Summary The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary post-natal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen rich postnatal environment is the upstream signal that results in cell cycle arrest of cardiomyocytes. Here we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, while hyperoxemia and ROS generators shorten it. These findings uncover a previously unrecognized protective mechanism that mediates cardiomyocyte cell cycle arrest in exchange for utilization of oxygen dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be important component of cardiomyocyte proliferation-based therapeutic approaches.

The adult mammalian heart is incapable of regeneration following cardiomyocyte loss, which underpins the lasting and severe effects of cardiomyopathy. Recently, it has become clear that the mammalian heart is not a post-mitotic organ. For example, the neonatal heart is capable of regenerating lost myocardium, and the adult heart is capable of modest self-renewal. In both of these scenarios, cardiomyocyte renewal occurs via the proliferation of pre-existing cardiomyocytes, and is regulated by aerobic-respiration-mediated oxidative DNA damage. Therefore, we reasoned that inhibiting aerobic respiration by inducing systemic hypoxaemia would alleviate oxidative DNA damage, thereby inducing cardiomyocyte proliferation in adult mammals. Here we report that, in mice, gradual exposure to severe systemic hypoxaemia, in which inspired oxygen is gradually decreased by 1% and maintained at 7% for 2 weeks, results in inhibition of oxidative metabolism, decreased reactive oxygen species production and oxidative DNA damage, and reactivation of cardiomyocyte mitosis. Notably, we find that exposure to hypoxaemia 1 week after induction of myocardial infarction induces a robust regenerative response with decreased myocardial fibrosis and improvement of left ventricular systolic function. Genetic fate-mapping analysis confirms that the newly formed myocardium is derived from pre-existing cardiomyocytes. These results demonstrate that the endogenous regenerative properties of the adult mammalian heart can be reactivated by exposure to gradual systemic hypoxaemia, and highlight the potential therapeutic role of hypoxia in regenerative medicine.

DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Although the kinase activity of DNA-PKcs is essential for NHEJ, thus far, no in vivo substrate has been conclusively identified except for an autophosphorylation site on DNA-PKcs itself (threonine 2609). Here we report the ionizing radiation (IR)-induced autophosphorylation of DNA-PKcs at a novel site, serine 2056, the phosphorylation of which is required for the repair of DSBs by NHEJ. Interestingly, IR-induced DNA-PKcs autophosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs resulted in DNA-PKcs autophosphorylation and localization to DNA damage sites. These results indicate that although IR-induced DNAPKcs phosphorylation is attenuated in the S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis.Repair of DNA double strand breaks (DSBs) 1 is critical for the maintenance of genome integrity, cell survival, and prevention of tumorigenesis (1, 2). In higher eukaryotes, non-homologous end joining (NHEJ) and homologous recombination (HR) are the two major pathways for DSB repair (3). HR requires the presence of a sister chromatid and is operational in the late S and G 2 phases of the cell cycle because of the availability of an optimally positioned sister chromatid (4). NHEJ, on other hand, does not depend on the presence of homologous DNA sequences and is the predominant pathway for DSB repair in mammalian cells (5). It was proposed that NHEJ is preferentially used in G 1 and early S phases of the cell cycle (6, 7). However, a recent report indicating that NHEJ-deficient cell lines are radiation-sensitive in all phases of the cell cycle suggests that NHEJ is important throughout the cell cycle (8). Clearly, the exact contribution of NHEJ in different phases of the cell cycle needs to be defined further.The NHEJ pathway of DSB repair requires both the DNAdependent protein kinase (DNA-PK) complex and the XRCC4/ DNA ligase IV complex, as well as possible additional accessory factors (5, 9, 10). DNA-PK, the key component of the NHEJ pathway, is composed of the Ku70/80 heterodimer and the catalytic subunit DNA-PKcs (11). Ku binds to DNA ends with very high affinity and is believed to function as the DNAbinding and regulatory subunit that recruits DNA-PKcs to breaks and stimulates its kinase activity (12, 13). DNA-PKcs is a member of the phosphatidylinositol-3-like kinase family that includes ATM (ataxia-telangiectasia mutated) and 15). Although the biochemical properties of DNA-PK have been extensively studied in vitro, it is still not clear how it functions in vivo in the context of NHEJ. Wild type DNA-PKcs, but not a kinase-dead mutant, is able to rescue the radiation sensitivity and DSB repair defect of DNA-PKcs-defective V3 cells demonstrati...

We have previously shown that RNA polymerase II (Pol II) pause release and transcriptional elongation involve phosphorylation of the factor TRIM28 by the DNA damage response (DDR) kinases ATM and DNA-PK. Here we report a significant role for DNA breaks and DDR signalling in the mechanisms of transcriptional elongation in stimulus-inducible genes in humans. Our data show the enrichment of TRIM28 and γH2AX on serum-induced genes and the important function of DNA-PK for Pol II pause release and transcriptional activation-coupled DDR signalling on these genes. γH2AX accumulation decreases when P-TEFb is inhibited, confirming that DDR signalling results from transcriptional elongation. In addition, transcriptional elongation-coupled DDR signalling involves topoisomerase II because inhibiting this enzyme interferes with Pol II pause release and γH2AX accumulation. Our findings propose that DDR signalling is required for effective Pol II pause release and transcriptional elongation through a novel mechanism involving TRIM28, DNA-PK and topoisomerase II.

Although the adult mammalian heart is incapable of meaningful functional recovery following substantial cardiomyocyte loss, it is now clear that modest cardiomyocyte turnover occurs in adult mouse and human hearts, mediated primarily by proliferation of pre-existing cardiomyocytes. However, fate mapping of these cycling cardiomyocytes has not been possible thus far owing to the lack of identifiable genetic markers. In several organs, stem or progenitor cells reside in relatively hypoxic microenvironments where the stabilization of the hypoxia-inducible factor 1 alpha (Hif-1α) subunit is critical for their maintenance and function. Here we report fate mapping of hypoxic cells and their progenies by generating a transgenic mouse expressing a chimaeric protein in which the oxygen-dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen-inducible CreERT2 recombinase. In mice bearing the creERT2-ODD transgene driven by either the ubiquitous CAG promoter or the cardiomyocyte-specific α myosin heavy chain promoter, we identify a rare population of hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage. Notably, these hypoxic cardiomyocytes contributed widely to new cardiomyocyte formation in the adult heart. These results indicate that hypoxia signalling is an important hallmark of cycling cardiomyocytes, and suggest that hypoxia fate mapping can be a powerful tool for identifying cycling cells in adult mammals.

The concept of DNA "repair centers" and the meaning of radiationinduced foci (RIF) in human cells have remained controversial. RIFs are characterized by the local recruitment of DNA damage sensing proteins such as p53 binding protein (53BP1). Here, we provide strong evidence for the existence of repair centers. We used live imaging and mathematical fitting of RIF kinetics to show that RIF induction rate increases with increasing radiation dose, whereas the rate at which RIFs disappear decreases. We show that multiple DNA double-strand breaks (DSBs) 1 to 2 μm apart can rapidly cluster into repair centers. Correcting mathematically for the dose dependence of induction/resolution rates, we observe an absolute RIF yield that is surprisingly much smaller at higher doses: 15 RIF∕Gy after 2 Gy exposure compared to approximately 64 RIF∕Gy after 0.1 Gy. Cumulative RIF counts from time lapse of 53BP1-GFP in human breast cells confirmed these results. The standard model currently in use applies a linear scale, extrapolating cancer risk from high doses to low doses of ionizing radiation. However, our discovery of DSB clustering over such large distances casts considerable doubts on the general assumption that risk to ionizing radiation is proportional to dose, and instead provides a mechanism that could more accurately address risk dose dependency of ionizing radiation. DNA damage-sensing proteins localize at sites of DNA double-strand breaks (DSBs) within seconds to minutes following ionizing radiation (IR) exposure, resulting in the formation of immunofluorescently stainable nuclear domains referred to as radiation-induced foci (RIF) (1-3). RIF numbers are routinely used to assess the amount of DNA damage and repair kinetics after different treatments (4). However, there is a controversy surrounding the question of whether there is a 1∶1 correspondence between RIF and DSBs. For example, pulse field gel electrophoresis (PFGE) analysis suggests that DSBs decay exponentially with time immediately after exposure (5). In contrast, DNA damage-sensing proteins do not instantaneously detect DSBs, leading to delayed kinetics for both detection and resolution. More specifically, the maximum number of 53BP1 or γH2AX RIF is not reached until 15 to 30 min after exposure, and the yield of DSBs predicted by RIF is typically lower than the expected 25-40 DSB∕Gy measured by PFGE (4).Dose response provides another assay for assessing the relationship between DSBs and RIF. Based on theoretical Monte Carlo simulations and PFGE measurements (6, 7), the frequency of DSBs should be highly correlated with radiation dose. Confirming this prediction, two research groups reported that RIF number is proportional to radiation dosage from 1 mGy to 2 Gy (8, 9). In both studies, methods were applied to identify "real" RIF at low doses, where frequencies may be close to background levels before IR (e.g., 10 mGy would lead to about 0.3 DSB∕cell). They either used cells with very low γH2AX background foci (i.e., 0.05 background foci∕cell in primary human l...

Clustered DNA damage induced by ionizing radiation is refractory to repair and may trigger carcinogenic events for reasons that are not well understood. Here, we used an in situ method to directly monitor induction and repair of clustered DNA lesions in individual cells. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the subnuclear domains, determined the cellular ability to repair the damage. We then examined checkpoint arrest mechanisms and yield of gross chromosomal aberrations. Induction of nonrepairable clustered damage affected only G2 accumulation but not the early G2/M checkpoint. Further, cells that were released from the G2/M checkpoint with unrepaired clustered damage manifested a spectrum of chromosome aberrations in mitosis. Difficulties associated with clustered DNA damage repair and checkpoint release before the completion of clustered DNA damage repair appear to promote genome instability that may lead to carcinogenesis.heterochromatin | high linear energy transfer | high charge and energy particles | 53BP1 | ionizing radiation induced foci I onizing radiation (IR) may induce cancer and loss of neural function or death in humans. Low (e.g., γ-and X-rays) and high [i.e., high charge and energy (HZE)] linear energy transfer (LET) IR induces a plethora of DNA damage, and the damage complexity increases with an increase in the LET of the radiation (1-3). Isolated DNA lesions (mainly induced by low-LET radiation), including double-strand breaks (DSBs), single-strand breaks (SSBs), and damaged bases located at a distance from other damage, are generally repaired efficiently. Substantial evidence indicates that high-LET radiation induces complex DNA damage, a unique class of DNA lesions that includes two or more individual lesions within one or two helical turns of the DNA (4). These lesions can be abasic sites (apurinic/apyrimidinic sites or APs), damaged bases (oxidized purines or pyrimidines), SSBs, or DSBs (5). Convincing evidence indicates that complex DNA lesions are more difficult to repair than isolated lesions and in some instances are irreparable; however, it is unclear why clustered lesions are difficult to repair.The biological consequences of complex DNA damage range from point mutations and loss of genetic material to cellular lethality due to repair impairment and lesion or repair-intermediate persistency. Clustered lesions induce intra-and interchromosomal insertions, and inversions often in association with large deletions (6). FISH-painting methodologies were used to show that high-LET IR induces a high fraction of chromosome rearrangements (7). Recently, it has been suggested that non-DSB clusters, if unrepaired, can lead to the formation of mutations and chromosome abnormalities (8). After exposure to high-LET radiation, immortalized...

Background-Circadian rhythm abnormalities are strongly associated with bipolar disorder, however the role of circadian genes in mood regulation is unclear. Previously, we reported that mice with a mutation in the Clock gene (ClockΔ19) display a behavioral profile that is strikingly similar to bipolar patients in the manic state.