Mechanism of radial chromosome formation in human premature aging syndrome cells

人类早衰综合征细胞放射状染色体形成机制

• 批准号: 10592123
• 负责人: Anja-Katrin Bielinsky
• 金额: --
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2022-12-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10592123
• 关键词: Aging; Amaze; Appearance; Applications Grants; Belief; Bioinformatics; Biological Models; Bloom Syndrome; Bone marrow failure; Cell Aging; Cell Line; Cell physiology; Cells; Centromere; Cessation of life; Chromosomal translocation; Chromosomes; Cisplatin; Conscious; Cross-Linking Reagents; Cytogenetics; DNA Crosslinking; DNA Crosslinking Agent; DNA Damage; DNA Double Strand Break; DNA Repair; DNA crosslink; DNA ligase III; DNA-Directed DNA Polymerase; Data; Diagnostic; Diagnostic tests; Double Strand Break Repair; Exposure to; Fanconi Anemia pathway; Fanconi' s Anemia; Frequencies; Genes; Genetic Recombination; Genomic DNA; Goals; Grant; Human; Human Cell Line; Individual; Knowledge; Lasers; Literature; Mediating; Metaphase; Microdissection; Mission; Mitomycin C; Modeling; Molecular; Molecular Profiling; Nonhomologous DNA End Joining; Normal Cell; Outcome; Pathology; Pathway interactions; Patients; Phenotype; Polymerase; Premature aging syndrome; Process; Production; Protocols documentation; Radial; Radiation; Regulation; Reporting; Research Personnel; Role; System; Testing; Therapeutic; United States National Institutes of Health; bioinformatics pipeline; cancer predisposition; chromosome fusion; clinically relevant; crosslink; experimental study; gene synthesis; helicase; insight; nanopore; new therapeutic target; novel; repaired; response; telomere; ubiquitin-protein ligase; Aging; Amaze; Appearance; Applications Grants; Belief; Bioinformatics; Biological Models; Bloom Syndrome; Bone marrow failure; Cell Aging; Cell Line; Cell physiology; Cells; Centromere; Cessation of life; Chromosomal translocation; Chromosomes; Cisplatin; Conscious; Cross-Linking Reagents; Cytogenetics; DNA Crosslinking; DNA Crosslinking Agent; DNA Damage; DNA Double Strand Break; DNA Repair; DNA crosslink; DNA ligase III; DNA-Directed DNA Polymerase; Data; Diagnostic; Diagnostic tests; Double Strand Break Repair; Exposure to; Fanconi Anemia pathway; Fanconi' s Anemia; Frequencies; Genes; Genetic Recombination; Genomic DNA; Goals; Grant; Human; Human Cell Line; Individual; Knowledge; Lasers; Literature; Mediating; Metaphase; Microdissection; Mission; Mitomycin C; Modeling; Molecular; Molecular Profiling; Nonhomologous DNA End Joining; Normal Cell; Outcome; Pathology; Pathway interactions; Patients; Phenotype; Polymerase; Premature aging syndrome; Process; Production; Protocols documentation; Radial; Radiation; Regulation; Reporting; Research Personnel; Role; System; Testing; Therapeutic; United States National Institutes of Health; bioinformatics pipeline; cancer predisposition; chromosome fusion; clinically relevant; crosslink; experimental study; gene synthesis; helicase; insight; nanopore; new therapeutic target; novel; repaired; response; telomere; ubiquitin-protein ligase

PROJECT SUMMARY Radial chromosomes (hereafter “radials”) have been part of the scientific consciousness for more than 50 years. Radials are operationally defined as fusions involving two or more nonhomologous chromosomes and as having more than one centromere. In normal cells, radials are quite rare, and they almost never form spontaneously. Radials have an important clinical relevance, however, since their appearance is a diagnostic hallmark of the bone marrow failure, cancer predisposition and premature aging syndrome, Fanconi anemia (FA). Interestingly though, even in FA cells, radial chromosomes do not appear spontaneously with high frequency but usually need to be induced by exposure to clinically-relevant DNA crosslinking reagents, such as mitomycin C (MMC) and cisplatin. Despite their diagnostic use for many decades, radial formation is still poorly understood, and the literature is filled with conflicting reports about what is required mechanistically for their production. In our first Specific Aim, we describe the construction of a human cell line that is defective for the radiation sensitive 18 (RAD18) gene. RAD18 encodes an E3 ubiquitin ligase and is required for translesion synthesis (TLS), an error-prone DNA damage tolerance pathway, which is a subpathway in the FA-mediated DNA crosslink repair process. Consistent with this role, RAD18-null human cells generated high levels of MMC- induced radials. Unexpectedly, the absence of DNA ligase III (LIGIII) and DNA polymerase theta (POLQ; both key components of alternative non-homologous end joining; A-EJ)) dramatically reduced RAD18-dependent, MMC-induced radial formation. Thus, the radials generated by the absence of RAD18 appear to require the A- EJ pathway. We propose to confirm these observations and extend the generality of these studies by determining the requirement for A-EJ in MMC-induced radial formation in Bloom syndrome (BLM) (another cancer predisposition and premature aging syndrome) and FA-defective cells. Amazingly, there is not a single report in the literature regarding the molecular makeup of a radial chromosome fusion junction. This is unfortunate, since an analysis of fusion junctions for a wide variety of other types of translocations (e.g., canonical reciprocal chromosomal translocations and telomere fusions) has provide a wealth of data regarding the molecular signatures of such junctions as well as insight into the repair mechanisms responsible for their formation. In our Specific Aim 2, we propose to rectify this egregious omission. Specifically, we describe a protocol (Radial-Seq) for isolating, sequencing and bioinformatically analyzing radial chromosome fusions. We expect this analysis to confirm the formation of such junctions by A-EJ and to greatly extend our understanding of the molecular makeup of a radial fusion. In toto, these experiments should mechanistically define the requirements for radial formation which should benefit basic researchers; in addition, the assignment of A-EJ for their formation may help clinicians therapeutically hinder their formation.

项目摘要 径向染色体（以下是“ radials”）已成为50多个科学意识的一部分 年。径向在操作上被定义为涉及两个或多个非同源染色体的融合体，并且 作为一个以上的集中码。在正常细胞中，径向非常罕见，它们几乎永远不会形成 但是，径向具有重要的临床相关性，因为它们的外观是诊断 骨髓衰竭，癌症易感性和过早衰老综合症的标志 （FA）。有趣的是，即使在FA细胞中，径向染色体也不会在很高的情况下显得 频率，但通常需要通过暴露于临床上的DNA交联试剂，例如 丝裂霉素C（MMC）和顺铂。尽管它们诊断了数十年，但径向的形成仍然很差 理解，文献充满了有关其机械需要的矛盾的报告 生产。在我们的第一个特定目标中，我们描述了人类细胞系的结构，该细胞系有缺陷 辐射敏感18（RAD18）基因。 RAD18编码E3泛素连接酶，是translesion所必需的 合成（TLS），一种容易出错的DNA损伤耐受性途径，它是FA介导的子路口 DNA交联修复过程。与此作用一致，rad18-null人类细胞产生高水平的MMC- 诱导的径向。出乎意料的是，不存在DNA连接酶III（LIGIII）和DNA聚合酶theta（Polq;均为DNA聚合酶 替代非理论结束的关键组成部分； a-ej））大大降低了rad18依赖性， MMC诱导的径向形成。那就是没有Rad18产生的径向似乎需要A- EJ途径。我们建议确认这些观察结果，并通过 确定在MMC诱导的Bloom综合征（BLM）中对A-EJ的需求（另一个 癌症易感性和过早衰老综合征）和FA缺陷细胞。 令人惊讶的是，文献中没有关于径向的分子构成的报告 染色体融合结。这是不幸的，因为对其他各种各样的融合连接处的分析 易位的类型（例如，已提供了典型的相互染色体易位和端粒融合） 有关此类连接的分子特征的大量数据以及对维修的见解 负责其形成的机制。在我们的特定目标2中，我们建议纠正这种严重的遗漏。 特别是，我们描述了用于隔离，测序和生物信息分析径向的方案（radial-seq） 染色体融合。我们希望该分析能够确认A-ej的形成，并大大确认 扩展我们对径向融合的分子构成的理解。在Toto中，这些实验应该 机械学上定义了应该使基础研究人员受益的径向形成的要求；此外， A-ej成立的分配可以帮助临床医生治疗上阻碍他们的形成。