Investigating the link between DNA damage, chromatin and nuclear dysfunction

研究 DNA 损伤、染色质和核功能障碍之间的联系

• 批准号: 9556510
• 负责人: Philipp Oberdoerffer
• 金额: $ 80.69万
• 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/9556510
• 关键词: Acute; Acute Erythroblastic Leukemia; Adverse effects; Age; Agreement; Alternative Splicing; BRCA1 gene; Cell Aging; Cell Cycle Arrest; Cell physiology; Cells; Chromatin; Chromosome Fragile Sites; Chromosome abnormality; Chronic; Collaborations; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA replication fork; Data; Deposition; Dimensions; Disease; Environment; Epigenetic Process; Exhibits; Fibroblasts; Functional disorder; Gamma-H2AX; Genes; Genome; Genomics; Heart; Histones; Human; Hybrids; Investigation; K-562; Knockout Mice; Light; Link; Maintenance; Malignant - descriptor; Manuscripts; Mediator of activation protein; Molecular; Molecular Chaperones; Nuclear; Nucleic Acids; Oncogene Deregulation; Oncogenes; Pathway interactions; Pattern; Phosphorylation; Physiological; Play; Population; Predisposition; Process; RNA; RNA Splicing; Recruitment Activity; Reporting; Research; Resolution; Role; Signal Transduction; Site; Stress; Structure; Telomere Maintenance; Testing; Tissues; Variant; Work; age related; base; cell growth; chromatin immunoprecipitation; deep sequencing; homologous recombination; improved; in vivo; mouse model; novel; prevent; programs; repaired; response; senescence; tool; transcriptome

We have recently identified the repressive histone variant macroH2A1.2 as a critical modulator of BRCA1-dependent genome maintenance during DSB repair via homologous recombination (HR) (Khurana et al., Cell Reports, 2014). Given that both HR and BRCA1 function are required for the efficient resolution of stalled and/or collapsed replication forks, we asked if macroH2A1.2 may act to modulate this process, invoking chromatin as a paradigm for the manipulation of the cellular response to replication stress. Using chromatin immunoprecipitation combined with deep sequencing (ChIP-Seq) in K562 erythroleukemia cells, in which both fragile sites an DNA replication patterns have been extensively characterized, we were able to show that macroH2A1.2 localizes to sites of replication stress-induced DNA damage. Notably, macroH2A1.2 peak coverage was most prominent at common fragile sites and was further positively correlated with CFS susceptibility to DNA breaks. Consistent with an active role during replication stress, we observed a fragile-site specific increase in macroH2A1.2 beyond its basal level of enrichment, which required DNA damage signaling and concomitant H2AX phosphorylation to coordinate FACT histone chaperone-dependent deposition of macroH2A1.2. Other H2A variants, including macroH2A1.1 and H2A.Z, remained unaltered, indicating that macroH2A.1.2 plays a unique role in the cellular response to replication stress. Demonstrating functional significance of this observation, we identified a protective role for macroH2A1.2 during replication stress, preventing excessive DNA damage accumulation at fragile DNA. Consistent with our finding that macroH2A1.2 promotes BRCA1-dependent genome maintenance at DSBs, we observed a significant reduction in BRCA1 localization at nascent replication forks using iPOND. Moreover, and in agreement with our ChIP results at fragile sites, replication stress resulted in a pronounced increase in gamma-H2AX at stalled forks in macroH2A1.2-depleted cells. Together, these findings demonstrate macroH2A1.2-dependent recruitment and/or stabilization of BRCA1 at replication forks, which is in turn protects from replication stress. While beneficial for genome maintenance in the short term, replication stress-induced chromatin reorganization may be a driver of progressive epigenetic dysfunction, particularly in the context of replicative age. To test this possibility, we used human primary fibroblasts, which can be cultured for a finite number of population doublings (PDs) and exhibit profound, yet poorly understood chromatin changes in late passage cells. We observed a robust, replication-dependent increase in macroH2A1.2 at seven of eight tested CFSs, that correlated with both age and replication stress-associated gene deregulation. Importantly, the same fragile sites were also subject to macroH2A1.2 and gamma-H2AX accumulation in response to replication stress. Together this work establishes macroH2A1.2 as a bona fide epigenetic modulator of replication stress with implications for age-associated epigenetic change. A manuscript describing this work currently in revision. Notably, replication stress and the resulting DNA damage response (DDR) are important drivers of cellular senescence, thus presenting an important barrier to malignant transformation. Consistent with a role for macroH2A1.2 in this process, our preliminary data indicate that loss of macroH2A1.2 can cause a near complete cell cycle arrest in primary fibroblasts, followed by hallmarks of cellular senescence. The molecular basis for this finding is under investigation. Of note, ongoing work directed at the identification of novel mediators of macroH2A1.2 function at stalled replication forks implicated macroH2A1.2 in the resolution of RNA/DNA hybrids (R-loops), which will be a focus of future research. Finally, macroH2A1.2 represents one of two alternative splice variants of the macroH2A1 (H2AFY) gene, which, based on our preliminary data, have seemingly opposing roles during DNA repair and cell growth, and the elucidation of splice variant-specific macroH2A1 functions is an important aspect of our current research program. As an orthogonal approach to study the impact of DNA damage on epigenetic integrity, we have generated a mouse model that allows for inducible, tissue-specific DSB formation at 140 defined genomic loci. Using this tool, we recently uncovered an unexpected capacity of primary cells to maintain transcriptome integrity in response to acute DSB exposure (Kim et al., Nucleic Acids Research, 2016). However, the epigenetic consequences of chronic DSB induction remain unclear. Moreover, little is known about the impact of DSBs on chromatin beyond the site of damage. The latter is particularly relevant in light of the highly structured mammalian chromatin environment, which may potentiate the adverse effects of DNA damage. Here, we will dissect DNA damage-induced epigenetic dysfunction in three-dimensional nuclear space, using of a mouse model that allows for chronic, spatially controlled DSB induction. Together, this work is expected to have significant implications for our understanding of the epigenetic pathways that control genome maintenance and malignant transformation.

我们最近将抑制性组蛋白变体MacRoH2A1.2确定为通过同源重组（HR）在DSB修复过程中BRCA1依赖性基因组维持的关键调节剂（HR）（Khurana等，Cell Reports，2014）。鉴于HR和BRCA1函数都需要有效地解决失速和/或折叠的复制叉，因此我们询问MacRoH2A1.2是否可以调节此过程，并将染色质作为对复制应力的细胞反应操纵的范例。使用染色质免疫沉淀与K562红血病细胞中的深层测序（CHIP-SEQ）结合在一起，其中两个脆弱的位点和DNA复制模式都得到了广泛的特征，我们能够表明MacroH2A1.2在复制应力诱导DNA损伤的位置局部。值得注意的是，MacroH2A1.2峰值覆盖率在常见的脆弱部位最突出，并且与CFS对DNA断裂的敏感性进一步相关。与在复制应力中的积极作用一致，我们观察到麦克罗2A1.2的脆弱点特异性增加超出了其基础富集水平，这需要DNA损伤信号传导和伴随的H2AX磷酸化，以协调事实的事实型组酮伴侣依赖性麦克罗H2A1.2。其他H2A变体（包括MacRoH2A1.1和H2A.Z）仍然没有改变，表明Macroh2a.1.2在细胞对复制应力的反应中起着独特的作用。证明了这一观察结果的功能意义，我们确定了在复制应力期间MacroH2A1.2的保护作用，从而防止了DNA脆弱的DNA的过度DNA损伤积累。与我们发现MacroH2A1.2在DSBS促进BRCA1依赖性基因组维持的发现一致，我们观察到使用IPOND在新生的复制叉处的BRCA1定位显着降低。此外，与我们在脆弱部位的芯片结果一致，复制应力导致在MacroH2A1.2耗尽细胞中停滞的叉子处的γ-H2AX明显增加。这些发现共同证明了MacroH2A1.2依赖性募集和/或在复制叉时BRCA1的稳定，这反过来又可以保护复制应力。尽管在短期内对基因组维持有益，但复制应力诱导的染色质重组可能是进行性表观遗传功能障碍的驱动力，尤其是在复制年龄的情况下。为了测试这种可能性，我们使用了人类的原代成纤维细胞，可以为有限数量的人口加倍（PD）培养，并在晚期细胞中表现出深刻但知之甚少的染色质变化。我们观察到在八个测试CFS中的七个中，MacRoH2A1.2的强大，复制依赖性增加与年龄和复制应力相关基因失控相关。重要的是，同一脆弱的位点还要响应复制应力而受到MacroH2A1.2和γ-H2AX的积累。这项工作共同建立了MacroH2A1.2作为复制应力的真正表观遗传调节剂，对年龄相关的表观遗传变化有影响。目前正在修订中描述这项工作的手稿。值得注意的是，复制应力和所得的DNA损伤反应（DDR）是细胞衰老的重要驱动因素，因此具有恶性转化的重要障碍。与MacRoH2A1.2在此过程中的作用一致，我们的初步数据表明，MacroH2A1.2的丧失会导致原代成纤维细胞中几乎完全完整的细胞周期停滞，然后是细胞衰老的标志。这一发现的分子基础正在研究中。值得注意的是，正在进行的工作是针对识别MacroH2A1.2新型介质功能在停滞的复制叉中的函数，该功能与MacroH2A1.2有关RNA/DNA混合体（R-Loops）的分辨率（R-Loops）涉及的MacroH2A1.2，这将是未来研究的重点。最后，MacRoH2A1.2代表了MacRoH2A1（H2AFY）基因的两个替代剪接变体之一，基于我们的初步数据，在DNA修复和细胞生长过程中似乎具有相反的作用，并且阐明了剪接特异性MacroH2A1功能是我们当前研究计划的重要方面。作为研究DNA损伤对表观遗传完整性的影响的正交方法，我们生成了一种小鼠模型，该模型允许在140定义的基因组基因座时诱导，组织特异性的DSB形成。使用此工具，我们最近发现了原代细胞的意外能力，以响应急性DSB暴露而维持转录组完整性（Kim等人，核酸研究，2016年）。然而，慢性DSB诱导的表观遗传后果尚不清楚。此外，对于DSB对损伤部位以外的染色质的影响知之甚少。鉴于高度结构化的哺乳动物染色质环境，后者特别相关，这可能会增强DNA损伤的不良反应。在这里，我们将使用小鼠模型在三维核空间中剖析DNA损伤诱导的表观遗传功能障碍，该模型允许慢性，空间控制的DSB诱导。预计这项工作将对我们对控制基因组维持和恶性转化的表观遗传途径的理解产生重大影响。