Transcription stress at telomeres leads to cytosolic DNA release and paracrine senescence.

Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1-/- cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1-/- cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging.

XPF interacts with TOP2B for R-loop processing and DNA looping on actively transcribed genes.

Co-transcriptional RNA-DNA hybrids can not only cause DNA damage threatening genome integrity but also regulate gene activity in a mechanism that remains unclear. Here, we show that the nucleotide excision repair factor XPF interacts with the insulator binding protein CTCF and the cohesin subunits SMC1A and SMC3, leading to R-loop-dependent DNA looping upon transcription activation. To facilitate R-loop processing, XPF interacts and recruits with TOP2B on active gene promoters, leading to double-strand break accumulation and the activation of a DNA damage response. Abrogation of TOP2B leads to the diminished recruitment of XPF, CTCF, and the cohesin subunits to promoters of actively transcribed genes and R-loops and the concurrent impairment of CTCF-mediated DNA looping. Together, our findings disclose an essential role for XPF with TOP2B and the CTCF/cohesin complex in R-loop processing for transcription activation with important ramifications for DNA repair-deficient syndromes associated with transcription-associated DNA damage.

SpotitPy: a semi-automated tool for object-based co-localization of fluorescent labels in microscopy images.

BACKGROUND: In fluorescence microscopy, co-localization refers to the spatial overlap between different fluorescent labels in cells. The degree of overlap between two or more channels in a microscope may reveal a physical interaction or topological functional interconnection between molecules. Recent advances in the imaging field require the development of specialized computational analysis software for the unbiased assessment of fluorescently labelled microscopy images. RESULTS: Here we present SpotitPy, a semi-automated image analysis tool for 2D object-based co-localization. SpotitPy allows the user to select fluorescent labels and perform a semi-automated and robust segmentation of the region of interest in distinct cell types. The workflow integrates advanced pre-processing manipulations for de-noising and in-depth semi-automated quantification of the co-localized fluorescent labels in two different channels. We validated SpotitPy by quantitatively assessing the presence of cytoplasmic ribonucleoprotein granules, e.g. processing (P) bodies, under conditions that challenge mRNA translation, thus highlighting SpotitPy benefits for semi-automatic, accurate analysis of large image datasets in eukaryotic cells. SpotitPy comes in a command line interface or a simple graphical user interphase and can be used as a standalone application. CONCLUSIONS: Overall, we present a novel and user-friendly tool that performs a semi-automated image analysis for 2D object-based co-localization. SpotitPy can provide reproducible and robust quantifications for large datasets within a limited timeframe. The software is open-source and can be found in the GitHub project repository: ( https://github.com/alexiaales/SpotitPy ).

R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage.

How DNA damage leads to chronic inflammation and tissue degeneration with aging remains to be fully resolved. Here, we show that DNA damage leads to cellular senescence, fibrosis, loss-of-tissue architecture, and chronic pancreatitis in mice with an inborn defect in the excision repair cross complementation group 1 (Ercc1) gene. We find that DNA damage-driven R-loops causally contribute to the active release and buildup of single-stranded DNAs (ssDNAs) in the cytoplasm of cells triggering a viral-like immune response in progeroid and naturally aged pancreata. To reduce the proinflammatory load, we developed an extracellular vesicle (EV)-based strategy to deliver recombinant S1 or ribonuclease H nucleases in inflamed Ercc1−/− pancreatic cells. Treatment of Ercc1−/− animals with the EV-delivered nuclease cargo eliminates DNA damage-induced R-loops and cytoplasmic ssDNAs alleviating chronic inflammation. Thus, DNA damage-driven ssDNAs causally contribute to tissue degeneration, Ercc1−/− paving the way for novel rationalized intervention strategies against age-related chronic inflammation.