Functional crosstalk between the Fanconi Anemia and ATRX/DAXX histone chaperone pathways

范可尼贫血和 ATRX/DAXX 组蛋白伴侣通路之间的功能串扰

• 批准号: 10392385
• 负责人: Jeongsik Yong
• 金额: $ 32.23万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392385
• 关键词: ATRX gene; Aphidicolin; BRCA2 gene; Cell Line; Cells; Chemotherapy-Oncologic Procedure; Chromatin; Chromosome abnormality; Complex; DAXX gene; DNA; DNA Double Strand Break; DNA Interstrand Crosslinking; DNA Repair; DNA Repair Gene; DNA Replication Inhibition; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Death Domain; Defect; Disease; Double Strand Break Repair; Excision; Exhibits; FANCD2 protein; Fanconi Anemia pathway; Fanconi anemia protein; Fanconi' s Anemia; Gene Mutation; Genes; Genetic Models for Cancer; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Glioblastoma; Heterodimerization; Histone H3; Histones; Human; Hypersensitivity; Knock-out; Laboratories; Link; Maintenance; Malignant Neoplasms; Mediating; Modeling; Molecular; Molecular Chaperones; Mutation; NBS1 gene; Names; Neuroblastoma; Nucleosomes; Pathway interactions; Patients; Play; Predisposition; Proteins; Rad51 recombinase; Recovery; Role; S phase; Solid Neoplasm; Structure; Syndrome; Telomere Maintenance; Testing; Variant; alpha-Thalassemia; base; cancer type; chromatin remodeling; crosslink; high risk; homologous recombination; hydroxyurea; insight; leukemia; malignant breast neoplasm; novel; nuclease; protein complex; recombinational repair; recruit; repaired; tool

PROJECT SUMMARY The Fanconi Anemia (FA) pathway functions to maintain genomic stability and is crucial for the repair of DNA interstrand crosslinks (ICLs). During ICL repair, the central FANCD2 protein promotes the recruitment of downstream factors such as BRCA2[FANCD1] and RAD51[FANCR] that are crucial for homologous recombination (HR) repair of DNA double stranded breaks (DSBs), including those generated during ICL removal. Previous studies from our laboratory and others showed that FANCD2 has additional roles in promoting the HR-dependent recovery of hydroxyurea (HU) or aphidicolin (APH) stalled replication forks. Intriguingly, we recently identified a new constitutive FANCD2 interactor named ATRX (Alpha Thalassemia Retardation Syndrome X-linked). ATRX, in complex with DAXX (Death Domain-Associated Protein 6), acts as a chromatin remodeler and histone H3.3 chaperone that regulates chromatin compaction. While ATRX was originally not considered to be involved in mechanisms of genome maintenance, recent studies showed that acquired ATRX gene defects are strongly associated with a subset of cancers that exhibit excessive genome instability. However, the underlying mechanisms of ATRX-dependent genome stability are not known. To elucidate a potential functional cross-talk between the ATRX and FANCD2 pathways, we generated isogenic human knockout cell lines lacking ATRX, FANCD2, or both. We found that ATRX and FANCD2 form a constitutive protein complex with the MRE11-RAD50-NBS1 (MRN) nuclease. This complex acts as a functional unit to promote HR-dependent replication fork recovery and the repair of directly inducible DNA DSBs. Simultaneously, and paradoxically, we also observed that ATRX and FANCD2 gene defects had a synergistic effect on cellular ICL sensitivities. Based on our observations, we hypothesize that ATRX cooperates with FANCD2 and MRN to promote HR-mediated repair mechanisms, but also possesses additional activities that contribute to DNA ICL removal in the absence of a functional FA pathway. To test our hypothesis, we propose three Specific Aims: 1) Elucidate the molecular and structural makeup of the ATRX-MRN-FANCD2 protein complex 2) Determine molecular mechanisms of ATRX/FANCD2-mediated replication fork recovery. 3) Dissect FANCD2-dependent and -independent roles of ATRX during DNA ICL repair.

项目总结 Fanconi贫血(FA)途径起着维持基因组稳定性的作用，对DNA的修复至关重要 链间交联剂(ICL)。在ICL修复过程中，中央FANCD2蛋白促进 BRCA2[FANCD1]和RAD51[FANCR]等下游因子对同源基因 DNA双链断裂(DSB)的重组(HR)修复，包括ICL过程中产生的DSB 移走。我们实验室和其他实验室之前的研究表明，FANCD2在 促进停滞的羟基脲(HU)或磷脂(APH)复制叉的HR依赖性恢复。 有趣的是，我们最近发现了一种新的结构性FANCD2相互作用因子，名为ATRX(阿尔法地中海贫血 X连锁迟缓综合征)。ATRX在与DAXX(死亡结构域相关蛋白6)的复合体中发挥作用 染色质重构体和组蛋白H3.3伴侣调节染色质压缩。而ATRX是 最初不被认为与基因组维持机制有关，最近的研究表明 获得性ATRX基因缺陷与一组表现出过量基因组的癌症密切相关 不稳定。然而，依赖ATRX的基因组稳定性的潜在机制尚不清楚。 为了阐明ATRX和FANCD2通路之间潜在的功能串扰，我们产生了 缺乏ATRX和/或FANCD2的等基因人类基因敲除细胞系。我们发现ATRX和FANCD2形成了一个 Mre11-Rad50-NBS1(MRN)核酸酶的构成蛋白复合体。这个复合体起到了功能性的作用 促进HR依赖的复制分叉恢复和直接诱导的DNA DSB的修复的单位。 同时，矛盾的是，我们还观察到ATRX和FANCD2基因缺陷具有协同作用 对细胞ICL敏感性的影响。根据我们的观察，我们假设ATRX与 FANCD2和MRN促进HR介导的修复机制，但也具有额外的活性， 在缺乏功能正常的FA途径的情况下，有助于DNA ICL的去除。 为了验证我们的假设，我们提出了三个具体目标： 1)阐明ATRX-MRN-FANCD2蛋白复合体的分子和结构组成 2)确定ATRX/FANCD2介导的复制分叉恢复的分子机制。 3)剖析ATRX在DNA ICL修复中的FANCD2依赖和非依赖作用。