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 redistribution 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) overall chromatin (re)organization following DSB induction using ChIP-seq technology and (ii) DNA damage-associated changes in tissue function using histology and biochemical approaches. We are presently investigating DSB induction and accumulation in the presence and absence of Tamoxifen following tissue-specific induction of the I-PpoI endonuclease. To do so, we generated DSB-mice under the control of both a hematopoietic system-specific and a T cell specific Cre transgene, which allows us to investigate the impact of DSBs in all hematopoietic cells and T lineage cells, respectively. The hematopoietic system has been well studied in the context of DNA damage and concomitant functional decline. Hematopoietic cells further represent a tractable, experimental system that can be readily isolated and manipulated. Cells expressing the ERT2-I-PpoI fusion protein can be identified based on co-expression of a GFP reporter gene and we have confirmed that 80-90% of thymic T cells and T cell precursors are GFP, and thus I-PpoI-positive. Tamoxifen treatment results in DSB induction at I-PpoI target sites and promotes a cellular response to DSBs comparable to that observed in mice exposed to 3 Gy of ionizing radiation. DSB-bearing T cells are isolated by cell sorting of GFP-positive cells for downstream analyses. We are further in the process of establishing in vitro culture conditions for ex vivo isolated I-Ppo-expressing T cells to obtain cell numbers required for the analysis of genome-wide (epi)genomic changes in primary cells. Given the dual role of macroH2A1 and related heterochromatic features during DSB repair and cellular senescence (see Project 1), it will be of particular interest to follow the genomic distribution of these marks in response to DSB induction in vivo. This analysis will determine the relevance of repressive chromatin in DNA repair across the genome and will simultaneously address global chromatin reorganization in response to DNA damage. This analysis will be complemented by gene expression profiling in the presence or absence of DSBs. The identification of DSB-specific epigenetic changes will provide a molecular basis for DSB-associated nuclear dysfunction at and beyond sites of damage with implications for aging and malignant transformation. 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. Global DNA damage induced reorganization of chromatin 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. Interestingly, the histone variants identified in Project 1 are associated with heterochromatin alterations in senescent cells and have further been shown to protect from metastasis through epigenetic silencing of the tumor promoter CDK8. 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.
