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

基本信息

批准号：
10014589
负责人：
Philipp Oberdoerffer
金额：
$ 38.62万
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/10014589
关键词：
ATRX gene Acute Acute Erythroblastic Leukemia Age Alternative Splicing Anaphase Aneuploidy Animals BRCA1 gene Biology Cell Aging Cell Cycle Arrest Cell physiology Cells Chromatin Chromosomal Stability Chromosome Fragile Sites Chromosome abnormality Chronic Collaborations DNA DNA Damage DNA Repair DNA biosynthesis DNA replication fork Data Defect Deposition Dimensions Disease Environment Epigenetic Process Exhibits Female Fibroblasts Functional disorder Genes Genome Genomic Instability Growth Health Heart Histones K-562 Knockout Mice Link Maintenance Malignant - descriptor Malignant Neoplasms Mediator of activation protein Molecular Molecular Biology Molecular Chaperones Mus National Institute on Aging Nature Nuclear Nucleic Acids Nucleosomes Oncogenes Pathway interactions Pattern Phosphorylation Physiological Predisposition Process Proteomics Publishing RNA Splicing Reporting Research Resolution Role Signal Transduction Site Source Stem cells Telomere Maintenance Telomere Pathway Tumor Cell Line Tumor Suppressor Proteins Variant Work X Inactivation age related base cell growth chromatin immunoprecipitation comparative deep sequencing design epigenome genome integrity genomic aberrations homologous recombination improved in vivo mouse model new therapeutic target novel prevent programs repaired replication stress response senescence structural biology telomere tumor

项目摘要

We have 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). In this project, we aim at a detailed characterization of this novel epigenetic effector of genome maintenance in health and malignancy. Given that both HR and BRCA1 function are required for the efficient resolution of stalled and/or collapsed replication forks, we focused on macroH2A1.2 function in 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 and DNA replication patterns have been extensively characterized, we found that macroH2A1.2 preferentially 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. MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from replication stress-induced DNA damage. The concomitant fragile site-associated chromatin reorganization is a driver of progressive epigenetic change, particularly in the context of replicative age. Specifically, we observed a robust, replication-dependent increase in macroH2A1.2 at CFSs in primary fibroblasts upon extended culture. Consistent with the notion that replication stress and the resulting DNA damage response (DDR) are important drivers of cellular senescence in primary cells, which in turn counteracts malignant transformation, we show that loss of macroH2A1.2 can cause a DDR-dependent, near complete cell cycle arrest in primary fibroblasts associated hallmarks of cellular senescence. Together this work establishes macroH2A1.2 as a bona fide epigenetic modulator of replication stress with implications for age-associated epigenetic change, genome integrity and malignant transformation. These findings have recently been published (Kim et al., Mol Cell 2018). A role for macroH2A1.2 during malignancy appears particularly likely in the context of tumors that rely on alternative lengthening of telomeres (ALT) for their growth. ALT is a homology-directed telomere maintenance pathway, which is tightly linked to increased telomeric replication stress as a source for the underlying DSBs. ALT telomeres furthermore exhibit a unique chromatin environment and generally lack the nucleosome remodeler ATRX, which was previously found to modulate macroH2A1.2 chromatin occupancy. As part of their unique chromatin composition, we found ALT telomeres to be highly enriched for macroH2A1.2, consistent with their inherent susceptibility to replication stress. However, in contrast to ATRX-proficient cells, ALT telomeres transiently lose macroH2A1.2 during acute fork stalling to facilitate DSB formation, a process that is almost completely prevented by ectopic ATRX expression. Telomeric macroH2A1.2 is re-deposited in a DNA damage response-dependent manner to promote HR-associated ALT pathways. Our findings thus identify the dynamic exchange of macroH2A1.2 on chromatin as an epigenetic link between ATRX loss, ALT initiation via replication stress and ALT execution via HR. MacroH2A1.2 may, thereby, provide a novel therapeutic target in ALT-dependent cancers. This work was performed in collaboration with Dr. Yie Liu at the National Institute on Aging and has been published this year (Kim et al., Nature Structural and Molecular Biology, 2019). Of importance for our understanding of macro-histone biology, macroH2A1.2 represents one of two alternative and mutually exclusive splice variants of the macroH2A1-endoding H2AFY gene. Based on data from us and others, these two variants, macroH2A1.1 and macroH2A1.2, have seemingly opposing roles during DNA repair and cell growth, and the elucidation of splice variant-specific macroH2A1 functions is, thus, an important aspect of our ongoing research program. As part of these efforts, we have generated a macroH2A1.2 variant-specific knockout mouse. Our initial analyses have uncovered an unexpected role for macroH2A1.2 in chromosome stability specifically at the inactive X chromosome (Xi). The Xi is inherently susceptible to genomic aberrations. Replication stress has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that the replication stress-protective macroH2A1.2 histone variant, which is enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics revealed macroH2A1.1-specific association with alternative end-joining (alt-EJ) mediators that promote anaphase defects in the absence of macroH2A1.2. Depletion of macroH2A1.1 or alt-EJ factors reverses genomic instability upon macroH2A1.2 loss, and mice deficient for both variants harbor no overt female defects. Together, our findings uncover splicing-regulated genome maintenance as a long sought-after rationale for macroH2A1 accumulation on the Xi, with implications for aneuploidy-associated malignancies. This work is currently being submitted. Altogether, this project is designed to uncover the contribution of macro-histones to genome and epigenome integrity both in tumor cell lines and in animals. Given our published and ongoing research, we anticipate that these histone variants will emerge as critical and potentially druggable modulators of genome integrity, particularly in replicating cells, with consequences for both (stem) cell growth and malignant transformation.

我们已经通过同源重组（HR）（HR）确定了抑制性组蛋白变体MacRoH2A1.2是DSB修复过程中BRCA1依赖性基因组维持的关键调节剂（Khurana等，Cell Reports，2014年）。在这个项目中，我们旨在详细描述健康和恶性肿瘤中基因组维持的新表观遗传效应子。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，因此我们在此过程中专注于MacRoH2A1.2功能，将染色质作为操纵复制应力的细胞反应的范例。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，其中脆弱的位点和DNA复制模式都得到了广泛的特征，我们发现MacroH2A1.2优先地将其定位于复制应力诱导DNA损伤的位点。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。 MacRoH2A1.2反过来促进了复制叉时肿瘤抑制剂和HR效应子BRCA1的积累，以防止复制应力诱导的DNA损伤。伴随脆弱的位点相关染色质重组是进行性表观遗传变化的驱动力，特别是在复制年龄的背景下。具体而言，我们观察到在延伸培养的原代成纤维细胞中，在CFSS的Macroh2a1.2的强大，复制依赖性增加。与主细胞中细胞衰老的重要驱动因素（DDR）的重要驱动因素与原代细胞中的重要驱动因素相一致，这反过来反过来抵消了恶性转化，我们表明，麦克罗2a1.2的丧失会导致DDR依赖性，几乎依赖于细胞周期的细胞周期在原发性成纤维细胞中相关的细胞呈现的单个细胞周期停滞。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，基因组完整性和恶性转化有影响。这些发现最近已发表（Kim等，Mol Cell 2018）。在恶性肿瘤中，MacRoH2A1.2的作用在依赖端粒替代延长（ALT）生长的肿瘤的背景下似乎特别有可能。 ALT是一种以同源性为导向的端粒维护途径，它与增加的端粒复制应力紧密相关，作为基础DSB的来源。 Alt端粒还表现出独特的染色质环境，并且通常缺乏核小体重塑ATRX，以前发现它可以调节MacroH2A1.2染色质占用率。作为其独特的染色质组成的一部分，我们发现Alt端粒对MacroH2A1.2高度丰富，这与它们对复制应力的固有敏感性一致。但是，与ATTRX的细胞相比，Alt端粒在急性叉停滞期间瞬时失去了MacroH2A1.2，以促进DSB形成，这一过程几乎完全被异位ATRX表达完全阻止。以DNA损伤响应依赖性方式重新沉积了端粒型MacroH2A1.2，以促进与HR相关的ALT途径。因此，我们的发现确定了染色质上的MacroH2A1.2的动态交换是ATRX损失，通过复制应力和ALT执行通过HR之间的表观遗传联系。 MacroH2A1.2 5月，因此，在Alt依赖性癌症中提供了一种新型的治疗靶标。这项工作是与美国国家衰老研究所的Yie Liu博士合作进行的，并于今年发表（Kim等，《自然结构和分子生物学》，2019年）。对于我们对宏观生物学的理解至关重要，MacRoH2A1.2代表了MacroH2A1端子的H2AFY基因的两个替代性和相互排斥的剪接变体之一。根据我们和其他人的数据，这两个变体MacRoH2A1.1和MacRoH2A1.2在DNA修复和细胞生长过程中似乎具有相反的作用，并且阐明了我们正在进行的研究计划的剪接变体特异性MacroH2A1函数。作为这些努力的一部分，我们生成了MacroH2A1.2特定于变异的敲除鼠标。我们的初步分析发现了MacroH2A1.2在染色体稳定性中的意外作用，特别是在非活性X染色体（XI）上。 XI固有地容易受到基因组畸变的影响。已经提出了复制应力作为根本原因，但是保护XI不稳定性的机制仍然未知。在这里，我们表明，Xi完整性和女性生存所必需的复制应力保护型MacroH2A1.2组蛋白变体是富含XI的。从机械上讲，MacroH2A1.2抵消了其同样富含Xi的替代剪接变体MacroH2A1.1。比较蛋白质组学揭示了MacroH2A1.1特异性与替代性最终连接（ALT-EJ）介质的关联，这些介体在没有MacRoH2A1.2的情况下促进了后期缺陷。 MacRoH2A1.1或Alt-EJ因子的耗竭会逆转MacRoH2A1.2损失的基因组不稳定性，而两种变体缺乏的小鼠都没有明显的女性缺陷。我们的发现共同发现了剪接调节的基因组维持，这是对XI上的MacroH2A1积累的长期追求的基本原理，对非整倍性相关的恶性肿瘤有影响。目前正在提交这项工作。总的来说，该项目旨在发现肿瘤细胞系和动物中宏观调子对基因组和表观基因组完整性的贡献。鉴于我们发表和正在进行的研究，我们预计这些组蛋白变异将成为基因组完整性的关键且潜在的可药物调节剂，尤其是在复制细胞方面，对（STEM）细胞生长和恶性转化都会产生影响。