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：
8938037
负责人：
Philipp Oberdoerffer
金额：
$ 48.88万
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/8938037
关键词：
Accounting Affect Aging BRCA1 gene Cell physiology Cells Chromatin Chromatin Structure DNA DNA Damage DNA Double Strand Break DNA Repair DNA biosynthesis Defect Deubiquitinating Enzyme Double Strand Break Repair Environment Enzymes Equilibrium Eukaryotic Cell Excision Family Foundations Functional disorder Future Gene Expression Regulation Genes Genome Genomic Instability Genomics Goals Histone H2A Histone H3 Histones Learning Light Link Lysine Maintenance Measures Mediating Mediator of activation protein Methylation Methyltransferase Modification Nature Nonhomologous DNA End Joining Normal Cell Nuclear Ontology Outcome PARP inhibition Pathway interactions Play Process Proteins RNA Interference Regulatory Pathway Reporter Reporting Resistance Role Site Stress System Tumor Suppressor Proteins Ubiquitin Ubiquitination Variant Work base cancer cell epigenomics high throughput screening histone modification homologous recombination in vivo insight mouse model novel overexpression promoter repaired restoration senescence tumor tumor initiation tumorigenesis

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. Specifically, Here, we identified two repressive chromatin components, the macro-histone variant macroH2A1 and the H3K9 methyltransferase and tumor suppressor PRDM2, which together modulate the choice between the antagonistic DSB repair mediators BRCA1 and 53BP1. The macroH2A1/PRDM2 module mediates an unexpected shift from accessible to condensed chromatin that requires the ATM-dependent accumulation of both proteins at DSBs to promote DSB-flanking H3K9-dimethylation. Remarkably, loss of macroH2A1 or PRDM2 as well as experimentally induced chromatin decondensation impairs BRCA1, but not 53BP1 retention at DSBs. As a result, macroH2A1 and/or PRDM2 depletion causes epistatic defects in DSB end resection, homology-directed repair and the resistance to PARP inhibition - all hallmarks of BRCA1-deficient tumors. Together, these findings identify dynamic, DSB-associated chromatin reorganization as a critical modulator of BRCA1-dependent genome maintenance. We are currently investigating the consequences of macroH2A1-associated repressive chromatin formation during replication stress, which involves HR-dependent genome maintenance pathways and has been linked to cellular dysfunction and, ultimately, senescence. Prompted by the potential of chromatin changes to selectively modulate DSB repair factor choice, we further 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 a USP-family deubiquitinating enzyme that was identified as a promoter of HR in our RNAi screen. We are currently investigating how loss or overexpression of this enzyme 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. Finally, we are analyzing the impact of USP loss on genome maintenance in vivo using a previpously established mouse model. 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与DNA断裂修复有关。具体而言，在这里，我们确定了两个抑制性染色质成分，即宏观 - 源变体MacRoH2A1和H3K9甲基转移酶和肿瘤抑制剂PRDM2，它们共同调节了拮抗DSB修复介质BRCA1和53BP1之间的选择。 MacRoH2A1/PRDM2模块介导了从可访问到凝结的染色质的意外转移，该染色质需要两种蛋白在DSBS上的ATM依赖性积累，以促进DSB-频率的H3K9-二甲基化。值得注意的是，MacRoH2A1或PRDM2的丢失以及实验诱导的染色质反应会损害BRCA1，但在DSBS处保留了53BP1。结果，MacRoH2A1和/或PRDM2的耗竭会导致DSB终端切除，同源指导的修复以及对PARP抑制的耐药性 - BRCA1缺陷型肿瘤的所有特征。这些发现一起，将动态的，与DSB相关的染色质重组是BRCA1依赖性基因组维持的关键调节剂。我们目前正在研究复制应力期间与MacRoH2A1相关的抑制性染色质形成的后果，该抑制蛋白涉及HR依赖性基因组维持途径，并与细胞功能障碍和最终衰老有关。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，我们进一步启动了一个新的目标，以研究其他DSB维修相关的染色质修饰剂在募集BRCA1和/或53BP1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的启动子的USP-家庭去泛素化酶。我们目前正在研究该酶的损失或过表达如何影响53BP1和BRCA1通过NHEJ募集到DSB的位点以及随之而来的DSB修复。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。最后，我们正在使用精确建立的小鼠模型分析USP损失对体内基因组维持的影响。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。