Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro‐inflammatory and pro‐oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent‐associated changes are dependent on mitochondria, particularly the pro‐inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC‐1β‐dependent mitochondrial biogenesis, contributing to a ROS‐mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC‐1β deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.

ObjectiveThe aim of this study was to characterize the genome-wide DNA methylation profile of chondrocytes from knee and hip cartilage obtained from patients with osteoarthritis (OA) and hip cartilage obtained from patients with femoral neck fracture, providing the first comparison of DNA methylation between OA and non-OA hip cartilage, and between OA hip and OA knee cartilage.MethodsThe study was performed using the Illumina Infinium HumanMethylation450 BeadChip array, which allows the annotation of ∼480,000 CpG sites. Genome-wide methylation was assessed in chondrocyte DNA extracted from 23 hip OA patients, 73 knee OA patients, and 21 healthy hip control patients with femoral neck fracture.ResultsAnalysis revealed that chondrocytes from the hip cartilage of OA patients and healthy controls have unique methylation profiles, with 5,322 differentially methylated loci (DMLs) identified between the 2 groups. In addition, a comparison between hip and knee OA chondrocytes revealed 5,547 DMLs between the 2 groups, including DMLs in several genes known to be involved in the pathogenesis of OA. Hip OA samples were found to cluster into 2 groups. A total of 15,239 DMLs were identified between the 2 clusters, with an enrichment of genes involved in inflammation and immunity. Similarly, we confirmed a previous report of knee OA samples that also clustered into 2 groups.ConclusionWe demonstrated that global DNA methylation using a high-density array can be a powerful tool in the characterization of OA at the molecular level. Identification of pathways enriched in DMLs between OA and OA-free cartilage highlight potential etiologic mechanisms that are involved in the initiation and/or progression of the disease and that could be therapeutically targeted.

Cells accumulate single-stranded DNA (ssDNA) when telomere capping, DNA replication, or DNA repair is impeded. This accumulation leads to cell cycle arrest through activating the DNA–damage checkpoints involved in cancer protection. Hence, ssDNA accumulation could be an anti-cancer mechanism. However, ssDNA has to accumulate above a certain threshold to activate checkpoints. What determines this checkpoint-activation threshold is an important, yet unanswered question. Here we identify Rif1 (Rap1-Interacting Factor 1) as a threshold-setter. Following telomere uncapping, we show that budding yeast Rif1 has unprecedented effects for a protein, inhibiting the recruitment of checkpoint proteins and RPA (Replication Protein A) to damaged chromosome regions, without significantly affecting the accumulation of ssDNA at those regions. Using chromatin immuno-precipitation, we provide evidence that Rif1 acts as a molecular “band-aid” for ssDNA lesions, associating with DNA damage independently of Rap1. In consequence, small or incipient lesions are protected from RPA and checkpoint proteins. When longer stretches of ssDNA are generated, they extend beyond the junction-proximal Rif1-protected regions. In consequence, the damage is detected and checkpoint signals are fired, resulting in cell cycle arrest. However, increased Rif1 expression raises the checkpoint-activation threshold to the point it simulates a checkpoint knockout and can also terminate a checkpoint arrest, despite persistent telomere deficiency. Our work has important implications for understanding the checkpoint and RPA–dependent DNA–damage responses in eukaryotic cells.