The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.

PurposeDNA double-strand breaks (DSBs) generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair.Methods and MaterialsUsing transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent) and heterochromatin (electron-dense) in cortical neurons of irradiated mouse brain.ResultsWhile DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer) are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥6 gold beads) may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥10 gold beads), occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage.DiscussionHigher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more complex, with multiple breaks in spatial vicinity inducing severe chromatin disruptions. Imperfect restoration of chromatin configurations may leave DSB-induced epigenetic memory of damage with potentially pathological repercussions.

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Regeneration processes in the periodontium occur by the interaction of different cell populations. It is known that these cells are also capable of forming new periodontal tissue after culture in vitro. The present study investigated whether replanted cultured cells from the periodontium could contribute to attachment formation. Primary cell cultures from alveolar bone and periodontal ligament were obtained from 11 minipigs. Experimentally induced furaction and interdental defects (n = 168) were treated in groups: (a) flap surgery, replantation of alveolar bone cells, and covering of the defects with Teflon membranes (ABC group); (b) flap surgery, replantation of periodontal ligament cells and membranes (PLC group); (c) flap surgery, bone gelatin (carrier material) and membranes (BG group); (d) flap surgery and membranes (NBG group); (e) flap surgery (FS group); and (f) no treatment (NT group). The defects were clinically and histologically (polyfluorochrome labeling) assessed after 10, 30, and 90 days. In the ABC group, initial calcified tissue formation at the roots was apparent after only 8 days. Marked new formation of cementum and alveolar bone and the development of a new attachment were observed after 90 days. In the BG and the NBG groups, wound healing varied depending on membrane healing and the morphology of the defects, which led to significantly poorer and variable results. Similar results were found in the PLC group, although some defects showed extensive cementum and bone formation. Defects in the FS and the NT groups healed largely by epithelialization. The study shows that replantation of cultured alveolar bone cells leads to formation of new cementum and bone, which, in turn, leads to formation of a new attachment. It is likely that the cells stabilize the tissue formation in the defect or on the root surface in the early phase of wound healing and prevent epithelial downgrowth. Results also show that regeneration in the periodontium is determined by the availability of (precursor) cells capable of forming calcified tissues.

Accumulation of DNA damage leading to stem cell exhaustion has been proposed to be a principal mechanism of aging. Using 53BP1-foci as a marker for DNA double-strand breaks (DSBs), hair follicle stem cells (HFSCs) in mouse epidermis were analyzed for age-related DNA damage response (DDR). We observed increasing amounts of 53BP1-foci during the natural aging process independent of telomere shortening and after protracted low-dose radiation, suggesting substantial accumulation of DSBs in HFSCs. Electron microscopy combined with immunogold-labeling showed multiple small 53BP1 clusters diffusely distributed throughout the highly compacted heterochromatin of aged HFSCs, but single large 53BP1 clusters in irradiated HFSCs. These remaining 53BP1 clusters did not colocalize with core components of non-homologous end-joining, but with heterochromatic histone modifications. Based on these results we hypothesize that these lesions were not persistently unrepaired DSBs, but may reflect chromatin rearrangements caused by the repair or misrepair of DSBs. Flow cytometry showed increased activation of repair proteins and damage-induced chromatin modifications, triggering apoptosis and cellular senescence in irradiated, but not in aged HFSCs. These results suggest that accumulation of DNA damage-induced chromatin alterations, whose structural dimensions reflect the complexity of the initial genotoxic insult, may lead to different DDR events, ultimately determining the biological outcome of HFSCs. Collectively, our findings support the hypothesis that aging might be largely the remit of structural changes to chromatin potentially leading to epigenetically induced transcriptional deregulation.