The inability of adult mammalian cardiomyocytes to proliferate underpins the development of heart failure following myocardial injury. Although the newborn mammalian heart can spontaneously regenerate for a short period of time after birth, this ability is lost within the first week after birth in mice, partly due to increased mitochondrial reactive oxygen species (ROS) production which results in oxidative DNA damage and activation of DNA damage response. This increase in ROS levels coincides with a postnatal switch from anaerobic glycolysis to fatty acid (FA) oxidation by cardiac mitochondria. However, to date, a direct link between mitochondrial substrate utilization and oxidative DNA damage is lacking. Here, we generated ROS-sensitive fluorescent sensors targeted to different subnuclear compartments (chromatin, heterochromatin, telomeres, and nuclear lamin) in neonatal rat ventricular cardiomyocytes, which allowed us to determine the spatial localization of ROS in cardiomyocyte nuclei upon manipulation of mitochondrial respiration. Our results demonstrate that FA utilization by the mitochondria induces a significant increase in ROS detection at the chromatin level compared to other nuclear compartments. These results indicate that mitochondrial metabolic perturbations directly alter the nuclear redox status and that the chromatin appears to be particularly sensitive to the prooxidant effect of FA utilization by the mitochondria.
Several studies have suggested that disruptions in circadian rhythms contribute to the pathophysiology of multiple psychiatric diseases, including drug addiction. In fact, a number of the genes involved in the regulation of circadian rhythms are also involved in modulating the reward value for drugs of abuse, like cocaine. Thus, we wanted to determine the effects of chronic cocaine on the expression of several circadian genes in the Nucleus Accumbens (NAc) and Caudate Putamen (CP), regions of the brain known to be involved in the behavioral responses to drugs of abuse. Moreover, we wanted to explore the mechanism by which these genes are regulated following cocaine exposure. Here we find that after repeated cocaine exposure, expression of the Period (Per) genes and Neuronal PAS Domain Protein 2 (Npas2) are elevated, in a somewhat regionally selective fashion. Moreover, NPAS2 (but not CLOCK (Circadian Locomotor Output Cycles Kaput)) protein binding at Per gene promoters was enhanced following cocaine treatment. Mice lacking a functional Npas2 gene failed to exhibit any induction of Per gene expression after cocaine, suggesting that NPAS2 is necessary for this cocaine-induced regulation. Examination of Per gene and Npas2 expression over twenty-four hours identified changes in diurnal rhythmicity of these genes following chronic cocaine, which were regionally specific. Taken together, these studies point to selective disruptions in Per gene rhythmicity in striatial regions following chronic cocaine treatment, which are mediated primarily by NPAS2.
Fanconi Anemia (FA) is a cancer predisposition syndrome and the factors defective in FA are involved in DNA replication, DNA damage repair and tumor suppression. Here, we show that FANCD2 is critical for genome stability maintenance in response to high-linear energy transfer (LET) radiation. We found that FANCD2 is monoubiquitinated and recruited to the sites of clustered DNA double-stranded breaks (DSBs) specifically in S/G2 cells after high-LET radiation. Further, FANCD2 facilitated the repair of clustered DSBs in S/G2 cells and proper progression of S-phase. Furthermore, lack of FANCD2 led to a reduced rate of replication fork progression and elevated levels of both replication fork stalling and new origin firing in response to high-LET radiation. Mechanistically, FANCD2 is required for correct recruitment of RPA2 and Rad51 to the sites of clustered DSBs and that is critical for proper processing of clustered DSBs. Significantly, FANCD2-decifient cells exhibited defective chromosome segregation, elevated levels of chromosomal aberrations, and anchorage-independent growth in response to high-LET radiation. These findings establish FANCD2 as a key factor in genome stability maintenance in response to high-LET radiation and as a promising target to improve cancer therapy.
Abstract Purpose of the study: Eukaryotic cells accrue DNA damage as a result of endogenous metabolic activities such as DNA replication, recombination errors or environmental exposures such as ionizing radiation, ultra-violet light and chemical mutagens. Unrepaired DNA damage leads to tumorigenesis. Rad51 is a multifunctional protein that plays a central role in DNA replication and homologous recombination repair. It is known that defects in Rad51 function can cause cancer. The goal of this study is to identify a novel role for Rad51 outside of its known functions in DSB repair and replication fork processing. Methods: Since Rad51 knockout is lethal to cells, we generated an inducible system in which we can down-regulate Rad51 expression in HT1080 cells after Doxycycline treatment. To determine the effect of Rad51-knockdown in global gene expression pattern, we carried out unbiased microarray gene expression analysis and after induction of DNA damage and replication stress by radiation. ssDNA and dsDNA in the cytosolic fractions were quantified using Quant-iT OliGreen and PicoGreen Assay Kits. For cytoplasmic BrdU detection, exponentially grown cells were labeled with BrdU for 18-20 h and then immunostained with anti-BrdU antibody. Additionally, we measured the expression and post-translational modification of proteins involved in innate immune signaling by western blotting. We also employed DNA fiber assay to determine the role of Rad51 in replication fork processing. Results: We found that defects in Rad51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. Mechanistically, the unprotected newly replicated genome in the absence of Rad51 is degraded by the exonuclease activity of Mre11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data revealed that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, Rad51 is also implicated in the suppression of innate immunity. Conclusion: Rad51 plays a novel role in immunity outside its known functions in DSB repair and replication fork processing. We discovered that the lack of Rad51 leads to the upregulation of innate immune response pathway genes upon DNA damage and replication induced by irradiation. We found that in the absence of Rad51 the newly replicated genome is degraded by the exonuclease activity of Mre11. We also showed that these degraded nascent DNA fragments are exported to the cytoplasm, triggering innate immune response signaling. Our study reveals a previously unidentified role for Rad51 in triggering an innate immune response, and places Rad51 at the hub of new interconnections between DNA replication, DNA repair, and immunity. Funding: This work was supported by NIH R01AG053341 grants. Citation Format: Kalayarasan Srinivasan, Souparno Bhattacharya, Salim Abdisalaam, Shibani Mukherjee, Asaithamby Aroumougame. Rad51 suppresses innate immune response by blocking MRE11-mediated degradation of newly replicated genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2017-2490
