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，是高保真染色体传递必不可少的 在细胞分裂期间。在有丝分裂中，微管附着在动粒上，有助于确保细胞分裂的可靠性。 姐妹染色单体转化为子细胞。我们的目标是了解支配着丝粒功能的机制。 着丝粒身份依赖于着丝粒特异性组蛋白H3变体CENP-A的沉积，但不依赖于 特定的DNA序列。在DNA复制后，"旧的"着丝粒核小体被转移到新的着丝粒核小体上。 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。的 CENP-A的沉积和维持机制对于正确的着丝粒遗传和功能至关重要 但仍待划定。我们最近发现EWSR1（尤文肉瘤断点区域1）是必需的 用于CENP-A在着丝粒的沉积和维持。我们发现EWSR1和EWSR1-FLI1（ Ewing肉瘤中的致癌融合蛋白）通过其朊病毒样蛋白内的SYGQ 2区域结合CENP-A 已知对于相分离是重要的域。我们发现EWSR 1是CENP-A沉积所必需的 在着丝粒上。CENP-A与着丝粒RNA相关，这对着丝粒功能很重要。我们 证明EWSR1在促进这种关联中起着关键作用。我们还有生化证据 EWSR1通过其RNA识别基序结合R环，一种三链RNA-DNA杂交体。基于 根据这些发现，我们推测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中。我们将测试这个假设。目标1： 确定EWSR1和RNA/R环在着丝粒维持中的功能。我们生成了EWSR1 条件KO细胞使用AID（生长素诱导降解决定子）系统，这使我们能够去除EWSR 1后， 生长素添加。我们将测试生长素诱导的EWSR 1降解是否消除了着丝粒上的CENP-A信号 在不同的细胞周期阶段。我们将在不同的温度下诱导SETX的表达，SETX可以清除RNA-DNA杂合体， 细胞周期阶段，以询问是否着丝粒RNA/R环是CENP-A维持所必需的。 着丝粒。目的2：研究EWSR1与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EWSR 1的SYGQ 2区域而在与CENP-A的结合上有缺陷。我们会研究 表型的EWSR1 KO细胞表达CENP-A结合缺陷突变体。我们还将确定 CENP-A的EWSR1结合结构域。然后，我们将分离EWSR1结合受损的CENP-A突变体。我们将 在CENP-A KO细胞中表达Flag标记的突变体，以检查CENP-A的定位和 突变细胞我们将研究EWSR 1与CENP-A的关联是否是间期唯一的。我们将 探讨EWSR1-CENP-A相互作用调控细胞周期特异性的机制。目标3：测试是否 EWSR1通过RNA结合将CENP-A锚定在着丝粒处。为了测试这个模型，我们将直接检查 通过构建和测试具有工程化功能结构域的CENP-A和EWSR1变体来构建模型。