent Epigenome Alterations Relevant to Human Lung Cancer

Human deep space and planetary travel is limited by uncertainties regarding the health risks associated with exposure to galactic cosmic radiation (GCR), and in particular the high linear energy transfer (LET), heavy ion component. Here we assessed the impact of two high-LET ions 56Fe and 28Si, and low-LET X rays on genome-wide methylation patterns in human bronchial epithelial cells. We found that all three radiation types induced rapid and stable changes in DNA methylation but at distinct subsets of CpG sites affecting different chromatin compartments. The 56Fe ions induced mostly hypermethylation, and primarily affected sites in open chromatin regions including enhancers, promoters and the edges (“shores”) of CpG islands. The 28Si ion-exposure had mixed effects, inducing both hyper and hypomethylation and affecting sites in more repressed heterochromatic environments, whereas X rays induced mostly hypomethylation, primarily at sites in gene bodies and intergenic regions. Significantly, the methylation status of 56Fe ion sensitive sites, but not those affected by X ray or 28Si ions, discriminated tumor from normal tissue for human lung adenocarcinomas and squamous cell carcinomas. Thus, high-LET radiation exposure leaves a lasting imprint on the epigenome, and affects sites relevant to human lung cancer. These methylation signatures may prove useful in monitoring the cumulative biological impact and associated cancer risks encountered by astronauts in deep space.

Overexpression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer.

Abstract Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.

Exosomes, Their Biogenesis and Role in Inter-Cellular Communication, Tumor Microenvironment and Cancer Immunotherapy

Exosomes are extracellular vesicles ranging from 30 to 150 nm in diameter that contain molecular constituents of their host cells. They are released from different types of cells ranging from immune to tumor cells and play an important role in intercellular communication. Exosomes can be manipulated by altering their host cells and can be loaded with products of interest such as specific drugs, proteins, DNA and RNA species. Due to their small size and the unique composition of their lipid bilayer, exosomes are capable of reaching different cell types where they alter the pathophysiological conditions of the recipient cells. There is growing evidence that exosomes are used as vehicles that can modulate the immune system and play an important role in cancer progression. The cross communication between the tumors and the cells of the immune system has gained attention in various immunotherapeutic approaches for several cancer types. In this review, we discuss the exosome biogenesis, their role in inter-cellular communication, and their capacity to modulate the immune system as a part of future cancer immunotherapeutic approaches and their potential to serve as biomarkers of therapy response.

Exposure to galactic cosmic radiation compromises DNA repair and increases the potential for oncogenic chromosomal rearrangement in bronchial epithelial cells

Participants in deep space missions face protracted exposure to galactic cosmic radiation (GCR). In this setting, lung cancer is a significant component of the overall risk of radiation-exposure induced death. Here we investigate persistent effects of GCR exposure on DNA repair capacity in lung-derived epithelial cells, using an enzyme-stimulated chromosomal rearrangement as an endpoint. Replicate cell cultures were irradiated with energetic 48Ti ions (a GCR component) or reference γ-rays. After a six-day recovery, they were challenged by expression of a Cas9/sgRNA pair that creates double-strand breaks simultaneously in the EML4 and ALK loci, misjoining of which creates an EML4-ALK fusion oncogene. Misjoining was significantly elevated in 48Ti-irradiated populations, relative to the baseline rate in mock-irradiated controls. The effect was not seen in γ-ray irradiated populations exposed to equal or higher radiation doses. Sequence analysis of the EML4-ALK joints from 48Ti-irradiated cultures showed that they were far more likely to contain deletions, sometimes flanked by short microhomologies, than equivalent samples from mock-irradiated cultures, consistent with a shift toward error-prone alternative nonhomologous end joining repair. Results suggest a potential mechanism by which a persistent physiological effect of GCR exposure may increase lung cancer risk.

Cell-type specific role of the RNA-binding protein, NONO, in the DNA double-strand break response in the mouse testes

The tandem RNA recognition motif protein, NONO, was previously identified as a candidate DNA double-strand break (DSB) repair factor in a biochemical screen for proteins with end-joining stimulatory activity. Subsequent work showed that NONO and its binding partner, SFPQ, have many of the properties expected for bona fide repair factors in cell-based assays. Their contribution to the DNA damage response in intact tissue in vivo has not, however, been demonstrated. Here we compare DNA damage sensitivity in the testes of wild-type mice versus mice bearing a null allele of the NONO homologue (Nono gt). In wild-type mice, NONO protein was present in Sertoli, peritubular myoid, and interstitial cells, with an increase in expression following induction of DNA damage. As expected for the product of an X-linked gene, NONO was not detected in germ cells. The Nono gt/0 mice had at most a mild testis developmental phenotype in the absence of genotoxic stress. However, following irradiation at sublethal, 2-4 Gy doses, Nono gt/0 mice displayed a number of indicators of radiosensitivity as compared to their wild-type counterparts. These included higher levels of persistent DSB repair foci, increased numbers of apoptotic cells in the seminiferous tubules, and partial degeneration of the blood-testis barrier. There was also an almost complete loss of germ cells at later times following irradiation, evidently arising as an indirect effect reflecting loss of stromal support. Results demonstrate a role for NONO protein in protection against direct and indirect biological effects of ionizing radiation in the whole animal.