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

基本信息

批准号：
8763431
负责人：
Philipp Oberdoerffer
金额：
$ 61.96万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/8763431
关键词：
Accounting Affect Aging Agreement BRCA1 gene Binding Cell physiology Cells Chromatin Chromatin Structure DNA DNA Damage DNA Double Strand Break DNA Repair Data Defect Deubiquitinating Enzyme Double Strand Break Repair Environment Enzymes Equilibrium Eukaryotic Cell Excision Family Foundations Future Gene Expression Regulation Genes Genome Genomic Instability Genomics Goals Histone H2A Histone H3 Histones Learning Light Link Lysine Measures Mediating Mediator of activation protein Methylation Methyltransferase Modification Molecular Nature Nonhomologous DNA End Joining Normal Cell Nuclear Ontology Outcome Pathway interactions Peptides Phosphorylation Physical condensation Play Poly(ADP-ribose) Polymerases Process Proteins RNA Interference Regulatory Pathway Relaxation Reporter Reporting Role SS DNA BP Signal Transduction Site System Tail Transcription Repressor/Corepressor Tumor Suppressor Proteins Ubiquitin Ubiquitination Variant Work base cancer cell epigenomics high throughput screening histone modification homologous recombination insight knock-down neoplastic cell novel overexpression promoter repaired response restoration tumor initiation tumorigenesis

Accounting Affect Aging Agreement BRCA1 gene Binding Cell physiology Cells Chromatin Chromatin Structure DNA DNA Damage DNA Double Strand Break DNA Repair Data Defect Deubiquitinating Enzyme Double Strand Break Repair Environment Enzymes Equilibrium Eukaryotic Cell Excision Family Foundations Future Gene Expression Regulation Genes Genome Genomic Instability Genomics Goals Histone H2A Histone H3 Histones Learning Light Link Lysine Measures Mediating Mediator of activation protein Methylation Methyltransferase Modification Molecular Nature Nonhomologous DNA End Joining Normal Cell Nuclear Ontology Outcome Pathway interactions Peptides Phosphorylation Physical condensation Play Poly(ADP-ribose) Polymerases Process Proteins RNA Interference Regulatory Pathway Relaxation Reporter Reporting Role SS DNA BP Signal Transduction Site System Tail Transcription Repressor/Corepressor Tumor Suppressor Proteins Ubiquitin Ubiquitination Variant Work base cancer cell epigenomics high throughput screening histone modification homologous recombination insight knock-down neoplastic cell novel overexpression promoter repaired response restoration tumor initiation tumorigenesis

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项目摘要

BACKGROUND AND OBJECTIVE: DNA damage-induced chromatin reorganization is emerging to be a key aspect of eukaryotic DNA repair. Much has been learned about the role of histone modifications as landing pads for repair effectors, thereby modulating or directing their recruitment to sites of damage. Recent work suggests that structural changes in the break-surrounding chromatin environment may be equally important in directing the repair process, with implications ranging from repair factor accessibility to break-proximal transcriptional silencing. In light of this complexity, we decided to take an unbiased approach to dissect the role of chromatin in DNA repair, identify its most critical components and explore their functional relevance. RESULTS AND FUTURE DIRECTIONS: To gain insight into the role of chromatin in DSB repair, we performed RNAi-based high-throughput screening of a comprehensive list of 400 Gene Ontology-annotated chromatin modifiers. Repair efficiency was determined using the previously established, U2OS cell-based DR-GFP reporter system, in which DNA repair by homologous recombination (HR) results in restoration of a functional GFP gene and HR efficiency can, thus, be measured as the fraction of GFP+ cells. We found a large number of chromatin-modifying enzymes to be involved in DNA break repair, and in contrast to previous reports suggesting DSB-induced chromatin relaxation, many of these proteins were transcriptional repressors and/or associated with the formation of silent chromatin. Specifically, we identified a DNA repair module consisting of two macro-histone variants and the histone 3 lysine 9 methyltransferase (H3K9MT) PRDM2, which has not previously been implicated in DNA break repair. Both macroH2A1 and PRDM2 promote a biphasic change in the DSB-proximal chromatin microenvironment. In agreement with DSB-induced chromatin decondensation reported previously, we observe initial depletion of macroH2A along with repressive chromatin marks from the break site, and initiation of the DNA damage response is not affected by either PRDM2 or the macro-histone variants. Following this initial expansion, we detect DNA damage signaling-dependent, DSB-proximal enrichment of macro-histone variants as well as the silent chromatin mark H3-dimethyl-K9 and PRDM2, which in turn promotes chromatin condensation. We show that macroH2A mediates PRDM2 recruitment to breaks and are currently investigating the molecular basis for macroH2A1 incorporation at DSB sites as well as its relevance for PRDM2 recruitment. Importantly, macroH2A or PRDM2 depletion causes a significant reduction in H3K9 dimethylation and concomitant break-associated chromatin reorganization. Consistent with a role for macroH2A and PRDM2 during HR, loss of either protein significantly reduced DSB recruitment of the HR mediator and tumor suppressor BRCA1 but not the NHEJ-associated repair protein 53BP1. Moreover, both macroH2A1 and PRDM2 promote the phosphorylation of the single-stranded DNA binding protein RPA, which is a critical step in HR-associated end resection and repair. Moreover, we observed increased sensitivity to inhibition of poly(ADP-ribose) polymerase following knock-down of macroH2A or PRDM2, which is a hallmark of BRCA1-deficient tumor cells. Our data thus implicate the formation of repressive chromatin as a modulator of repair outcome, which is expected to have significant consequences for genomic integrity, specifically in the context of defective BRCA1 function. Further nderlining the functional relevance of structural changes in DSB-associated chromatin, we found that experimentally induced chromatin relaxation via the inhibition of histone deacetylases results in a selective decrease in BRCA1 recruitment following DSB induction. Consistent with this observation, BRCA1 preferably binds to H3K9me2 modified histone tails as opposed to H3K9 acetylated peptides, and future work is directed at identifying the mechanistic basis for this phenomenon. Prompted by the potential of chromatin changes to selectively modulate DSB repair factor choice, we initiated a new aim to investigate the role of other DSB repair-associated chromatin modifiers in the recruitment of BRCA1 and/or 53BP1. Based on recent reports describing ubiquitination of histone H2A as modification that is critical for 53BP1 recruitment to DSBs, we focused on two USP-family deubiquitinating enzymes that were identified as HR promoters in our RNAi screen. We are currently investigating how loss of these enzymes affects the recruitment of 53BP1 and BRCA1 to sites of DSBs and concomitant DSB repair via NHEJ. We further plan to determine possible consequences of USP loss or overexpression on H2A ubiquitination. Together, this aim is expected to shed light on a novel aspect of ubiquitin-dependent DSB repair factor recruitment with the potential to distinguish between 53BP1 and BRCA1-mediated DSB repair pathways. IMPLICATIONS: Defects in BRCA1 function have been linked to tumor initiation and genomic instability. More recently, it has been suggested that a key role for BRCA1 may be to prohibit aberrant 53BP1 recruitment to sites of DNA damage, which accounts for many of the detrimental genomic effects of BRCA1 loss. Determining the factors that control the balance between BRCA1 and 53BP1 at DNA breaks is, thus, critical for our understanding of DSB repair in general and BRCA1-associated tumorigenesis in particular. Both 53BP1 and BRCA1 occupy expansive, DSB-surrounding subnuclear domains, and the identification of selective, chromatin-based modulators of BRCA1/53BP1 recruitment to sites of damage, therefore, has implications for the targeted manipulation of repair outcome and possibly tumor initiation.

背景和目标：DNA损伤诱导的染色质重组已成为真核DNA修复的关键方面。关于组蛋白修饰作为维修效应子的着陆垫的作用，已经了解了很多了解，从而调节或指导其招募到损害部位。最近的工作表明，断裂染色质环境的结构变化在指导修复过程中同样重要，其影响范围从修复因子的可及性到断裂 - 差异转录沉默。鉴于这种复杂性，我们决定采用一种公正的方法来剖析染色质在DNA修复中的作用，确定其最关键的成分并探索其功能相关性。结果和未来方向：为了深入了解染色质在DSB修复中的作用，我们对400个基因本体学宣布的染色质修饰剂的综合列表进行了基于RNAi的高通量筛选。使用先前确定的基于U2OS细胞的DR-GFP报告基因系统确定修复效率，其中通过同源重组（HR）修复DNA可导致功能性GFP基因和HR效率的恢复，因此可以作为GFP+细胞的分数来测量。我们发现大量染色质修饰酶参与了DNA断裂修复，并且与先前的报道相反，与表明DSB诱导的染色质松弛相比，其中许多蛋白质是转录抑制剂和/或与静音染色质的形成有关。具体而言，我们鉴定了一个由两个宏观历史变体和组蛋白3赖氨酸9甲基转移酶（H3K9MT）PRDM2组成的DNA修复模块，该模块以前尚未与DNA断裂修复有关。 MacRoH2A1和PRDM2均促进了DSB-proximal染色质微环境的双相变化。与先前报道的DSB诱导的染色质脱敏性一致，我们观察到MacRoH2A的初始耗竭以及抑制性染色质标记中断位点的抑制性染色质标记，而DNA损伤响应的启动不受PRDM2或宏观增强剂的影响。在这一初始扩展之后，我们检测到DNA损伤信号依赖性，DSB-Proximal富集宏观 - 内酮变体以及静音染色质H3-二甲基-K9和PRDM2，这又促进了染色质凝结。我们表明，MacRoH2A介导PRDM2募集到中断，目前正在研究DSB站点上MacRoH2A1掺入的分子基础，以及其与PRDM2募集的相关性。重要的是，MacRoH2A或PRDM2耗尽会导致H3K9二甲基化和随之而来的断裂相关染色质重组的显着降低。与HR期间的MacRoH2A和PRDM2的作用一致，蛋白质的损失显着降低了HR介质和肿瘤抑制剂BRCA1的DSB募集，而不是NHEJ相关的维修蛋白53BP1。此外，MacRoH2A1和PRDM2都促进了单链DNA结合蛋白RPA的磷酸化，这是与HR相关的终端切除和修复的关键步骤。此外，我们观察到对抑制MacRoH2A或PRDM2的抑制（ADP-核糖）聚合酶的敏感性增加，这是BRCA1缺陷型肿瘤细胞的标志。因此，我们的数据暗示了抑制性染色质作为修复结果的调节剂的形成，这预计将对基因组完整性产生重大影响，特别是在有缺陷的BRCA1功能的背景下。进一步了解与DSB相关染色质中结构变化的功能相关性，我们发现通过抑制组蛋白脱乙酰基酶可以实验诱导的染色质松弛导致DSB诱导后BRCA1募集的选择性降低。与该观察结果一致，BRCA1优选地与H3K9ME2修饰的组蛋白尾巴结合，而不是H3K9乙酰化的肽，并且未来的工作旨在确定这种现象的机械基础。在染色质变化以选择性调节DSB修复因子选择的潜力的推动下，我们启动了一个新的目标，以研究其他DSB修复相关的染色质修饰剂在BRCA1和/或53BP1募集中的作用。基于最新的报道，将组蛋白H2A的泛素化为53BP1募集到DSB至关重要的修饰，我们专注于在RNAi筛查中被鉴定为HR启动子的两个USP-元家庭去泛素化酶。我们目前正在研究这些酶的损失如何影响53BP1和BRCA1通过NHEJ募集到DSB和伴随DSB修复的位置。我们进一步计划确定USP损失或过表达对H2A泛素化的可能后果。总之，这一目标有望阐明泛素依赖性DSB修复因子募集的新方面，具有区分53BP1和BRCA1介导的DSB修复途径的潜力。含义：BRCA1功能的缺陷已与肿瘤起始和基因组不稳定性有关。最近，有人提出，BRCA1的关键作用可能是禁止53BP1募集到DNA损伤部位，这构成了BRCA1损失的许多有害基因组效应。因此，确定控制DNA断裂时BRCA1和53BP1之间平衡的因素对于我们对DSB修复的理解至关重要，尤其是与BRCA1相关的肿瘤发生。 53BP1和BRCA1均占据了膨胀的DSB刺激下核域，以及鉴定基于染色体的选择性，基于染色质的调节剂，招募了损害部位，对靶向修复结果的目标操纵和可能的肿瘤启动具有含义。