Project_2_AR2015 BACKGROUND: Genomic instability is a conserved hallmark of eukaryotic aging and increased DNA damage load has been shown to promote both cancer and age-related diseases. Moreover, epigenetic changes such as altered histone modification patterns appear sufficient to modulate life span in model organisms. We have previously uncovered a potential link between DNA damage and age-related epigenomic changes, suggesting that DNA damage can alter chromatin structure at DNA breaks and beyond. Specifically, the histone modifier SIRT1 is redistributed across chromatin in response to DNA damage, moving away from SIRT1-regulated promoters to sites of DNA damage. This reorganization may be critical for efficient DNA repair, but comes at the cost of deregulation of SIRT1 target genes, which mirrors aspects of age-associated transcriptional deregulation. This work led to the more general hypothesis that a DNA damage-induced reorganization of chromatin modifiers may underlie the alterations in gene expression and genomic stability that characterize eukaryotic aging. More recently we have identified a repressive chromatin module, consisting of the macro-histone variant macroH2A1 and the H3K9 methyltransferase PRDM2, as a modulator of DSB repair (see Project 1). Given that both proteins are involved in epigenome maintenance in the absence of DNA damage and have further been linked to malignant transformation and age-related chromatin reorganization, we speculate that their recruitment to DSBs may (transiently) affect the latter. In light of these findings, it will be of great interest to determine if DNA damage-induced epigenetic changes can induce age-related organ pathologies. OBJECTIVE AND RESULTS: To better understand how DNA damage and its repair affect the (epi-)genome over a lifetime, and how these changes impinge on tissue homeostasis, disease progression and mammalian aging, we generated a mouse model that allows for the conditional induction of DNA double-strand breaks (DSB-mice). In this model, the DSB-inducing homing endonuclease I-PpoI is under the control of both Cre-recombinase and a Tamoxifen-activated nuclear translocation domain (ERT2), and optimal DSB induction requires both Cre and Tamoxifen. DSB-mice will be analyzed for (i) epigenetic changes following DSB induction both at DSB containing loci and at ostensibly undamaged regions and (ii) DNA damage-associated changes in tissue function using histology and biochemical approaches. To follow the consequences of DSB induction in a tractable cell type, we generated DSB-mice under the control of the T cell specific lck-Cre transgene. Hematopoietic cells have been well studied in the context of DNA damage and concomitant functional decline and can be readily isolated and manipulated. Cells expressing the ERT2-I-PpoI fusion protein were identified based on co-expression of a GFP reporter gene and we have confirmed that up to 95% of T cells are GFP-, and thus I-PpoI-positive. Tamoxifen treatment resulted in DSB induction at all tested I-PpoI target sites and promoted a cellular response to DSBs comparable to that observed in mice exposed to 3 Gy of ionizing radiation. DSB-bearing T cells were isolated by cell sorting of GFP-positive cells for downstream analyses. We have further established in vitro culture conditions for ex vivo isolated I-Ppo-expressing T cells. Using this model, we were able to dissect the impact of DSB induction on DSB-associated genes in primary cells. Specifically, we found that DSBs promote a transient, DNA damage signaling-dependent repression of break-associated genes both in vivo and in vitro. Importantly, we observed no evidence for persistent changes in the expression of these genes, consistent with an efficient restoration of DSB-induced gene deregulation following DSB repair. Together, these findings reveal an unexpected capacity of primary cells to maintain transcriptome integrity in response to DSBs, and, thus, point to alimited role for DNA damage as a mediator of epigenetic dysfunction often associated with aging and disease. In depth analyses of possible indirect epigenetic defects at non-DSB associated genomic loci as well as a dissection of physiological consequences of DSB induction for T cell differentiation and function are ongoing. IMPLICATIONS: The potential of DNA damage to affect cell function both through direct DNA alterations and through indirect, epigenetic changes in chromatin structure puts it at a critical position to influence eukaryotic aging and disease progression. Our recent data suggest that mammalian cells have evolved a surprising capacity to maintain transcriptome integrity and ,thus, balance the potentially harmful epigenetic impact of DSB repair. Nevertheless, a global DNA damage-induced reorganization of chromatin remains a possibility, particularly in the presence of persisting DSBs, which may explain epigenetic changes observed with age and/or during malignant transformation. A disturbance of nuclear integrity has been tightly linked to aging, cancer and degenerative diseases, and our work is expected to shed light on the molecular drivers of DNA repair associated chromatin reorganization. Together, this work may thus improve our understanding of the functional interplay between DNA damage, age-related (epi)genomic reorganization and tissue homeostasis with implications for cancer development as well as therapeutic intervention.