The role of EWSR1 at the centromere

EWSR1在着丝粒中的作用

• 批准号: 10659762
• 负责人: KATSUMI KITAGAWA
• 金额: $ 31万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659762
• 关键词: Auxins; Binding; Biochemical; Cell Cycle; Cell Cycle Stage; Cell division; Cells; Centromere; Chimeric Proteins; Chromatids; Chromatin; Chromosomal Instability; Chromosome Segregation; Chromosomes; Congenital Abnormality; DNA; DNA Sequence; DNA biosynthesis; Data; Defect; Deposition; Development; Disease; Down Syndrome; EWSR1 gene; Engineering; Ensure; Ewings sarcoma; FLI1 gene; Functional disorder; G1 Phase; Goals; Health; Histone H3; Human; Hybrids; Impairment; Interphase; Kinetochores; Maintenance; Malignant Neoplasms; Mammals; Microtubules; Mitosis; Modeling; Molecular Chaperones; Mutation; Nucleosomes; Oncogenic; Phase; Phenotype; Play; Proteins; RNA; RNA Binding; RNA Recognition Motif; Role; SETX gene; Signal Transduction; Sister Chromatid; Specific qualifier value; Specificity; Structure; System; TAF15 gene; Testing; Variant; centromere protein A; daughter cell; developmental disease; insight; mutant; prion-like; protein complex; segregation; transmission process

The centromere is a unique chromosomal region that is essential for high-fidelity chromosome transmission during cell division. In mitosis, microtubules attached to the kinetochore help ensure the faithful segregation of sister chromatids into daughter cells. Our goal is to understand the mechanisms that govern centromere function. Centromere identity relies on the deposition of the centromere-specific histone H3 variant, CENP-A but not on specific DNA sequences. After DNA replication, “old” centromeric nucleosomes are transferred onto the newly replicated chromatids. In mammals, deposition of CENP-A occurs in the G1 phase of the cell cycle. The mechanisms of CENP-A deposition and maintenance are crucial for proper centromere inheritance and function but remain to be delineated. We recently found that EWSR1 (Ewing Sarcoma Breakpoint Region 1) is required for CENP-A deposition and maintenance at the centromere. We show that EWSR1 and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma) bind to CENP-A through the SYGQ2 region within its prion-like domain known to be important for phase separation. We found that EWSR1 is required for CENP-A deposition at the centromere. CENP-A is associated with centromeric RNA, which is important for centromere function. We demonstrate that EWSR1 plays a key role in promoting this association. We also have biochemical evidence that EWSR1 binds R-loops, a three-stranded RNA-DNA hybrid, through its RNA-recognition motif. Based on these findings, we hypothesize that EWSR1 plays crucial roles in maintaining centromere structure and function by binding RNA and anchoring CENP-A in centromeric chromatin. We will test this hypothesis. Aim 1: To determine the function of EWSR1 and RNA/R-loops in centromere maintenance. We generated EWSR1 conditional KO cells using an AID (auxin-inducible degron) system, which allows us to remove EWSR1 upon auxin addition. We will test if auxin-induced degradation of EWSR1 abolishes CENP-A signals at the centromere at different cell cycle stages. We will induce the expression of SETX, which clears RNA-DNA hybrids, at different cell cycle stages to ask whether centromeric RNA/R-loops are required for CENP-A maintenance at the centromere. Aim 2: To investigate the function of EWSR1-CENP-A interaction. We will generate EWSR1 mutants defective in binding to CENP-A by introducing mutations into the SYGQ2 region of EWSR1. We will examine the phenotypes of the EWSR1 KO cells expressing CENP-A binding deficient mutants. We will also identify the EWSR1-binding domain of CENP-A. We will then isolate CENP-A mutants impaired for EWSR1 binding. We will express Flag-tagged mutants in CENP-A KO cells to examine the localization of CENP-A and the phenotypes of the mutant cells. We will investigate if EWSR1 association with CENP-A is unique for interphase. We will investigate the mechanism that regulates cell cycle specificity of EWSR1-CENP-A interaction. Aim 3: To test if EWSR1 anchors CENP-A at the centromere via RNA binding. To test this model, we will directly examine this model by constructing and testing CENP-A and EWSR1 variants with engineered functional domains.

着丝粒是一个独特的染色体区域，对于高保真的染色体传递是必不可少的。 在细胞分裂过程中。在有丝分裂中，附着在着丝粒上的微管有助于确保 姐妹染色单体形成子代细胞。我们的目标是了解控制着丝粒功能的机制。 着丝粒的同一性依赖于着丝粒特异的组蛋白H3变异体CENP-A的沉积，而不是 特定的DNA序列。DNA复制后，“旧的”着丝粒核小体转移到新的 复制的染色单体。在哺乳动物中，CENP-A的沉积发生在细胞周期的G1期。这个 CENP-A的沉积和维持机制对着丝粒的正常遗传和功能至关重要 但仍有待描述。我们最近发现EWSR1(尤文肉瘤断点区1)是必需的 用于CENP-A在着丝粒的沉积和维护。我们证明了EWSR1和EWSR1-FLI1( 尤文肉瘤中的癌基因融合蛋白)通过其蛋白样蛋白SYGQ2区域与CENP-A结合 已知对相分离很重要的结构域。我们发现CENP-A沉积需要EWSR1 在着丝粒。CENP-A与着丝粒RNA有关，着丝粒RNA对着丝粒功能具有重要作用。我们 证明EWSR1在促进这种联系方面发挥了关键作用。我们还有生化证据 EWSR1通过其RNA识别基序与R-环结合，R-环是一种三链RNA-DNA杂交体。基于 这些发现，我们假设EWSR1在维持着丝粒结构和功能方面起着至关重要的作用 通过结合RNA并将CENP-A锚定在着丝粒染色质上。我们将检验这一假设。目标1：实现 确定EWSR1和RNA/R环在着丝粒维持中的作用。我们生成了EWSR1 使用AID(生长素诱导降解)系统的条件性KO细胞，该系统允许我们在 外加生长素。我们将测试生长素诱导的EWSR1的降解是否会取消着丝粒上的CENP-A信号 处于不同的细胞周期阶段。我们将诱导SETX的表达，它清除RNA-DNA杂交物，在不同的 细胞周期阶段，询问着丝粒RNA/R环是否需要CENP-A在 着丝粒。目的：研究EWSR1-CENP-A相互作用的功能。我们将产生EWSR1突变体 通过将突变引入EWSR1的SYGQ2区域而导致与CENP-A结合的缺陷。我们将研究 表达CENP-A结合缺陷突变体的EWSR1 KO细胞的表型。我们还将确定 CENP-A的EWSR1结合域。然后，我们将分离EWSR1结合受损的CENP-A突变体。我们会 在CENP-A KO细胞中表达标志标记突变体以检测CENP-A的定位和CENP-A的表型 突变细胞。我们将调查EWSR1与CENP-A的关联是否在间期是唯一的。我们会 探讨EWSR1-CENP-A相互作用调节细胞周期特异性的机制。目标3：测试 EWSR1通过RNA结合将CENP-A锚定在着丝粒上。为了测试这个模型，我们将直接检查以下内容 通过构建和测试具有工程功能域的CENP-A和EWSR1变体来建立模型。