RNAi screen for chromatin modifiers in DNA repair and aging

RNAi 筛选 DNA 修复和衰老中的染色质修饰剂

• 批准号: 8349443
• 负责人: Philipp Oberdoerffer
• 金额: $ 45.88万
• 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/8349443
• 关键词: Accounting; Address; Affect; Age; Aging; Biological Assay; Cell physiology; Cells; Chromatin; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; Data; Deacetylase; Degenerative Disorder; Double Strand Break Repair; Environment; Enzymes; Epigenetic Process; Fluorescence; Foundations; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Genomics; Heterochromatin; Histone H3; Histones; Imaging Techniques; In Situ Hybridization; Light; Link; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Mediating; Mediator of activation protein; Metastatic Skin Cancer; Methylation; Methyltransferase; Modeling; Molecular; Molecular Biology; Mutation; Nuclear; Ontology; Phosphorylation; Process; Proteins; RNA Interference; RNA Polymerase II; Reporting; Role; Screening procedure; Signal Transduction; Site; System; Testing; Therapeutic Intervention; Tissues; Transcription Repressor/Corepressor; Tumor Promoters; Validation; Variant; Work; age related; base; chromatin remodeling; epigenomics; genome-wide; histone methyltransferase; improved; interest; knock-down; novel; repaired; response; restoration; senescence; theories; tumor progression

BACKGROUND AND OBJECTIVE: We found previously that the SIRT1 deacetylase provides a critical link between DNA repair, aging and genomic instability. Specifically, we found that DNA damage can cause the redistribution of SIRT1 on chromatin, resulting in its recruitment to DNA breaks and concomitant deregulation of otherwise SIRT1 regulated genomic loci. We were further able to demonstrate the direct involvement of SIRT1 in DNA break repair and propose SIRT1 redistribution as a molecular mechanism linking DNA damage to large-scale epigenetic deregulation, even of undamaged loci. This work further led to the more general hypothesis that an evolutionarily conserved, DNA damage-induced redistribution of chromatin modifiers (or 'RCM response') may underlie the alterations in gene expression and genomic stability that characterize eukaryotic aging. Elucidating the impact of the DNA damage response on chromatin organization is, thus, vital to improve our understanding of often detrimental, age-related (epi-)genomic changes and the associated organismal decline, and may eventually help to promote genomic stability and maintain a more youthful epigenetic profile. RESULTS AND FUTURE DIRECTIONS: To test if DNA damage can indeed result in a genome-wide remodeling of chromatin beyond the redistribution of SIRT1, we used an RNA interference based screening approach to identify additional DNA-repair linked chromatin modifiers from a comprehensive list of Gene Ontology annotated chromatin remodelers. Our preliminary results suggested that a large number of chromatin-modifying enzymes are involved in DNA break repair. Interestingly, many of these proteins are transcriptional repressors and associated with the formation of silent chromatin. While it has been postulated that chromatin surrounding DNA breaks is made accessible for efficient repair factor access, our data suggest large-scale chromatin compaction at sites of DNA damage, possibly a mechanism to help confine the site of damage and to aid the repair process. We are currently investigating the hypothesis that DNA breaks can induce break-associated chromatin compaction using a range of molecular biology and cell-based imaging techniques. Initial fluorescence-based in situ hybridization (FISH) analyses support our model and provide the basis for an in depth analysis of the role of DNA-repair related chromatin modifiers during this process. We have further established several independent cell-based systems to induce defined DNA breaks in mammalian cells, thereby enabling us to investigate direct association of said chromatin modifiers with DNA breaks. Using these systems, we validated the recruitment of two repressive histone variants that were amongst the top 5 hits in our screen, to sites of DSBs. This recruitment is downstream of early DNA damage signaling through H2AX phosphorylation and correlates with the acquisition of the repressive histone H3 K9 methyl-mark at the DSB site. Importantly, our screen identified an H3K9 methyltransferase with no known function in DNA break repair. Using RNAi based knock-down, we are currently investigating if this enzyme is responsible for H3K9 methylation of DSB proximal histones. We are further interested in understanding the role of both the histone variants and the H3K9 histone methyltransferase with regard to chromatin reorganization at DSBs and how these proteins may affect the proposed chromatin compaction. Together, these data point to a novel DNA repair module that may critically modulate the chromatin microenvironment surrounding a DSB. To study the latter we are currently performing genome wide circular chromatin configuration capture assays (4C), characterizing the three-dimensional chromatin microenvironment around at the DSB site before and after break induction. In combination with FISH validation, this approach will further allow us to address the impact of the DNA repair relevant repressive chromatin components on break-proximal structural changes. Consistent with far-reaching DSB-induced chromatin remodeling, we found that genes within up to 4 Mb from the break site can be transiently repressed upon induction of a single DSB. This finding implies that previously reported break-proximal interference with RNA polymerase II transcription can occur over megabase regions of the genome, highlighting the far-reaching impact of DSB-mediated chromatin remodeling. IMPLICATIONS: The implications of chromatin modifiers as critical mediators of DNA repair are two-fold: (1) Only a small number of chromatin modifiers has been conclusively linked to the DNA repair process. A comprehensive analysis will significantly improve our understanding of mammalian DNA repair and its impact on genomic instability and cancer. (2) DNA damage induced reorganization of chromatin may explain epigenetic changes observed with age and/or during malignant transformation. A global disturbance of nuclear integrity has been tightly linked to aging, cancer and degenerative diseases and this project is expected to shed light on the contribution of DNA repair associated chromatin reorganization to these processes. Interestingly, the histone variants identified in our screen are associated with heterochromatin alterations in senescent cells and have further been shown to protect from skin metastasis through epigenetic silencing of the tumor promoter CDK8. Together, these observations corroborate the link between DNA damage, age-related (epi)genomic reorganization and ultimatley cancer progression. Unlike mutations in DNA, epigenetic changes are, at least in theory, reversible and may prove to serve as a target for directed therapeutic intervention, aiming at the restoration of tissue-appropriate gene regulation.

背景和客观：我们以前发现SIRT1脱乙酰基酶在DNA修复，衰老和基因组不稳定性之间提供了关键的联系。具体而言，我们发现DNA损伤会导致SIRT1在染色质上的重新分布，从而导致其募集到DNA断裂，并同时放松SIRT1调节的基因组基因局基因局基因局基因局基因局。我们进一步证明了SIRT1在DNA断裂修复中的直接参与，并提出SIRT1将DNA损伤与大规模表观遗传消失的分子机制，甚至是未损坏的基因座连接起来。这项工作进一步导致了更一般的假设，即在进化保守的，DNA损伤引起的染色质修饰剂（或“ RCM响应”）的重新分布可能是基因表达和基因组稳定性的改变，这表征了真核老化的基因组稳定性。因此，阐明DNA损伤反应对染色质组织的影响对于提高我们对经常有害的，与年龄相关的（Epi-）基因组变化和相关的有机体下降的理解至关重要，并且最终可能有助于促进基因组稳定性并保持更年轻的表观遗传学特征。结果和未来的方向：为了测试DNA损伤是否确实可以导致染色质的全基因组重塑超出SIRT1的重新分布，我们使用了基于RNA干扰的筛选方法来识别来自基因学分解陈述蛋白重塑的全面列表的其他DNA固定蛋白修饰符。我们的初步结果表明，大量染色质修饰酶参与DNA断裂修复。有趣的是，这些蛋白质中的许多是转录阻遏物，并且与静音染色质的形成有关。虽然已经假设，使DNA断裂周围的染色质可用于有效的维修因子进入，但我们的数据表明，在DNA损伤部位的大规模染色质压实，可能是一种机制，可以帮助局限损伤部位并帮助修复过程。我们目前正在研究以下假设：DNA断裂可以使用一系列分子生物学和基于细胞的成像技术诱导断裂相关的染色质压实。基于初始荧光的原位杂交（FISH）分析支持我们的模型，并为在此过程中对DNA-REPAIR相关的染色质修饰剂的作用进行深入分析。我们进一步建立了几种基于细胞的独立系统，以诱导哺乳动物细胞中定义的DNA断裂，从而使我们能够研究所述染色质修饰剂与DNA断裂的直接关联。使用这些系统，我们验证了两个抑制性组蛋白变体的募集，这些变体是我们屏幕中前5个命中量之一，即DSB的位置。该募集是通过H2AX磷酸化的早期DNA损伤信号的下游，与DSB部位的抑制性组蛋白H3 K9甲基标记的获取相关。重要的是，我们的屏幕确定了H3K9甲基转移酶，在DNA断裂修复中没有已知功能。使用基于RNAi的敲除，我们目前正在研究该酶是否负责DSB近端组蛋白的H3K9甲基化。我们对了解组蛋白变异和H3K9组蛋白甲基转移酶在DSBS的染色质重组以及这些蛋白质如何影响拟议的染色质压实方面的作用进一步感兴趣。这些数据共同指出了一个新型的DNA修复模块，该模块可能会严重调节DSB周围的染色质微环境。为了研究后者，我们目前正在进行基因组宽圆形染色质构型捕获测定（4C），以在诱导前后的DSB位点表征了三维染色质微环境。结合鱼类的验证，这种方法将进一步使我们能够解决DNA修复相关的抑制性染色质成分对断裂 - 预抗结构变化的影响。与深远的DSB诱导的染色质重塑一致，我们发现在诱导单个DSB时，可以暂时抑制从断裂位点内最多4 MB的基因。这一发现表明，先前报道的对RNA聚合酶II转录的断裂 - 差异可能会发生在基因组的巨大区域上，这突出了DSB介导的染色质重塑的深远影响。含义：染色质修饰剂作为DNA修复的关键介质的含义是两个方面：（1）只有少量的染色质修饰剂与DNA修复过程最终链接。全面的分析将显着提高我们对哺乳动物DNA修复及其对基因组不稳定性和癌症的影响。 （2）DNA损伤诱导的染色质的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。核完整性的全球干扰与衰老，癌症和退化性疾病紧密相关，预计该项目将揭示与这些过程相关的染色质重组的DNA修复的贡献。有趣的是，在我们筛选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，并已通过表观遗传沉默的肿瘤启动子CDK8的表观遗传沉默可保护免受皮肤转移的影响。总之，这些观察结果证实了DNA损伤，与年龄相关的（EPI）基因组重组和最后玛特利癌的进展之间的联系。与DNA中的突变不同，至少在理论上，表观遗传学变化是可逆的，可以证明是定向治疗干预的靶标，旨在恢复适合组织的基因调节。