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: 8763432
• 负责人: Philipp Oberdoerffer
• 金额: $ 26.55万
• 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/8763432
• 关键词: Address; Affect; Age; Aging; Animal Model; Apoptotic; Biochemical; Biological Assay; Cell Aging; Cell Count; Cell Death; Cell Lineage; Cell Separation; Cell physiology; Cells; ChIP-seq; Chimeric Proteins; Chromatin; Chromatin Structure; Chromosomal Rearrangement; Chronic; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; Degenerative Disorder; Development; Disease; Disease Progression; Distal; Endonuclease I; Epigenetic Process; Functional disorder; Gene Expression; Gene Mutation; Gene Targeting; Genome; Genome Stability; Genomic Instability; Genomics; Hematopoietic; Hematopoietic System; Heterochromatin; Histology; Histones; Homeostasis; Homing; In Vitro; Investigation; Light; Link; Location; Longevity; Maintenance; Malignant - descriptor; Malignant Neoplasms; Mammals; Mediating; Methyltransferase; Modeling; Molecular; Mus; Mutation; Neoplasm Metastasis; Nuclear; Nuclear Translocation; Organ; Pathology; Pattern; Physiological; Positioning Attribute; Process; Proteins; Reporter Genes; Role; Site; System; T-Lymphocyte; Tamoxifen; Technology; Testing; Therapeutic Intervention; Tissues; Transgenes; Tumor Promoters; Variant; Work; age related; base; chromatin immunoprecipitation; chromatin remodeling; cost; endonuclease; epigenome; epigenomics; functional decline; genome-wide; genome-wide analysis; histone modification; improved; in vivo; interest; mammalian genome; mouse model; promoter; recombinase; repaired; response; senescence; theories; transcriptome sequencing

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. More recently we have identified a repressive chromatin module, consisting of the macro-histone variant macroH2A1 and an H3K9 methyltransferase, 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 both 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 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 (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. We are currently analyzing I-PpoI mediated DSB induction in T cells using the T cell specific Cre driver. 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. Following Tamoxifen treatment and concomitant DSB induction, we observed phosphorlation of H2AX as well as a moderate increase in apoptotic cells, suggesting DSB induction in the absence of excessive cell death. Together, these observations show that we can induce I-PpoI-mediated DSBs in vivo, resulting in a significant fraction of DSB-bearing T cells that can be 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. To distinguish between break-distal and break-proximal epigenomic changes, we will identify DSB-flanking chromatin both in cis and in trans using genome-wide circular chromatin configuration capture assays (4C). This analysis is further expected to reveal the impact of DSBs on three-dimensional chromatin integrity. The identification of DSB-induced epigenomic remodeling will provide a molecular basis for DSB-associated transcriptional silencing and concomitant epigenetic deregulation beyond sites of damage with implications for age-related epigenomic changes. 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.

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。最近，我们确定了一个抑制性染色质模块，该模块由宏观历史变体MacroH2A1和H3K9甲基转移酶组成，是DSB修复的调节剂（请参阅项目1）。鉴于两种蛋白在没有DNA损伤的情况下都参与了表观基因组的维持，并且与恶性转化和与年龄有关的染色质重组既有联系，因此我们推测它们对DSB的募集可能会（暂时）影响后者。鉴于这些发现，确定DNA损伤引起的表观遗传变化是否确实会引起与年龄相关的器官病理，这将引起极大的兴趣。目的和结果：为了更好地了解DNA损伤及其修复如何影响（Epi-）基因组在一生中，以及这些变化如何影响组织稳态，疾病进展和哺乳动物衰老，我们产生了一种小鼠模型，允许有条件地诱导DNA双链断裂（DSB-MITE）。在此模型中，诱导DSB的归核核酸内切酶I-PPOI在Cre-recombinase和他莫昔芬激活的核转运结构域（ERT2）的控制之下，并且最佳DSB诱导需要CRE和Tamoxifen。使用芯片seq技术诱导DSB诱导后，将对（i）总体染色质（RE）组织分析DSB - 小鼠，并使用组织学和生化方法进行组织功能的DNA损伤相关变化。目前，在组织特异性诱导I-PPOI核酸内切酶后，我们正在研究和不存在他莫昔芬的诱导和积累。为此，我们在造血系统特异性和T细胞特异性CRE转基因的控制下产生了DSB - 小鼠，这使我们能够分别研究DSB在所有造血细胞和T谱系细胞中的影响。在DNA损伤和伴随功能下降的背景下，造血系统已经进行了很好的研究。造血细胞进一步代表了一个可以容易隔离和操纵的可进行的实验系统。我们目前正在使用T细胞特异性CRE驱动器分析I-PPOI介导的T细胞中DSB诱导。可以根据GFP报告基因的共表达来鉴定表达ERT2-I-PPOI融合蛋白的细胞，我们已经确认80-90％的胸腺T细胞和T细胞前体是GFP，因此I-PPPOI阳性。他莫昔芬治疗和伴随DSB诱导后，我们观察到H2AX的磷酸化以及凋亡细胞的中等增加，表明在没有过多细胞死亡的情况下，DSB诱导。总之，这些观察结果表明，我们可以在体内诱导I-PPOI介导的DSB，从而导致大量的含DSB的T细胞，可以通过对GFP阳性细胞的细胞分选进行下游分析来分离，以进行下游分析。我们进一步在建立离体分离的表达I-PPO的T细胞的体外培养条件下，以获得分析全基因组（EPI）基因组变化所需的细胞数量。鉴于在DSB修复和细胞衰老过程中MacroH2A1和相关的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于Vivo的DSB诱导而特别感兴趣。该分析将确定抑制性染色质在整个基因组中的DNA修复中的相关性，并将同时解决响应DNA损伤的全局染色质重组。为了区分突破性和断裂 - 抗晶格的表观基因组变化，我们将使用全基因组圆形染色质构型捕获测定法（4C）识别CIS和TRENS中的DSB频率染色质。进一步期望该分析揭示DSB对三维染色质完整性的影响。 DSB诱导的表观基因组重塑的鉴定将为DSB相关的转录沉默和随之而来的表观遗传学放松管制提供分子基础，超出了损伤部位，对年龄相关的表观基因组变化的影响。含义：DNA损伤通过直接DNA改变以及通过间接的染色质结构的表观遗传变化造成影响细胞功能的潜力，使其处于影响真核老化和疾病进程的关键位置。全球DNA损伤诱导的染色质重组可能解释了随着年龄和/或恶性转化期间观察到的表观遗传变化。核完整性的干扰与衰老，癌症和退化性疾病紧密相关，并且我们的工作有望揭示DNA修复的分子驱动因素相关的染色质重组。有趣的是，项目1中鉴定出的组蛋白变体与衰老细胞中的异染色质改变有关，并进一步证明可以通过肿瘤启动子CDK8的表观遗传沉默来防止转移。因此，这项工作可能会提高我们对DNA损伤，与年龄相关（EPI）基因组重组和组织稳态之间的功能相互作用的理解，对癌症发展以及治疗性干预的影响。