Investigating the role of DNA breaks in mammalian aging

研究 DNA 断裂在哺乳动物衰老中的作用

• 批准号: 8553080
• 负责人: Philipp Oberdoerffer
• 金额: $ 10.08万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553080
• 关键词: Address; Affect; Age; Aging; Animal Model; Apoptosis; Biochemical; Brain; Brain Pathology; Cell Aging; Cell Lineage; Cell physiology; Cells; ChIP-seq; Chromatin; Chromatin Structure; Chromosomal Rearrangement; Chronic; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; Disease; Disease Progression; Endonuclease I; Epigenetic Process; Functional disorder; Gene Expression; Gene Mutation; Gene Targeting; Genome; Genome Stability; Genomic Instability; Genomics; Gliosis; Hematopoietic System; Hippocampus (Brain); Histology; Histones; Homeostasis; Homing; In Vitro; Intervention; Investigation; Learning; Light; Link; Location; Longevity; Malignant Neoplasms; Mammals; Mature Lymphocyte; Mediating; Mediator of activation protein; Memory; Modeling; Mus; Mutation; Nuclear; Nuclear Translocation; Organ; Pathology; Pattern; Physiological; Positioning Attribute; RNA; Role; Site; System; Tamoxifen; Technology; Testing; Time; Tissues; Transgenes; Transgenic Organisms; Work; age related; aging brain; base; behavior test; cerebral atrophy; chromatin immunoprecipitation; chromatin remodeling; cost; design; endonuclease; epigenomics; genome-wide; histone modification; improved; in vivo; interest; irradiation; mammalian genome; mouse model; prevent; programs; promoter; recombinase; repaired; response; theories

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 redistribution of chromatin modifiers (or 'RCM response') may underlie the alterations in gene expression and genomic stability that characterize eukaryotic aging. In light of these findings, it will be of great interest to determine if DNA damage-induced epigenetic changes can indeed 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, 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. We further generated DSB-mice under the control of a ubiquitous Tamoxifen-inducible Cre, which allows us to investigate the impact of DSBs on a variety of cell lineages, particularly the irradiation sensitive hematopoetic system. Using these mice, we will further be able to isolate mature lymphocytes and induce DSBs in vitro to study (epi)genomic changes in primary cells. Given the dual role of macroH2A 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 beyond a single transgenic break site and will simultaneously address global chromatin reorganization in response to DNA damage. The latter further allows for a direct comparison with known, age-related epigenomic changes.Regarding the physiological consequences of DSB induction, we will initially focus on brain-specific DSB induction and perform a detailed histopathological analysis assessing hallmarks of brain aging such as brain atrophy, apoptosis and gliosis. We will focus on cortex and hippocampus, which show efficient transgene activation and serve as central mediators of many aspects of learning and memory. Brain sections will be analyzed at different time points after (chronic) 4OHT administration. Should we detect signs of DSB-induced brain pathology, we will determine possible changes in learning and memory formation using a variety of behavioral tests, which are established in our program.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. A better understanding of how DNA breaks and their repair impinge on tissue homeostasis, cancer and aging is, therefore, critical for the design of targeted intervention, which may be able to prevent or improve many of the negative aspects of DNA break induced chromatin reorganization.

背景：基因组不稳定性是真核老化的保守标志，DNA损伤负荷已显示可促进癌症和与年龄有关的疾病。此外，表观遗传学变化（例如改变组蛋白修饰模式）似乎足以调节模型生物的寿命。以前，我们已经发现了DNA损伤与年龄相关的表观基因组变化之间的潜在联系，这表明DNA损伤可以改变DNA断裂及以后的染色质结构。具体而言，将组蛋白修饰剂SIRT1响应于DNA损伤的染色质重新分布，从SIRT1调节的启动子转移到DNA损伤部位。这种重组对于有效的DNA修复可能至关重要，但以SIRT1靶基因放松管制为代价，这反映了与年龄相关的转录放松管制的各个方面。这项工作导致了更一般的假设，即DNA损伤引起的染色质修饰剂的重新分布（或“ RCM响应”）可能是表征真核老化的基因表达和基因组稳定性的改变。 In light of these findings, it will be of great interest to determine if DNA damage-induced epigenetic changes can indeed 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 - 小鼠）。在此模型中，诱导DSB的归核核酸内切酶I-PPOI在CRE-成年酶和他莫昔芬激活的核转运结构域的控制之下，最佳DSB诱导需要CRE和Tamoxifen。 DSB-mice will be analyzed for (i) overall chromatin (re)organization following DSB induction using ChIP-seq technology and (ii) DN​​A 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.我们进一步在无处不在的他莫昔芬诱导的CRE的控制下产生了DSB - 小鼠，这使我们能够研究DSB对各种细胞谱系的影响，尤其是辐射敏感的造血系统。使用这些小鼠，我们将能够进一步分离成熟的淋巴细胞并在体外诱导DSBS研究（EPI）基因组变化。鉴于在DSB修复和细胞衰老过程中，MacRoH2A和相关杂色特征的双重作用（请参阅项目1），遵循这些标记的基因组分布以响应Vivo中的DSB诱导而特别感兴趣。该分析将确定抑制性染色质在DNA修复中的相关性，而不是单个转基因断裂位点，并将同时解决全局染色质重组，以响应DNA损伤。后者进一步允许与已知的，与年龄相关的表观基因组变化进行直接比较。为了确定DSB诱导的生理后果，我们最初将专注于脑特异性DSB诱导，并进行详细的组织病理学分析，评估脑老化的标志，例如脑萎缩，凋亡，凋亡和胶质病。我们将专注于皮层和海马，它们显示出有效的转基因激活，并作为学习和记忆许多方面的中心介体。 （慢性）4OHT给药后，将在不同时间点分析脑部切片。如果我们检测到DSB诱导的脑病理学的迹象，我们将使用各种行为测试来确定学习和记忆形成的可能变化，这些行为测试是在我们的程序中确定的。插入：DNA损伤通过直接DNA的影响通过直接DNA改变的细胞功能的潜力，以及通过染色质结构中的间接，表观遗传变化使其在其上的关键位置，从而影响Euk euk euk euk eucaritotity agarytotity agareation agareation agareation agareation agary of eucaritotic ohary of eucaritotity agary at e eucaritotic agareation agryation agryation agryation。因此，更好地了解DNA破裂及其修复对组织稳态，癌症和衰老的影响对于靶向干预的设计至关重要，这可能能够预防或改善DNA断裂诱导的染色质重组的许多负面方面。