RAG-2 deficiency results in fewer phosphorylated histone H2AX foci, but increased retinal ganglion cell death and altered axonal growth.
ABSTRACT: DNA double-strand breaks (DSBs), selectively visualized as ?-H2AX+ foci, occur during the development of the central nervous system, including the retina, although their origin and biological significance are poorly understood. Mutant mice with DSB repair mechanism defects exhibit increased numbers of ?-H2AX+ foci, increased cell death during neural development, and alterations in axonogenesis in the embryonic retina. The aim of this study was to identify putative sources of DSBs. One of the identified DSBs sources is LINE-1 retrotransposition. While we did not detect changes in LINE-1 DNA content during the early period of cell death associated with retinal neurogenesis, retinal development was altered in mice lacking RAG-2, a component of the RAG-1,2-complex responsible for initiating somatic recombination in lymphocytes. Although ?-H2AX+ foci were less abundant in the rag2-/- mouse retina, retinal ganglion cell death was increased and axonal growth and navigation were impaired in the RAG-2 deficient mice, a phenotype shared with mutant mice with defective DNA repair mechanisms. These findings demonstrate that RAG-2 is necessary for proper retinal development, and suggest that both DSB generation and repair are genuine processes intrinsic to neural development.
Project description:Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB) repair complex of ?H2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30) in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months) retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of ?H2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and ?H2AX staining was a very rare event (< 1% of ?H2AX foci in the ONL and < 3% in the INL), suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted.
Project description:Ionizing radiation (IR) induces a variety of DNA lesions among which DNA double-strand breaks (DSBs) are the biologically most significant. It is currently unclear if DSB repair is equally efficient after low and high doses. Here, we use gamma-H2AX, phospho-ATM (pATM), and 53BP1 foci analysis to monitor DSB repair. We show, consistent with a previous study, that the kinetics of gamma-H2AX and pATM foci loss in confluent primary human fibroblasts are substantially compromised after doses of 10 mGy and lower. Following 2.5 mGy, cells fail to show any foci loss. Strikingly, cells pretreated with 10 microM H(2)O(2) efficiently remove all gamma-H2AX foci induced by 10 mGy. At the concentration used, H(2)O(2) produces single-strand breaks and base damages via the generation of oxygen radicals but no DSBs. Moreover, 10 microM H(2)O(2) up-regulates a set of genes that is also up-regulated after high (200 mGy) but not after low (10 mGy) radiation doses. This suggests that low radical levels induce a response that is required for the repair of radiation-induced DSBs when the radiation damage is too low to cause the induction itself. To address the in vivo significance of this finding, we established gamma-H2AX and 53BP1 foci analysis in various mouse tissues. Although mice irradiated with 100 mGy or 1 Gy show efficient gamma-H2AX and 53BP1 foci removal during 24 h post-IR, barely any foci loss was observed after 10 mGy. Our data suggest that the cellular response to DSBs is substantially different for low vs. high radiation doses.
Project description:Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G1-like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.
Project description:Ovarian aging is closely tied to the decline in ovarian follicular reserve and oocyte quality. During the prolonged reproductive lifespan of the female, granulosa cells connected with oocytes play critical roles in maintaining follicle reservoir, oocyte growth and follicular development. We tested whether double-strand breaks (DSBs) and repair in granulosa cells within the follicular reservoir are associated with ovarian aging.Ovaries were sectioned and processed for epi-fluorescence microscopy, confocal microscopy, and immunohistochemistry. DNA damage was revealed by immunstaining of ?H2AX foci and telomere damage by ?H2AX foci co-localized with telomere associated protein TRF2. DNA repair was indicated by BRCA1 immunofluorescence.DSBs in granulosa cells increase and DSB repair ability, characterized by BRCA1 foci, decreases with advancing age. ?H2AX foci increase in primordial, primary and secondary follicles with advancing age. Likewise, telomere damage increases with advancing age. In contrast, BRCA1 foci in granulosa cells of primordial, primary and secondary follicles decrease with monkey age. BRCA1 positive foci in the oocyte nuclei also decline with maternal age.Increased DSBs and reduced DNA repair in granulosa cells may contribute to ovarian aging. Discovery of therapeutics that targets these pathways might help maintain follicle reserve and postpone ovarian dysfunction with age.
Project description:H2AX is a core histone H2A variant that contains an absolutely conserved serine/glutamine (SQ) motif within an extended carboxy-terminal tail. H2AX phosphorylation at the SQ motif (gamma-H2AX) has been shown to increase dramatically upon exogenously introduced DNA double-strand breaks (DSBs). In this study, we use quantitative in situ approaches to investigate the spatial patterning and cell cycle dynamics of gamma-H2AX in a panel of normally growing (unirradiated) mammalian cell lines and cultures. We provide the first evidence for the existence of two distinct yet highly discernible gamma-H2AX focal populations: a small population of large amorphous foci that colocalize with numerous DNA DSB repair proteins and previously undescribed but much more abundant small foci. These small foci do not recruit proteins involved in DNA DSB repair. Cell cycle analyses reveal unexpected dynamics for gamma-H2AX in unirradiated mammalian cells that include an ATM-dependent phosphorylation that is maximal during M phase. Based upon similarities drawn from other histone posttranslational modifications and previous observations in haplo-insufficient (H2AX-/+) and null mice (H2AX-/-), gamma-H2AX may contribute to the fidelity of the mitotic process, even in the absence of DNA damage, thereby ensuring the faithful transmission of genetic information from one generation to the next.
Project description:CHD1 is a conserved chromatin remodeling enzyme required for development and linked to prostate cancer in adults, yet its role in human cells is poorly understood. Here, we show that targeted disruption of the CHD1 gene in human cells leads to a defect in early double-strand break (DSB) repair via homologous recombination (HR), resulting in hypersensitivity to ionizing radiation as well as PARP and PTEN inhibition. CHD1 knockout cells show reduced H2AX phosphorylation (?H2AX) and foci formation as well as impairments in CtIP recruitment to the damaged sites. Chromatin immunoprecipitation following a single DSB shows that the reduced levels of ?H2AX accumulation at DSBs in CHD1-KO cells are due to both a global reduction in H2AX incorporation and poor retention of H2AX at the DSBs. We also identified a unique N-terminal region of CHD1 that inhibits the DNA binding, ATPase, and chromatin assembly and remodeling activities of CHD1. CHD1 lacking the N terminus was more active in rescuing the defects in ?H2AX formation and CtIP recruitment in CHD1-KO cells than full-length CHD1, suggesting the N terminus is a negative regulator in cells. Our data point to a role for CHD1 in the DSB repair process and identify a novel regulatory region of the protein.
Project description:The binding of DNA-dependent protein kinase catalytic subunit (DNA-PKcs, also known as PRKDC) to Ku proteins at DNA double-strand breaks (DSBs) has long been considered essential for non-homologous end joining (NHEJ) repair, providing a rationale for use of DNA-PKcs inhibitors as cancer therapeutics. Given lagging clinical translation, we reexamined mechanisms and observed instead that DSB repair can proceed independently of DNA-PKcs. While repair of radiation-induced DSBs was blocked in cells expressing shRNAs targeting Ku proteins or other NHEJ core factors, DSBs were repaired on schedule despite targeting DNA-PKcs. Although we failed to observe a DSB repair defect, the ?H2AX foci that formed at sites of DNA damage persisted indefinitely after irradiation, leading to cytokinesis failure and accumulation of binucleated cells. Following this mitotic slippage, cells with decreased DNA-PKcs underwent accelerated cellular senescence. We identified downregulation of ataxia-telangiectasia mutated kinase (ATM) as the critical role of DNA-PKcs in recovery from DNA damage, insofar as targeting ATM restored ?H2AX foci resolution and cytokinesis. Considering the lack of direct impact on DSB repair and emerging links between senescence and resistance to cancer therapy, these results suggest reassessing DNA-PKcs as a target for cancer treatment.
Project description:BACKGROUND:Phosphorylated histone H2AX, also known as ?H2AX, forms ?m-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, ?H2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of ?H2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of ?H2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected. RESULTS:To overcome the limitations of 2D foci counting, we present a freely available ImageJ-based plugin (FocAn) for automated 3D analysis of ?H2AX foci in z-image stacks acquired by confocal fluorescence microscopy. The image-stack processing algorithm implemented in FocAn is capable of automatic 3D recognition of individual cell nuclei and ?H2AX foci, as well as evaluation of the total foci number per cell nucleus. The FocAn algorithm consists of two parts: nucleus identification and foci detection, each employing specific sequences of auto local thresholding in combination with watershed segmentation techniques. We validated the FocAn algorithm using fluorescence-labeled ?H2AX in two glioblastoma cell lines, irradiated with 2?Gy and given up to 24?h post-irradiation for repair. We found that the data obtained with FocAn agreed well with those obtained with an already available software (FoCo) and manual counting. Moreover, FocAn was capable of identifying overlapping foci in 3D space, which ensured accurate foci counting even at high DSB density of up to ~?200 DSB/nucleus. CONCLUSIONS:FocAn is freely available an open-source 3D foci analyzer. The user-friendly algorithm FocAn requires little supervision and can automatically count the amount of DNA-DSBs, i.e. fluorescence-labeled ?H2AX foci, in 3D image stacks acquired by laser-scanning microscopes without additional nuclei staining.
Project description:Phosphorylation of histone H2AX on serine 139 (?H2AX) is an early step in cellular response to a DNA double-strand break (DSB). ?H2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of ?H2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 ?M camptothecin, 10 ?M etoposide or 0.2 ?M mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual ?H2AX foci induced by these drugs. Also, investigated was spatial association between ?H2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that ?H2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation ?H2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of ?H2AX sites of different sizes and intensities.
Project description:The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of gamma-H2AX foci at a single DSB was independent of ATM or Ku70. ATM-deficient cells showed normal kinetics of 53Bp1 recruitment to DSBs, but Rad51 localization was retarded. 53Bp1 and Rad51 foci formation at a single DSB was greatly reduced in H2AX-null DT40 cells. We also observed decreased inter-sister chromatid distances after DSB induction, suggesting that cohesin loading at DSBs causes elevated sister chromatid cohesion. Loss of ATM reduced DSB-induced cohesion, consistent with cohesin being an ATM target in the DSB response. These data show that the same genetic pathways control how cells respond to single DSBs and to multiple lesions induced by whole-cell DNA damage.