Irreparable DNA damage following ionizing radiation (IR) triggers prolonged DNA damage response and induces premature senescence. Cellular senescence is a permanent state of cell-cycle arrest characterized by chromatin restructuring, altered nuclear morphology and acquisition of secretory phenotype, which contributes to senescence-related inflammation. However, the mechanistic connections for radiation-induced DNA damage that trigger these senescence-associated hallmarks are poorly understood. In our in vitro model of radiation-induced senescence, mass spectrometry-based proteomics was combined with high-resolution imaging techniques to investigate the interrelations between altered chromatin compaction, nuclear envelope destabilization and nucleo-cytoplasmic chromatin blebbing. Our findings confirm the general pathophysiology of the senescence-response, with disruption of nuclear lamin organization leading to extensive chromatin restructuring and destabilization of the nuclear membrane with release of chromatin fragments into the cytosol, thereby activating cGAS-STING-dependent interferon signaling. By serial block-face scanning electron microscopy (SBF-SEM) whole-cell datasets were acquired to investigate the morphological organization of senescent fibroblasts. High-resolution 3-dimensional (3D) reconstruction of the complex nuclear shape allows us to precisely visualize the segregation of nuclear blebs from the main nucleus and their fusion with lysosomes. By multi-view 3D electron microscopy, we identified nanotubular channels formed in lamin-perturbed nuclei of senescent fibroblasts; the potential role of these nucleo-cytoplasmic nanotubes for expulsion of damaged chromatin has to be examined.

Purpose: Radiation-induced senescence is characterized by profound changes in chromatin organization with the formation of Senescence-Associated-Heterochromatin-Foci (SAHF) and DNA-Segments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-Associated-Secretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in establishing radiation-induced senescence. Experimental Design: Primary and genetically-modified lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy. Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry. Results: Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory regions decisively modulates transcription factor recruitment and inflammatory gene expression, resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent H2A.J expression in response to radiation-induced DNA damage, therefore contributing to pro-inflammatory tissue reactions. Conclusions: The fine-tuned incorporation of H2A.J defines the epigenetic landscape for driving the senescence programme in response to radiation-induced DNA damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into DNA-damage triggered epigenetic mechanisms governing the biological processes of radiation-induced injury.

The global continued decline in coral reefs is intensifying the need to understand the response of corals to local environmental stressors. Coral-associated bacterial communities have been suggested to have a swift response to environmental pollutants. This study aims to determine the variation in the bacterial communities associated with the mucus of two coral species, Pocillopora damicornis (Linnaeus, 1758) and Stylophora pistillata (Esper, 1792), and the coral-surrounding seawater from three areas exposed to contamination at the Jordanian coast of the Gulf of Aqaba (Red Sea), and also explores the antibacterial activity of these bacteria. Corals were collected from three contaminated zones along the coast, and the bacteria were quantified and identified by conventional morphological and biochemical tests, as well as 16S rRNA gene sequencing. The average number of bacteria significantly varied among the coral mucus from the sampling zones and between the coral mucus and the surrounding seawater. The P. damicornis mucus-associated bacterial community was dominated by members of the classes Gammaproteobacteria, Cytophagia, and Actinomycetia, while the mucus of S. pistillata represented higher bacterial diversity, with the dominance of the bacterial classes Gammaproteobacteria, Actinomycetia, Alphaproteobacteria, and Bacilli. The effects of local anthropogenic impacts on coral mucus bacterial communities were represented in the increased abundance of bacterial species related to coral diseases. Furthermore, the results demonstrated the existence of bacterial isolates with antibacterial activity that possibly acted as a first line of defense to protect and maintain the coral host against pathogens. Indeed, the dynamics of coral-associated microbial communities highlight the importance of holistic studies that focus on microbial interactions across the coral reef ecosystem.

Background: Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. Methods: Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. Results: Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. Conclusions: The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns.

The impact of ionizing radiation (IR) with induction of various DNA damage is based not only on genetic but also on epigenetic effects. Epigenetic modifications determine the chromatin structure and DNA accessibility, thereby regulating cellular functions through the expression of individual genes or entire groups of genes. However, the influence of DNA repair processes on the restoration of local chromatin structures and global nuclear architectures is still insufficiently understood. In multicellular organisms, epigenetic mechanisms control diverse cellular functions of specific cell types through precise temporal and spatial regulation of gene expression and silencing. How altered epigenetic mechanisms regulate the pathophysiological function of cells, tissues, and ultimately entire organs following IR exposure remains to be investigated in detail. Radiation-induced epigenetic processes are particularly critical for immature cell populations such as tissue-specific stem and progenitor cells during development and differentiation of organ tissues. Genome-wide patterns of DNA and histone modifications are established cell types—specifically during the development and differentiation of organ tissues but can also be fundamentally altered in adult organism by stress responses, such as radiation-induced DNA damage. Following IR exposure, epigenetic factors are not always fully restored to their original state, resulting in epigenetic dysfunction that causes cells to lose their original identity and function. Moreover, severe radiation-induced DNA damage can induce premature senescence of cells in complex tissues, which ultimately leads to signs of aging and age-related diseases such as cancer. In this work, we provide an overview of the most important epigenetic changes following IR exposure and their pathophysiological significance for the development of acute and chronic radiation reactions.

Background: Cellular senescence in response to ionizing radiation (IR) limits the replication of damaged cells by causing permanent cell cycle arrest. However, IR can induce pro-survival signaling pathways that reduce the extent of radiation-induced cytotoxicity and promote the development of radioresistance. The differential incorporation of histone variant H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of radiation-induced senescence. However, the precise epigenetic mechanism and function of H2A.J overexpression in response to IR exposure still needs to be elucidated. Methods: Primary (no target, NT) and genetically modified fibroblasts overexpressing H2A.J (H2A.J-OE) were exposed to 20 Gy and analyzed 2 weeks post-IR for radiation-induced senescence by immunohistochemistry and immunofluorescence microscopy. Transcriptome signatures were analyzed in (non-)irradiated NT and H2A.J-OE fibroblasts by RNA sequencing. Since H2A.J plays an important role in the epidermal homeostasis of human skin, the oncogenic potential of H2A.J was investigated in cutaneous squamous cell carcinoma (cSCC). The tissue microarrays of cSCC were analyzed for H2A.J protein expression pattern by automated image analysis. Results: In response to radiation-induced DNA damage, the overexpression of H2A.J impairs the formation of senescence-associated heterochromatin foci (SAHF), thereby inhibiting the SAHF-mediated silencing of proliferation-promoting genes. The dysregulated activation of cyclins and cyclin-dependent kinases disturbs cell cycle arrest in irradiated H2A.J-OE fibroblasts, thereby overcoming radiation-induced senescence. Comparative transcriptome analysis revealed significantly increased WNT16 signaling in H2A.J OE fibroblasts after IR exposure, promoting the fundamental mechanisms of tumor development and progression, including the activation of the epithelial–mesenchymal transition. The quantitative analysis of cSCCs revealed that undifferentiated tumors are associated with high nuclear H2A.J expression, related with greater oncogenic potential. Conclusion: H2A.J overexpression induces radioresistance and promotes oncogenic transformation through the activation of WNT16 signaling pathway functions. H2A.J-associated signatures may improve risk stratification by identifying patients with more aggressive cSCC who may require radiotherapy with increased doses.