Environmental signals can be translated into chromatin changes, which alter gene expression. Here we report a novel concept that cells can signal chromatin damage from the nucleus back to the surrounding tissue through the cytokine interleukin-1alpha (IL-1α). Thus, in addition to its role as a danger signal, which occurs when the cytokine is passively released by cell necrosis, IL-1α could directly sense DNA damage and act as signal for genotoxic stress without loss of cell integrity. Here we demonstrate localization of the cytokine to DNA-damage sites and its subsequent secretion. Interestingly, its nucleo-cytosolic shuttling after DNA damage sensing is regulated by histone deacetylases (HDAC) and IL-1α acetylation. To demonstrate the physiological significance of this newly discovered mechanism, we used IL-1α knockout mice and show that IL-1α signaling after UV skin irradiation and DNA damage is important for triggering a sterile inflammatory cascade in vivo that contributes to efficient tissue repair and wound healing.
ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein–protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo. ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo. Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.
Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments.
Xeroderma pigmentosum group C (XPC) protein is a sensor of helix-distorting DNA lesions, the function of which is to trigger the global genome repair (GGR) pathway. Previous studies demonstrated that XPC protein operates by detecting the single-stranded character of non-hydrogen-bonded bases opposing lesion sites. This mode of action is supported by structural analyses of the yeast Rad4 homologue that identified critical side chains making close contacts with a pair of extrahelical nucleotides. Here, alanine substitutions of the respective conserved residues (N754, F756, F797, F799) in human XPC were tested for DNA-binding activity, accumulation in tracks and foci of DNA lesions, nuclear protein mobility, and the induction of downstream GGR reactions. This study discloses a dynamic interplay between XPC protein and DNA, whereby the association with one displaced nucleotide in the undamaged strand mediates the initial encounter with lesion sites. The additional flipping-out of an adjacent nucleotide is necessary to hand over the damaged site to the next GGR player. Surprisingly, this mutagenesis analysis also reveals that the rapid intranuclear trafficking of XPC protein depends on constitutive interactions with native DNA, implying that the search for base damage takes place in living cells by a facilitated diffusion process. Xeroderma pigmentosum group C (XPC) protein is a sensor of helix-distorting DNA lesions, the function of which is to trigger the global genome repair (GGR) pathway. Previous studies demonstrated that XPC protein operates by detecting the single-stranded character of non-hydrogen-bonded bases opposing lesion sites. This mode of action is supported by structural analyses of the yeast Rad4 homologue that identified critical side chains making close contacts with a pair of extrahelical nucleotides. Here, alanine substitutions of the respective conserved residues (N754, F756, F797, F799) in human XPC were tested for DNA-binding activity, accumulation in tracks and foci of DNA lesions, nuclear protein mobility, and the induction of downstream GGR reactions. This study discloses a dynamic interplay between XPC protein and DNA, whereby the association with one displaced nucleotide in the undamaged strand mediates the initial encounter with lesion sites. The additional flipping-out of an adjacent nucleotide is necessary to hand over the damaged site to the next GGR player. Surprisingly, this mutagenesis analysis also reveals that the rapid intranuclear trafficking of XPC protein depends on constitutive interactions with native DNA, implying that the search for base damage takes place in living cells by a facilitated diffusion process.
Understanding the cellular response to DNA strand breaks is crucial to decipher the mechanisms maintaining the integrity of our genome. We present a novel method to visualize how the mobility of nuclear proteins changes in response to localized DNA damage. DNA strand breaks are induced via nonlinear excitation with femtosecond laser pulses at λ = 1050 nm in a 3D‐confined subnuclear volume. After a time delay of choice, protein mobility within this volume is analysed by two‐photon photoactivation of PA‐GFP fusion proteins at λ = 775 nm. By changing the position of the photoactivation spot with respect to the zone of lesion the influence of chromatin structure and of the distance from damage are investigated. As first applications we demonstrate a locally confined, time‐dependent mobility increase of histone H1.2, and a progressive retardation of the DNA repair factor XRCC1 at damaged sites. This assay can be used to map the response of nuclear proteins to DNA damage in time and space. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
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